Accademia dei Georgofili
The First Symposium on Plant Neurobiology
May 17-20, 2005 Florence, ITALY
BOOK OF ABSTRACTS
Programme
Tuesday, May 17
Historical Perspectives and General Topics Chairman: Dieter Volkmann
09.00-09.30 Greetings from the President of the Accademia dei Georgofili 09.30-10.00 Peter Barlow: Charles Darwin and the plant root brain: closing the gap in plant living systems theory 10.00-10.30 Tony Trewavas: The green plant as an intelligent organism 10.30-10.50 Eric Davies & Bratislav Stankovic: Electrical signals, the cytoskeleton, and gene expression: current hypotheses on the coherence of the cellular responses to environmental insult
10.50-11.20 Refreshment
11.20-11.40 František Baluška: Neurobiological view of plants and their body plan 11.40-12.00 Ariel Novoplansky: Self/non-self discrimination in plants 12.00-12.20 Tsvi Sachs: How plants choose the most promising organ? 12.20-12.40 Peter Neumann: The role of root apices in shoot growth regulation 12.40-13.00 General Discussion
13.00-14.00 Lunch
Chairman: Eric Davies
14.00-14.30 Keith A. Mott: Information processing by stomatal networks 14.30-14.50 Michel Thellier: Memory effects in the response of plants to environmental signals 14.50-15.10 Jiri Friml: Auxin distribution network as a means to integrate and process signals in plant development 15.10-15.30 Markus Geisler: Active export of auxin by MDR-type ATP-binding cassette transporters
15.30-16.00 Refreshment
Molecules 16.00-16.20 Susan Murch: The brain of plants: neurotransmitters, neuroregulators and neurotoxins 16.20-16.50 Bazbek Davletov: Synaptotagmins and cell-to-cell communication at neuronal synapse 16.50-17.10 Molly Craxton: Synaptotagmin genes in plants and other eukaryotes 17.10-17.30 Günther Witzany: Serial Endosymbiotic Theory: the biosemiotic update 2005 17.30-17.50 Bratislav Stankovic: Intellectual property rights in smarty plants 17.50-18.10 General Discussion
2 Wednesday, May 18
Chairman: Tony Trewavas
09.00-09.30 Matthew Gilliham: Plant glutamate receptors – progress to date 09.30-09.50 Dirk Becker: Keeping in touch with plant glutamate receptors 09.50-10.10 Eric Brenner: Pharmacological approaches to understand plant glutamate receptors 10.10-10.30 Janet Braam: Touch-responsive gene expression
10.30-11.00 Refreshment
11.00-11.20 Petra Dietrich: Ca2+-permeable channels in the plant pathogen response 11.20-11.40 Bert de Boer: 14-3-3 brain proteins in plants: master regulators that couple signalling to ion transport 11.40-12.00 Jozef Šamaj: MAP kinases in neurons and plants 12.00-12.20 Tomohiro Uemura: SNARE molecules indicate the complexity of the post-Golgi traffic in plant cells 12.20-12.40 Gian-Pietro Di Sansebastiano: Syntaxin 1 as the central element of regulated exocytosis in plants 12.40-13.00 Viktor Žársky: Rab GTPases and Exocyst in plants
13.00-14.00 Lunch
Chairman: Peter Barlow
14.00-14.20 Jack C. Schultz: Receptor-like kinases: how plants sense their environment and can tell us what they "see" 14.20-14.40 Massimo Delledonne: Nitric oxide functions in plant disease resistance 14.40-15.00 Lorenzo Lamattina: Nitric oxide and auxin interactions in root formation
Cell Biology and Plant Physiology
15.00-15.30 Stefano Mancuso: Oxygen/Auxin influx and NO efflux: neural-like features of the transition zone of the root apex 15.30-15.50 Thomas Berleth: Polar signals in plant vascular development 15.50-16.10 Enrico Scarpella: Leaf vascular patterning in monocots and dicots
16.10-16.30 Refreshment
16.30-16.50 Noni Franklin-Tong: Signals and targets triggered by self-incompatibility: recognition of "self" can be deadly 16.50-17.10 Thorsten Nürnberger: Signal perception and transduction in plant innate immunity 17.10-17.30 Manfred Heinlein: TMV as a model for the analysis of RNA transport via plasmodesmata 17.30-17.50 Gladys Cassab: Hydrotropism: root growth responses to water 17.50-18.20 Thorsten Hamann: Cell wall integrity signalling in Arabidopsis thaliana 18.20-18.50 General Discussion
3 Thursday 19 May
Chairman: Keith Mott
09.00-09.30 Elison Blancaflor: N-acylethanolamines: emerging lipid mediators of seed and seedling development 09.30-09.50 Tobias Baskin: A Phytosynapse? The plant’s use of glutamate receptors to respond to aluminium 09.50-10.10 Pia Walch-Liu: Glutamate signalling and root development in Arabidopsis 10.10-10.30 Hillel Fromm: Genetic and pharmacological evidence for a role of the GABA shunt in maintaining the levels of reactive oxygen intermediates in plant cells
10.30-10.50 Refreshment
10.50-11.10 Frank Turano: Glutamate receptors and GABA in plant responses to environmental stress 11.10-11.30 Frank Ludewig: Arabidopsis knock out mutants of GABA metabolism and their response to different growth conditions 11.30-11.50 Erwan Le Deunff: Does GABA act as a long-distance signal in the regulation of nitrate uptake in plants? 11.50-12.10 Vadim Demidchik: Is ATP a signaling agent in plants? 12.10-12.40 Stanley Roux: Signaling role of ATP in growth control and in wound responses 12.40-13.00 Benoit Lacombe: Ion channels in plants: from DNA sequence to integrative biology 13.00-13.20 General Discussion
13.20-14.20 Lunch
Electrophysiology
Chairman: Thomas Berleth
14.20-14.50 Ivana Macháčková: Developmental effects of electric current in thermo- and photoperiodic plants 14.50-15.10 Kazimierz Trebacz: Action potentials – from mechanisms to functions 15.10-15.30 Sergey Shabala: Oscillations in plants 15.30-15.50 Rainer Stahlberg: Slow wave potentials – higher plants' very own propagating electrical signals
15.50-16.10 Refreshment
16.10-16.30 Edgar Wagner: Hydro-electrochemical integration of the higher plant – basis for electrogenic flower induction 16.30-16.50 Aart von Bel: Forisomes as sensors and aphids as reporters of Ca2+influx and efflux during depolarization waves along sieve tubes 16.50-17.10 Jörg Fromm: Heat-induced electrical signals change photosynthesis in poplar 17.10-17.30 General Discussion
4 Friday 20 May
Chairman: Janet Braam
09.00-09.20 Alexander George Volkov: Electrophysiology and phototropism 09.20-09.40 David Wildon: Signals and signalling pathways in plant wound responses 09.40-10.00 Alain Vian: Effects of high frequency electromagnetic stimulation on plants 10.00-10.20 Thijs Pons: Shade avoidance without photoreceptors
10.20-10.40 Refreshment
Plant-to-Plant Communication and Ecophysiology
Chairman: Janet Braam
10.40-11.00 Laura Perry: Root exudation and rhizosphere biology: allelochemicals and cell death 11.00-11.20 Ragan Callaway: The transmogrification of plant invaders: biogeographic differences in allelopathic effects and native evolutionary responses 11.20-11.40 Jürgen Engelberth: Interplant communication: from induced volatiles to signal transduction pathways 11.40-12.00 Ninkovic Velemir: Communication between undamaged plants by volatiles: the role of allelobiosis 12.00-12.20 Manuela Giovannetti: Self/non-self recognition in mycorrhizal – root system networks 12.20-12.40 Wilhelm Boland: Early chemical and electrical signals in herbivore stressed plants 12.40-13.00 General Discussion
13.00-14.00 Lunch
14.00-15.00
‘Plant Neurobiology’: where to go now?
Chairmen: Stefano Mancuso & František Baluška
1. Journal
2. Society
3. The next meeting
5
Abstracts
6 Charles Darwin and the plant root brain: closing the gap in plant living systems theory
Peter W. Barlow
School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK Email: [email protected]
Charles Darwin was “always pleased to exalt plants in the scale of organised beings” and “felt an especial pleasure in showing how many and what admirably well adapted movements the tip of a root possesses.” (1) Indeed, Darwin went so far as to say that the root tip acts like a brain, located within the anterior end (sic) of the plant body, “receiving impressions from the sense organs and directing the root’s several movements.” (2) What clearly impressed Darwin was the ability of the root to perceive, often simultaneously, multiple vectorial stimuli – touch, moisture, light, gravity – and then make a “decision” about which “final purpose”, or bending response, to follow. In connection with these growth responses, Darwin had also said that “plants do not of course possess nerves or a central nervous system.” (2) According to J.G. Miller’s “Living Systems Theory” (LST), developed over the last three decades mainly for human organisms and human societies, (3) there are sets of 20 subsystems which, within each level of organisation, from cellular to supranational, are functionally equivalent and govern each level’s activity. LST can be adapted, apparently with success, to plant organisms also. (4) About half of the LST subsystems concern the processing of information. Of interest in the present plant-neurobiological context is the subsystem “Channel and Net”. (5) In an earlier formulation of plant LST, (4) this subsystem was equated, at the cellular level of organisation, with cytoskeleton and endomembranes, and with the symplasm at the organ level. Now, in the light of recent discoveries from plant cell biology, these designations appear to be confirmed, reinforcing the idea that plants do possess a form of nervous system which, moreover, makes use of molecules and organelles similar to those found in animal systems. As a result, the systems-analytical approach to the constructional hierarchy of plant life converges upon that already recognised for animals, (3) hence providing a truly coherent LST for the two major living systems of Plants and Animals.
Darwin C. 1880. The Power of Movements in Plants. London: John Murray Darwin C. 1876/1983. Autobiography. In G de Beer, ed; Charles Darwin and TH Huxley, Autobiographies. Oxford: Oxford University Press. p 8-88. Miller JG. 1978. Living Systems. New York: McGraw-Hill Publishing Co. Barlow PW. 1999. Living plant systems: How robust are they in the absence of gravity? Adv Space Res 23:1975-1986. Miller JL, Miller JG. 1995. Greater than the sum of its parts III. Information processing subsytems. Channel and net. Behav Sci 40:238-268.
7 The green plant as an intelligent organism
Tony Trewavas
Institute of Cell and Molecular Biology, Mayfield Road, University of Edinburgh, Edinburgh EH9 3JH, UK Email: [email protected]
Intelligent behaviour, a complex adaptive phenomenon, is designed to increase fitness in variable environmental circumstances particularly those involving resource foraging and competition. Biologists suggest that intelligence encompasses the characteristics of detailed sensory perception, information processing, learning, memory, choice, efficient optimisation of resource sequestration with minimal outlay, self-recognition, and foresight by predictive modelling. There is good evidence that individual plant species exhibit all of these intelligent behavioural capabilities. Plants should therefore be regarded as prototypical intelligent organisms, a concept that has considerable consequences for evolutionary convergence and investigations of whole plant communication, computation and signal transduction.
8 Electrical signals, the cytoskeleton, and gene expression: current hypotheses on the coherence of the cellular responses to environmental insult
Eric Davies* and Bratislav Stankovic Department of Botany, North Carolina State University, 1231 Gardner Hall Raleigh, NC 27695 USA *Email: [email protected]
When plant tissue is abiotically injured by crushing, cutting, flaming, electrical stimulation or several other means, the injured (perceiving) tissue generates electrical signals, including action potentials and variation potentials. These are transmitted to distant regions (responding tissue) where they evoke apparently disparate responses, including callose formation, closing of plasmodesmata, stoppage of cytoplasmic streaming, inhibition of ribosome movement along mRNA, and ultra-rapid but transient accumulation of several hundred transcripts, which are degraded without being translated. These apparently disparate responses can be reconciled by one fundamental hypothesis that assumes that “the plant does now know what hit it” and thus “expecting the worst” mounts a holistic defense response against its most potent nemesis, a putative viral invasion. The basis for this response is calcium influx into the cytoplasm via voltage-gated channels (action potential) associated with the microtubules, or via mechano-sensitive channels (variation potential) associated with microfilaments. The calcium interacts with calcium and/or calmodulin-dependent cytoskeleton-associated protein kinases. This causes the phosphorylation of myosin, which stops cytoplasmic streaming, and of elongation factor 2F, which slows elongation and causes ribosomes to pile up on polysomes, thereby decreasing protein synthesis, but protecting pre-existing “host” transcripts from degradation. The phosphorylation signal then passes into the nucleus where it phosphorylates RNA polymerase II, which goes into overdrive, (i.e., does not stop at accuracy check-points), thus causing the synthesis of large amounts of mis-made mRNA. The mRNA is transported into the cytoplasm where it is scanned (checked for accuracy) by ribosomes, and found to be incorrect. This surveillance mechanism stimulates ribonuclease activity which degrades the free (non- polysome-associated), mis-made RNA, but leaves the original, “host” mRNA unscathed since it is protected by ribosomes. The ribonuclease also (and here is the crux of the matter) attack other free mRNAs, including viral mRNAs, so these are disposed before they can be translated. Within minutes this reaction is over, cytoplasmic steaming resumes, translation continues, ribosomes are released and so can be used to translate new (correctly-made) transcripts.
9 Neurobiological view of plants and their body plan
František Baluška1,*, Stefano Mancuso2, Dieter Volkmann1, and Peter W. Barlow3
1 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany 2 Dept. Horticulture, University of Firenze, Viale delle idee 30, 50019 Sesto F.no (FI), Italy 3 School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK *Email: [email protected]
In the centuries following the time of Aristotle and his students, who made the first philosophical attempts to understand plants in their complexity, interest in herb plants was limited mainly to their medical usage. This changed in the sixteenth century when the first biological attempts were done to understand the basic principles of structure and function of plants. At first, studies were largely devoted to plant distribution, taxonomy, and morphology. Later, taking the lead from medicine, anatomy and cytology of plants were added to the curriculum of plant sciences, as studied in the early universities. In fact, the cellular nature of living organisms was first elaborated using plants (Hooke 1665). By the end of the 19th century, it was realised that plants were even more similar to animals than had been thought hitherto. Advances in physiology helped confirm this, especially with regard to some of the basic physiological processes, such as respiration, digestion, and cell growth, where plants often provided the material of choice for experimental studies. All principal metabolic biochemical pathways are conserved in both animal and plant cells. As a big surprise, plants have been shown to be identical to animals from several other rather unexpected perspectives. For their reproduction, plants use identical sexual processes based on fusing sperm cells and oocytes. Next, plants attacked by pathogens develop immunity using the corresponding processes and mechanisms operating in animals. Last but not least, both animals and plants use the same molecules and pathways to drive their circadian rhythms. Currently, plant science has reached another cross-road as the critical mass of new data has been accumulated, culminating in the emergence of plant neurobiology as the most recent area of plant sciences. Plants are intelligent organisms which perform complex information processing (Trewavas 2003, 2004) and which use not only action potentials but also synaptic modes of cell-cell communication(Baluška et al. 2003, 2004, 2005). Thus, the term ‘plant neurobiology’ appears to be justified. In fact, the word neuron was taken by animal neurobiologists from Greek where the original meaning of this word is vegetal fibre. Moreover, there are several surprises if we apply a ‘neurobiological’ perspective to how the plant tissues and body are organized. Auxin emerges as a plant-specific neurotransmitter (Baluška et al. 2003). Root apices are specialized not only for the uptake of nutrients but also seem to support neuronal-like activities based on plant synapses (Baluška et al. 2004, 2005). All this suggests that root apices represent the anterior pole of the plant body. In accordance with this perspective, shoot apices act as the posterior pole. They are specialized for sexual reproduction and the excretion of metabolic products via hydatodes, trichomes, and stomata. Next, vascular elements allow the rapid spread of hydraulic signals and action potentials, resembling nerves. As plants are capable of learning and they take decisions about their future activities according to the actual environmental conditions (Trewavas 2003, 2004), it is obvious that they possess a complex apparatus for the storage and processing of information.
Baluška F, Šamaj J, Menzel D (2003) Polar transport of auxin: carrier-mediated flux across the plasma membrane or neurotransmitter-like secretion? Trends Cell Biol 13: 282-285 Baluška F, Mancuso S, Volkmann D, Barlow PW (2004) Root apices as plant command centres: the unique ‘brain-like’ status of the root apex transition zone. Biologia 59: 7-19 Baluška F, Volkmann D, Menzel D (2005) Plant synapses: actin-based domains for cell-to-cell communication. Trends Plant Sci 10: 106-111 Hooke R (1665) Of the schematisme or texture of cork, and of the cells and pores of some other such frothy bodies. Observation 18. Micrographia, pp 112-116. London Trewavas A (2003) Aspects of plant intelligence. Ann Bot 92: 1-20 Trewavas A (2004) Aspects of plant intelligence: an answer to Firn. Ann Bot 93: 353-357
10 Competitive self/non-self discrimination in plants
Ariel Novoplansky
Mitrani Department of Desert Ecology, Blaustein Institute for Desert Research, Ben- Gurion University of the Negev, Midreshet Ben-Gurion, 84990, Israel Email: [email protected]
Competition usually involves the allocation of limiting resources to non-reproductive functions. Natural selection is expected to favour mechanisms that increase competition with nonself neighbours and limit wasteful competition with self. We compared the growth of plants that were grown in the presence of neighbors that belonged to the same physiological individual, were separated from each other for variable periods, or originated from adjacent or remote tillers on the same clone. The results demonstrate that Pisum sativum, Buchloe dactyloides and Trifolium repens plants are able to differentiate between self and nonself neighbors and develop fewer and shorter roots in the presence of other roots of the same plant. Furthermore, once Buchloe dactyloides cuttings that originate from the very same node are separated they become progressively alienated from each other and eventually relate to each other as genetically alien plants. The results suggest that the observed self/nonself discrimination is mediated by physiological coordination among roots that developed on the same plant rather than allogenetic recognition. The observed physiological coordination is based on, an as yet, unknown mechanism and has important ecological implications for it allows the avoidance of competition with self and the allocation of greater resources to alternative functions.
11 How can plants choose the most promising organs?
Tsvi Sachs
Plant Sciences, The Hebrew University, Jerusalem 91904, Israel Email: [email protected]
Almost all plants have numerous apical meristems, each with the potential of forming an entire shoot system. Since resources are limited, a plant must ‘choose’ the best alternatives, based on integrated though incomplete information from various sources and its predictions about future performance. An additional challenge is that the development of any meristem must be correlated with appropriate vascular differentiation and other developmental events, throughout the plant. The presentation will aim to show how, in the absence of a central decision organ, integration of both types can depend on the formation of, and responses to, auxin and other simple molecular signals. Experiments were carried out on a model system, pea seedlings with two shoots. In most plants only one of these shoots would continue growing. We studied the influence of both local and overall environmental conditions, the removal of leaves of different ages, and the previous developmental rate of individual shoots on the choice of which shoot will continue growing. The evidence of both experiments and comparative observations supports the following hypothesis. All components of a shoot are sources of auxin and presumably other signals. The synthesis of different amounts of the very same hormones depends on the immediate environment and developmental stage of individual organs. The responses to auxin occur throughout the plant and include the orientation of vascular differentiation towards organs that are its strongest source. This results in competition between alternative organs according to integrated information about their state. This information predicts their future contribution according to the adaptations and evolutionary experience of the species.
12 The role of root apices in shoot growth regulation
Peter M. Neumann
Department of Environmental , Water & Agricultural Engineering, Civil and Environmental Engineering Faculty, Technion-Israel Institute of Technology, Haifa 32000, Israel Email: [email protected]
Long distance communication between roots and shoots of higher plants is essential for regulating whole plant adaptations to environmental changes. For example, decreased availability of essential mineral nutrients or water in the soil is often followed by a seemingly adaptive inhibition of shoot growth. Chemical, hormonal, electrical or hydraulic signals may be used to transmit status information between plant organs. The roots will clearly be the first to generate such signals in response to rhizosphere changes. Whether active neuronal type changes in the root apices or simple passive changes are required in order to generate and transmit such information to the developing shoots, is the subject of this chapter. Several examples of laboratory experiments in which roots appear to respond actively in response to changes in nutrient or water availability are first discussed. However, other examples indicate that shoot responses to rhizosphere changes can occur without the active intervention of root apices. The conclusion is that while neuronal types of communication may occur in plants, they are not always essential for early plant growth responses to environmental changes.
13 Information processing by stomatal networks
Keith A. Mott
Biology Department, Utah State University, Logan, Utah, USA Email: [email protected]
Stomata must respond to environmental factors such that they open to admit enough CO2 for photosynthesis, yet close sufficiently to prevent excessive water loss. Much of this regulation occurs at the level of the individual pore, through signal transduction pathways in the guard cells. However there is increasing evidence that stomata interact with each other over short distances and can therefore be said to form a locally-connected network. We present evidence showing that stomatal networks may be processing information in a manner similar to artificial networks that perform distributed emergent computation. This information processing may allow stomata networks to optimize gas exchange for an entire leaf or plant despite the fact that each individual stoma has only local information.
14 Memory effects in the response of plants to environmental signals
Michel Thellier*, Marie-Claire Verdus, Vic Norris, Camille Ripoll
Laboratoire AMMIS, FRE CNRS 2829, Faculté des Sciences de l’Université de Rouen, F76821 Mont-Saint-Aignan Cedex, France *Email: [email protected]
Plants are sensitive to stimuli from the environment (e.g. wind, rain, contact, pricking, wounding). They usually respond to such stimuli by metabolic or morphogenetic changes. These changes may occur in tissues at a distance from the stimulated region, which implies that the stimulus must be transmitted in some form from the stimulated region to the region where it takes effect (i.e. the stimulus and the response then are spatially separate). Sometimes the information corresponding to a stimulus may be “stored” in the plant where it remains inactive until a second stimulus “recalls” this information and finally allows it to take effect (i.e. the stimulus and the response then are temporally separate). Two experimental systems have proved especially useful in unravelling the main features of these memory-like processes. In the system based on Bidens seedlings, an asymmetrical treatment (pricking, or gently rubbing one of the seedling cotyledons) causes the cotyledonary buds to grow asymmetrically after release of apical dominance by decapitation of the seedlings. This information may be stored within the seedlings, without taking effect, for at least two weeks; then the information may be recalled by subjecting the seedlings to a second, appropriate, treatment that permits transduction of the signal into the final response (differential growth of the buds). Whilst storage is an irreversible, all-or-nothing process, recall is sensitive to a number of factors, including the intensity of these factors, and can readily be enabled or disabled. In consequence, it is possible to recall the stored message several times successively. In the system based on flax seedlings, a mechanical stimulus (e.g. wind, touching) has no apparent effect. If, however, the plant is subjected at the same time to a 2-day calcium deprivation, numerous meristems form in the stem. When the calcium deprivation treatment is applied a few days after the mechanical treatment, t he time taken for the meristems to appear is increased by a number of days exactly equal to that between the application of the mechanical treatment and the beginning of the calcium deprivation treatment. This means that the information corresponding to the mechanical treatment has been stored in the plants, without any apparent effect, until the calcium deprivation treatment recalls this information to allow it to take effect. The response of flax seedlings to cold shock, to radiation from a GSM telephone and to low intensity electromagnetic radiation at 105 GHz is the same as to mechanical treatment, namely storage of the information and recall after calcium deprivation. The calcium status of the plants before, during and after the calcium deprivation treatment has been studied by SIMS (secondary ion mass spectrometry). Gel electrophoresis has shown that a few protein spots are shifted (compared to controls) in flax seedlings that have stored information corresponding to a cold shock signal but that are not in a state to recall it; a SIMS investigation has revealed that the shift of one of these spots is probably due to protein phosphorylation. Similar modifications of the proteome have been observed in Arabidopsis seedlings subjected to the same stimuli. We suggest that storage/recall processes in plants constitute an elementary form of « memory » and that, given their simplicity relative to the animal brain, these plant processes may be used as experimental systems for testing hypotheses about memory storage and evocation.
15 Auxin distribution network as a means to integrate and process signals in plant development
Marta Michniewicz, Jozef Mravec, Tomasz Paciorek, Kamil Ruzicka, Michael Sauer, Anne Vieten, Justyna Wisniewska, Jiri Friml*
Zentrum für Molekularbiologie der Pflanzen, Universität Tübingen, Auf der Morgenstelle 3, 72076 Tübingen, Germany *Email: [email protected]
Plant hormone auxin is a prominent intercellular signal in plants. Auxin distributed over long distances largely contributes to the coordination and integration of growth at the whole plant level. On the other hand, directional, active, cell-to-cell transport over short distances mediates local, differential auxin distributions (gradients), required for various patterning processes, including apical-basal axis formation and organogenesis. Also growth responses to environmental cues such as light or gravity utilize a similar mechanism involving auxin gradients. Differentially expressed auxin transport facilitators of the PIN family, each with specific polar, subcellular localization form a network for auxin distribution and formation of these local gradients. The activity of PIN proteins can be regulated at the single cell level by changes in their vesicle trafficking-dependent polar targeting in response to developmental and environmental cues. Thus, this auxin distribution network, whose directional throughput is modulated by both endogenous and exogenous signals, provides, by means of mediating auxin fluxes and creating local gradients, a common mechanism for the plasticity and adaptability of plant development.
16 Active export of auxin by MDR-type ATP-binding cassette transporters
Markus Geisler1,*, Joshua J. Blakeslee2, Rodolphe Bouchard1, Anindita Bandyopadhyay2, Wendy Ann Peer2, Robert Dudler1, Angus S. Murphy2, Enrico Martinoia1
1 Institute of Plant Biology, Basel-Zurich Plant Science Center, University of Zürich, CH-8007 Zurich, Switzerland 2 Department of Horticulture, Purdue University, West Lafayette, IN 47907 USA *Email: [email protected]
The plant hormone auxin regulates virtually all plant developmental processes. On the cellular level, a protein complex characterized by the PIN proteins is thought to mediate auxin efflux, however, PIN-mediated transport has not been conclusively demonstrated. Recent findings suggest that multidrug resistance (MDR)-like p-glycoproteins (PGPs) function in auxin transport, but the molecular basis remains elusive. Here we report decreased auxin efflux in atpgp mutant protoplasts as well as the export of IAA and synthetic auxins by AtPGP1 and AtPGP19 heterologously expressed in yeast and HeLa cell systems. In contrast to the gradient- driven model proposed for PIN-mediated transport, biochemical and pharmacological analysis demonstrates an energy-dependent, primary active transport mechanism for AtPGP-mediated auxin transport. Further, the auxin transport inhibitors NPA and quercetin that bind AtPGP1 and AtPGP19 were shown to inhibit AtPGP function. Basipetal auxin transport in atpgp1 roots is reduced, which correlates well with AtPGP1 basal plasma membrane localization in cortical root cells. Our data suggest the presence of a primary energized auxin transport system involved in polar auxin transport.
17 The brain of plants: neurotransmitters, neuroregulators and neurotoxins
Susan J. Murch
Institute for Ethnobotany, National Tropical Botanical Garden, 3530 Papalina Road, Kalaheo, 96741, HI, USA Email: [email protected]
The existence of humans in our world is dependent on plants and human culture is profoundly influenced by those plants which affect the function of our brains. Some plants are chosen and cultivated for their capacity to change our moods while other plants contain toxins that impair our cognitive functions. One of the more fascinating questions in plant biology is the role of these compounds in the growth and development of higher plants. It has been hypothesized that neurologically active secondary metabolites in plants function as anti-herbivory compounds or attractants. Additionally, recent evidence suggests that the compounds may play regulatory roles in plants similar to those observed in other species. The discovery of the mammalian neurohormone melatonin in growing plants and the correlation of this compound with photoregulated processes is one example of a human hormone that mediates plant growth. Further studies have uncovered a range of neurologically-active plant-based compounds with functions and activities in organisms throughout the ecosystem. As new techniques are developed to study these compounds and their activity in plant and animal systems, there is enormous potential to develop a greater understanding of plant life, human health and human diseases.
18 Synaptotagmins and cell-to-cell communication at neuronal synapse
Bazbek Davletov
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK Email: [email protected]
Intracellular membrane traffic is governed by a conserved set of proteins including SNARE fusion proteins and synaptotagmins (Syts). The mammalian Syt family consist of 16 isoforms. Syts are membrane proteins that possess tandem C2 domains (C2AB) implicated in calcium- dependent phospholipid binding. We performed a pair-wise amino acid sequence comparison together with functional studies of rat Syt C2ABs to examine common and divergent properties within the mammalian family. Sequence analysis indicates three different C2AB classes, the members of which share a high degree of sequence similarity. All the other C2ABs are highly divergent in sequence. Interestingly, nearly half of the Syt family does not exhibit calcium- dependent phospholipid binding. Syts do however possess a more conserved function, namely constitutive binding to target SNARE heterodimers. All tested isoforms bound the target SNARE dimer composed of syntaxin1 and SNAP-25. Our study suggests that both calcium-sensitive and insensitive Syt isoforms function in membrane traffic to interact with the target SNARE heterodimer on the pathway to membrane fusion.
19 Synaptotagmin genes in plants and other eukaryotes
Molly Craxton
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK Email: [email protected]
Synaptotagmin genes are not just present in animals with nervous systems, but also occur in plants. Sequence analysis indicates the presence of synaptotagmin genes in all animals and all land plants, but there is no evidence of synaptotagmin genes in unicellular organisms or those with simple forms of multicellularity. Animals posess diverse synaptotagmin genes, the number of which varies with organism complexity. There have been different patterns of synaptotagmin gene acquisition and loss in different animal lineages. All animals posess a Syt1 homologue which provides the function of fast, synchronous, calcium regulated synaptic vesicle exocytosis, essential for efficient neurotransmission. The diverse other synaptotagmin homologues appear to be involved in other types of specific vesicle trafficking within and beyond the nervous system. There appear to be three different types of synaptotagmin genes in plants, and varying numbers of each type in different species. Sequence analysis shows that these genes are related to groups of genes present in a wide variety of eukaryotes including animals, plants, fungi and protozoa. These relatives all have C2 domains, but none have the N-terminal transmembrane, linker, C2A, C2B arrangement which defines synaptotagmins. Unfortunately, these genes are all poorly functionally characterized. Although plants lack Syt1 homologues, and their synaptotagmin genes are clearly divergent from those of animals, it still remains to establish whether the functional roles of the plant and animal genes overlap. Functional studies of the synaptotagmin genes of the primitive moss Physcomitrella, and of Arabidopsis and other higher plants are now in progress.
20 Serial Endosymbiotic Theory (SET): the biosemiotic update 2005
Günther Witzany
Vogelsangstraße 18c, A-5111-Bürmoos, Austria Email: [email protected]
The serial endosymbiotic theory (SET) explains the origin of nucleated organisms through merging between archae-bacterial and eubacterial cells. The paradigmatic change to former evolutionary theories is that the driving force of evolution is not ramification but merging. Lynn Margulis describes the symbiogenetic processes in the language of the classical biology in termslike “merging”, “fusion”, etc. within natural laws. Biosemiotics has proved all cell-cell- interactions are rule-governed sign-mediated interac-tions (rsi’s). Rsi’s between and within cells and cell societies can be described better in terms of a biology as an understanding social science, than in terms of the classical biology. The decisive difference between natural laws and semiotic rules is that every living being is underlying natural laws in a strict sense, as opposed to semiotic rules may be followed or not, may be changed or not, may be generated or not; so living beings have a relationship to rsi’s but not to natural laws. The change from the classical biology to a biology as understanding social science may be also a change from the 3 rd person perspective (external observer) to the 1 st/2 nd person perspective (performative participant). It leaves behind the subject-object-dichotomy and integrates the Umwelt –concept (J.v.Uexkuell) into a Mitwelt- concept, in which all living beings are participants of a universal community of communicating life.
21 Intellectual property rights in smarty plants
Bratislav Stankovic
BRINKS HOFER GILSON & LIONE, NBC Tower - Suite 3600, 455 North Cityfront Plaza Drive, Chicago, IL 60611-5599 , USA Email: [email protected]
Advances in plant biology are being affected by the continuous strengthening of intellectual property protection for biological inventions. The acquisition of rights in intellectual property controls the research and commercial use of protected techniques, materials, genes, seeds, vectors, cell lines, etc. Efforts are underway to harmonize intellectual property right regimes in the international arena. The recognition of the existence of plant neurobiological phenomena creates new opportunities for the scientists that study smarty plants. This presentation will highlight the above issues and will educate scientists so that they can make informed decisions regarding their research practices and the licensing of their discoveries. Patented neurobiological inventions relating to human and animal model systems will be used as reference points. Parallels will be drawn, and the potential for obtaining plant-specific intellectual property rights will be presented.
22 Functional characterisation of Arabidopsis glutamate-like receptors
Matthew Gilliham1,*, Lai-hua Liu1, Pauline Essah1, Romola Davenport1, Mark Tester2, Julia Davies1
1 Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK 2 Australian Centre for Plant Functional Genomics & University of Adelaide, PMB1, Glen Osmond, South Australia, 5074, Australia *Email: [email protected]
There are twenty members of the Arabidopsis glutamate-like receptor family (AtGLR) that have been so named as they share significant similarity with the animal ionotropic glutamate receptors (iGLuR). Animal iGLuR are non-selective cation channels that have been demonstrated to function predominately as Ca2+ and Na+ influx pathways, particularly at synaptic junctions. Although it is predicted that AtGLR have the 3+1 channel-like structure of iGLuR, and they also share a ligand-binding domain of significant similarity to the animal iGluR, the amino acid sequence within the putative pore of AtGLR is unlike any other known channel. This makes predictions of AtGLR selectivity, if they are indeed ion channels, impossible. In an attempt to determine AtGLR function, the expression of several members of the family has been attempted in Xenopus oocytes, a popular expression system for iGLuR. Preliminary results indicating successful heterologous expression and characterisation of AtGLR as Ca2+, K+ and Na+ permeable ion channels have been reported at several conferences over the last five years. So far, however, these reports have not been successfully published in peer reviewed journals. iGLuR form functional channels by combining subunits into hetero- or homo-tetramers therefore, as well as the expression of single members of the AtGLR family we are undertaking co-expression studies to screen for AtGLR function. The utility and difficulty of using Xenopus oocytes as an heterologous expression system for plant ion channels and transport proteins will therefore be discussed with reference to our own attempts to characterise AtGLRs. In the light of difficulties in the definitive functional characterisation of the AtGLRs in heterologous systems, we are also turning our attention to the plant resource and conducting electrophysiological studies in planta in an attempt to elucidate AtGLR function.
23 Keeping in touch with plant glutamate receptors
Oliver Meyerhoff1, Katharina Müller1, M. Rob G. Roelfsema1, Andreas Latz1, Benoit Lacombe2, Rainer Hedrich1, Petra Dietrich3, Dirk Becker1,*
1 University of Wuerzburg , Molecular Plant Physiology & Biophysics, Julius-von- Sachs-Platz 2, 97082 Wuerzburg , Germany 2 Biochimie et Physiologie Moleculaires des Plantes, UMR 5004, Agro- M/CNRS/INRA/UM2, F-34060 Montpellier Cedex 1, France 3 University of Erlangen, Institut für Molekulare Pflanzenphysiologie, Staudtstr. 5, 91058 Erlangen, Germany * Email: [email protected]
The Arabidopsis genome encodes for 20 members of putative ligand-gated channels, termed glutamate receptors (GLR). Despite the fact that initial studies suggested a role of GLRs in various aspects of photomorphogenesis, calcium homeostasis or aluminium toxicity, their functional properties and physiological role in plants remain elusive. Here we focussed on AtGLR3.4, which is ubiquitously expressed in Arabidopsis including roots, vascular bundles, mesophyll cells and guard cells. AtGLR3.4 encodes a glutamate-, touch-, and cold-sensitive member of this gene family. Abiotic stress stimuli such as touch, osmotic stress or cold stimulated AtGLR3.4 expression in an abscisic acid independent, but calcium dependent manner. In plants expressing the Ca2+-reporter apoaequorin glutamate as well as cold elicited cytosolic calcium elevations. Upon glutamate treatment of mesophyll cells, the plasma membrane depolarised by about 120 mV. Both glutamate responses were transient in nature, sensitive to glutamate receptor antagonists, and were subject to desensitisation. One hour after eliciting the first calcium signal, a 50 % recovery from desensitisation was observed, reflecting the stimulus-induced fast activation of AtGLR3.4 transcription. We thus conclude that AtGLR3.4 in particular and GLRs in general could play an important role in the Ca2+-based, fast transmission of environmental stress.
24 Pharmacological approaches to understand plant glutamate receptors
Eric D. Brenner1,*, Dennis Stevenson1, Joanna Chiu2, Suzan Runko1, Nora Barboza3, Gloria Coruzzi3
1 The New York Botanical Garden 200th St. and Kazimiroff, Bronx, NY 10458, USA 2 Dept. of Biology, 1009 Main Building , New York University, New York , NY 10003, USA *Email: [email protected]
Plants produce a variety of compounds which affect the human nervous system. Although their effects on humans have been well studied, their role in plants is poorly understood. One such compound, BMAA [S(+)-beta-Methyl-alpha, beta-diaminopropionic acid], is common to all species of the most ancient, living seed plants, the cycads. BMAA in cycads has been incriminated as the etiological source of Guam’s dimentia, a disease in the South Pacific-- manifested often years after exposure, that results in an Alzheimer’s and Parkinson’s disease. In the animal nervous system, BMAA acts upon ionotropic and metabotropic glutamate receptors (iGluRs), which are ligand-gated ion channels that transmit synaptic signals necessary for a variety of functions including vision and memory. It is not known if BMAA has a physiological role in plants; however, genes with high sequence similarity to animal iGluRs have been identified in a number of plant species. We have taken a pharmacological approach to uncover the role of plant glutamate receptor (AtGLR) genes, by examining the effects of BMAA, a cycad-derived iGluR agonist, on Arabidopsis morphogenesis. When grown in the presence of BMAA, Arabidopsis seedlings show a two to three fold increase in hypocotyl length and a significant inhibition of cotyledon opening. The effect of BMAA on hypocotyl elongation is light-specific and can be reversed by the simultaneous application of glutamate, the native iGluR agonist in animals. A genetic screen was devised to isolate Arabidopsis mutants with a BMAA insensitive morphology (bim). When grown in the light on BMAA, bim mutants have shorter hypocotyls then wildtype. Analysis of the bim mutants, as well as the role of BMAA, will be discussed.
25 Touch-responsive gene expression
Janet Braam
Biochemistry and Cell Biology, Rice University, Houston, TX 77005-1892, USA Email: [email protected]
Plants can perceive environmental stimuli with exquisite sensitivity. Indeed, even the seemingly innocuous stimulus of touch can elicit elaborate and unexpected responses in plants (reviewed in (1)). Plants use the sense of touch to capture prey, enhance the likelihood of crosspollination and climb other objects to reach heights resplendent in sunshine. Even nonspecialized plants respond to mechanical perturbations, such as wind and gravity, through morphogenetic alterations. Darwin noted that plant roots turn and grow away from points of contact (2). Jaffe coined the term thigmomorphogenesis to describe the touch-induced decreased elongation and enhanced radial expansion of plant shoots (3). Single cells, in addition to organisms, are likely to perceive and respond to mechanical stimuli. To maintain turgor homeostasis, individual cells must sense turgor pressure and wall integrity. In addition, subcellular organelles can translocate in response to mechanical perturbations. Signaling molecules and hormones, including intracellular calcium, reactive oxygen species, octadecanoids and ethylene have been implicated in touch responses. Touch stimulation can also rapidly alter gene expression regulation. The Arabidopsis thaliana TCH genes were discovered as genes whose expression is upregulated in plants perturbed by touch or wind (4). The TCH genes encode proteins predicted to function in calcium signal transduction and cell wall modification. Recently, we investigated the prevalence of touch inducibility among plant genes (5) . Touch- induced gene expression is surprisingly widespread; more than 2.5% of Arabidopsis genes are rapidly upregulated in expression in touch-stimulated plants. In addition to encoding calcium- binding and cell wall modifying proteins, the induced genes include many encoding proteins predicted to function in defense, as transcription factors and as protein kinases. With these genes as tools, we are employing molecular genetic methods to begin to elucidate mechanisms of touch perception, signal transduction and response regulation.
Research supported by: the National Science Foundation (IBN 0313432, IBN 0235953, IBN 9982654), and the Department of Energy (DE-FG02-03ER15394)
Braam, J., In touch: plant responses to mechanical stimuli. New Phytol., 2005. : p. 373-389. Darwin, C., The power of movement in plants. 1880, London: William Clowes and Sons, Ltd. Jaffe, M.J., Thigmomorphogenesis: The response of plant growth and development to mechanical stimulation. Planta, 1973. 114: p. 143-157. Braam, J. and R.W. Davis, Rain-, wind- and touch-induced expression of calmodulin and calmodulin-related genes in Arabidopsis. Cell, 1990. 60: p. 357-364. Lee, D., D.H. Polisensky, and J. Braam, Genome-wide identification of touch- and darkness- regulated Arabidopsis genes: a focus on calmodulin-like and XTH genes. New Phytol., 2005. 165: p. 429-444.
26 Ca2+-permeable channels in the plant pathogen response
Petra Dietrich
University of Erlangen, Dept. of Molecular Plant Physiology, Staudstr. 5, 91058 Erlangen, Germany Email: [email protected]
Production of reactive oxygen species (ROS) and hypersensitive cell death are characteristic features of the induced pathogen resistance in plants. Ca2+-signals preceding the pathogen- induced cell death have been shown to involve Ca2+ entry via the plasma membrane. We therefore analyzed Ca2+-signals and Ca2+-channels involved in the responses to pathogen attack and ROS. For this purpose, we used the Ca2+-reporter protein aequorin as well as the combined single-cell fluorescence imaging and patch clamp technique. Moreover, we compared Ca2+-channel activities in Arabidopsis wild type and the pathogen-resistant mutant, dnd1 (Yu et al., 1998) . Cellular responses to ROS were significantly reduced in dnd1. Since DND1 was shown to encode the Ca2+-permeable and cyclic nucleotide-gated ion channel CNGC2 (Clough 2+ et al., 2000) , this channel represents a possible candidate for H2O2-dependent Ca entry via the plasma membrane. We thoroughly examined this hypothesis and will present recent results on how CNGC2 transmits cellular responses to reactive oxygen species, a step essential for the control of hypersensitive cell death.
Clough SJ, Fengler KA, Yu IC, Lippok B, Smith RK, Jr., Bent AF (2000) The Arabidopsis dnd1 "defense, no death" gene encodes a mutated cyclic nucleotide-gated ion channel. Proc. Natl. Acad. Sci. USA 97: 9323-9328 Yu IC, Parker J, Bent AF (1998) Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant. Proc. Natl. Acad. Sci. USA 23: 7824
27 14-3-3 brain proteins in plants: master regulators that couple signaling to ion transport
Bert de Boer*, Peter Schoonheim, Oleg Klychnikov
Vrije Universiteit, Fac. Earth & Life Sci., Dept. of Structural Biol., Section Mol. Plant Physiol. & Biophys. De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands *Email: [email protected]
14-3-3 proteins were originally identified during a systematic classification of brain proteins. The brain does contain high levels of 14-3-3 protein and it was thought initially that they were specific for neuronal tissue. Not surprisingly, diseases like Alzheimer, Parkinson and SCA1 have been linked to 14-3-3 function. It is now clear that 14-3-3 protein are ubiquitously expressed in almost all eukaryotic cells and that they have a wide range of functions due to the phosphorylation dependent interaction with numerous (>300) partner proteins (1). During evolution the basic structure of 14-3-3 proteins has been conserved and in contrast to the initial picture it looks like there is also functional conservation. Thus, it becomes clear that both in plant and in animals 14-3-3 proteins are essential for the activity of certain ion transporters, like pumps and channels (2). Here we report on the function of plant 14-3-3 proteins in coupling chemical (ABA) and light (blue/red) signals, to electrical signals and gene expression. Whether functional conservation goes as far as the demonstrated role of brain 14-3-3s in learning and memory formation remains to be seen (3).
Dougherty, M.K., and Morrison, D.K. (2004). Unlocking the code of 14-3-3. Journal of Cell Science 117, 1875-1884. Van den Wijngaard, P.W.J., Sinnige, M.P., Roobeek, I., Reumer, A., Schoonheim, P.J., Mol, J.N.M., Wang, M., and De Boer, A.H. (2005). Abscisic acid and 14-3-3 proteins control K + channel activity in barley embryonic root. Plant Journal 41, 43-55. Simsek-Duran, F., Linden, D.J., and Lonart, G. (2005). Adapter protein 14-3-3 is required for a presynaptic form of LTP in the cerebellum. Nature Neuroscience 7, 1296-1298.
28 MAP kinases in neurons and plants
Jozef Šamaj
Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D – 53115 Bonn, Germany Email: [email protected]
Mitogen activated-protein kinases (MAPKs) such as ERK and p38 play a crucial role in synaptic plasticity since they are involved both in neuritogenesis and in neuronal polarity, differentiation and survival (Howe et al. 2001, Krapivinski et al. 2003, Nakata et al. 2005). These MAPKs are polarly localized in tip-growing neuronal axons. ERK, as an essential component, participates on signalling induced by neuronal growth factor (NGF) during retrograde transport of NGF receptor from synapses to the cell body, as well as on the signalling of glutamate receptors of the NMDA subtype underlying memory processes (Howe et al. 2001, Krapivinski et al. 2003). Recently, a novel p38 MAPK pathway was shown to play a role in pre-synaptic development (Nakata et al.2005). Interestingly, scaffolded molecular members of ERK pathway including NGF recepror, TRK, RAS, RAF, MEK and ERK itself are associated with clathrin-coated vesicles and with signalling endosomes upon NGF activation (Howe et al. 2001). On the other hand, hydrogen peroxide induces p38 and accelerates endocytosis via stimulation of GDI:Rab5 complex (Cavalli et al. 2001). In plants, nothing is known about the function of MAPK membrane scaffolding. Previously, we revealed that plant MAPK, polarly localized to vesicular structures, is associated with tip-growth of root hairs (Šamaj et al. 2002). Our recent data suggest that MAPKs are enriched at transversal cross walls of root cell files (representing putative plant synapses), and some MAPKs are subcelularly targeted to clathrin-coated vesicles and endosomal membranes, a phenomenon which is enhanced by oxidative and other abiotic stresses.
Cavalli V, Vilbois F, Corti M, Marcote MJ, Tamura K, Karin M, Arkinstall S, Gruenberg J (2001) The stress-induced MAP kinase p38 regulates endocytic trafficking via GDI:Rab5 complex. Mol Cell 7: 421-432 Howe CL, Valletta JS, Rusnak AS, Mobley WC (2001) NGF signalling from clathrin-coated vesicles: evidence that signaling endosomes serve as a platform for the Ras-MAPK pathway. Neuron 32: 801-814 Krapivinski G, Krapivinski L, Manasian Y, Ivanov A, Tyzio R, Pellegrino C, Ben-Ari Y, Clapham DE, Medina I (2003) The NMDA receptor is coupled to the ERK pathway by a direct interaction between NRB2B and RasGRF1. neuron 40: 775-784 Nakata K, Abrams B, Grill B, Goncharov A, Huang X, Chisholm AD, Jin Y (2005) Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development. Cell 120: 407-420 Šamaj J, Ovecka M, Hlavacka A, Lecourieux F, Meskiene I, Lichtscheidl I, Lenart P, Salaj J, Volkmann D, Bogre L, Baluška F, Hirt H (2002) Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth. EMBO J 21: 3296-3306
29 SNARE molecules indicate the complexity of the post-Golgi traffic in plant cells
Tomohiro Uemura*, Masa Sato
Department of Dynamics of Natural Environment, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501 Japan Email: [email protected]
Although the endomembrane system is well conserved among all eukaryotic cells, the plant endomembrane system also has unique features. In mammals and yeast, each cell has only one type of vacuole, whereas plant cells possess two-types of functionally different vacuoles, a lytic vacuole and a protein storage vacuole. The lytic vacuole is comparable to the mammalian lysosome and the yeast vacuole with an acidic pH of inside of the compartment, while protein storage vacuole is the specialized vacuoles having the capacity to store proteins in seed or vegetative cells. Therefore, protein sorting in plant cells is likely to be more complicated than other eukaryotes because two separate pathways diverge from the Golgi apparatus or plasma membrane to two different vacuole destinations. In all eucaryotic cells, specific vesicle fusion during vesicular transport is mediated by membrane-associated proteins called SNAREs ( soluble N-ethyl-maleimide sensitive factor attachment protein receptors). In the Arabidopsis genome, 54 SNARE and 57 Rab GTPase genes have been identified. These numbers are greater than those of yeast and mammalians, indicating the complexity of the plant endomembrane system. SNAREs and Rab GTPase are necessary for plant-unique higher order physiological function, suggesting that plants have adopted the membrane trafficking system to plant specific phenomenon. A series of transient expression assays using green fluorescent protein (GFP) fused proteins revealed that most of SNARE proteins were located on specific intracellular compartments: 6 in the endoplasmic reticulum, 9 in the Golgi apparatus, 4 in the trans-Golgi network (TGN), 2 in endosomes, 17 on the plasma membrane, 7 in both the prevacuolar compartment (PVC) and vacuoles, 2 in TGN/PVC/vacuoles, and 1 in TGN/PVC/plasma membrane. S ome SNARE proteins showed multiple localization patterns in two or more different organelles, suggesting that these SNAREs shuttle between the organelles. Furthermore, the SYP41/SYP61-residing compartment, which we define as the TGN, was not always located along with the Golgi apparatus, suggesting that this compartment is an independent organelle distinct from the Golgi apparatus. We will discuss possible combinations of SNARE proteins on all subcellular compartments, and suggest the complexity of the post-Golgi membrane traffic in higher plant cells.
30 Syntaxin 1 as the central element of regulated exocytosis in plants
Gian-Pietro Di Sansebastiano*, Massimiliano Gigante, Maria Rosaria Leucci, Stefania De Domenico, Gabriella Piro, Giuseppe Dalessandro
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Università di Lecce, via prov.le Lecce-Monteroni, I-73100 Lecce, Italy *Email: [email protected]
The best-known secretory models in plants appear more closely allied to the idea of constitutive secretion. In contrast with the events of neurotransmitter release, in each of these cases secretion is prolonged or continuous, rather than transient; the secretory event is not preceded by a large accumulation of vesicles beneath the target membrane; and the exocytotic fusion does not appear tightly coupled to endocytosis and vesicle recycling. Nevertheless the secretion in plants is frequently targeted to specific regions of the cell or triggered by external stimuli, and now there is growing evidence pointing to vesicle pools that are released on stimulation. Specific interactions between SNAREs, regulated by specific GTPases (Rab proteins), represent a central event of vesicular traffic and modulate secretion. SNAREs study, carried out essentially with biochemical approaches, presents great difficulties because the specificity found in function and localization does not correspond to an equivalent differentiation of chemical and physical characteristics. All SNAREs share, to a certain extent, unspecific affinity for each other. Ten different syntaxins seem to contribute to the last steps of exocytosis and endocytosys. Their role is far from being fully characterised. Nicotiana tabacum Syntaxin 1 (Nt-Syr1 or SYP121) is a SNARE protein required for ABA control of ion channels and it seems to be involved in exocytosis. In fact the expression of a dominant negative fragment of Nt-Syr1 indicated that this syntaxin mediates the traffic between the Golgi complex and the plasma membrane. Using the approach of dominant negative mutants, we proved that this syntaxin is involved in a secretory process independent from the constitutive secretion of cell wall polysaccharides. By analogy, we expect this process to be essentially the same known in the synaptic transmission but used by plants for rapid variations of the plasma membrane surface (stomata movements and turgor). To study the sorting and recycling of this protein, we generated a collection of GFP-tagged variants. Even if GFP tagged SYP121 appeared to be localised essentially on the plasma membrane, other variants suggested that this protein was anchored on internal membranes and then sorted to the plasma membrane. These GFP-tagged variants may allow to visualise post-Golgi intermediate compartments of post-transcriptionally regulated secretory events, in this case ABA dependent. The pool of secretory vesicles ready to answer ABA stimulus and controlled by the tSNARE SYP121, could also assemble in structures similar to the secretory granules described in the animal systems but never observed in plants.
31 Rab GTPases and Exocyst in plants
Viktor Žársky
Department of Plant Physiology, Charles University, Vinicná 5, CZ-12844 Prague, Czech Republic Email: [email protected]
Oriented cell expansion in plant cells is regulated by members of Ras-related superfamily of GTPases as in other eukaryots. Functional cycle of Rab family of protens is dependent on C- terminal double-cysteine motif modification by geranylgeranyl hydrophobic moiety. We found plant-specific features of Rab-geranylgeranyltransferase (RabGGTase) subunits and using molecular phylogenetic analysis we proposed a new scenario of the GDI/REP superfamily of neurobiological proteins evolution. Analysis of a mutant in one of the RabGGTase subunits points to the feedback regulation of photomorphogenesis by cell expansion. We are studying exocyst complex in plants as a putative effector of small GTPases including Rab proteins. Our biochemical as well as genetic evidence points to the exocyst involvement in plant cell polarity regulation.
32 Receptor-like kinases: how plants sense their environment and can tell us what they "see"
Jack C. Schultz1,*, Heidi M. Appel1, Ramesh Raina2
1 Center for Chemical Ecology, Penn State University, University Park, PA, USA 2 Department of Biology, Syracuse University, Syracuse, NY, USA *Email: [email protected]
Because plants cannot physically change environments, they must be able to sense and respond dynamically to numerous environmental cues. This probably explains why an exceptionally large fraction of the Arabidopsis genome comprises genes encoding receptor proteins, particularly receptor-like kinases (RLKs), which initiate often complex local and systemic signaling networks. We have found that several RLKs are expressed in response to insect attack and may participate in "sensitizing" the plant for subsequent responses. We are attempting to discern the function of some of the approximately 600 RLKs in Arabidopsis by creating chimeric proteins having various extracellular (sensor) domains linked to a single intracellular (kinase) domain which produces a consistent visible report. We report the creation of the first such chimeric receptors expressed in planta, and discuss the value of the "RLK Kits" we will be producing to investigators in many disciplines.
33 Nitric oxide functions in plant disease resistance
Massimo Delledonne*, Alberto Ferrarini, Matteo De Stefano
Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada Le Grazie 15, Cà Vignal 1, 37134 Verona, Italy * Email: [email protected]
A widespread feature of plant disease r esistance is the hypersensitive response (HR), which is characterized by the formation of necrotic lesions at the infection site and by the restriction of pathogen growth and spread. Following this local resistance response, tissue distal to the infection site develops a systemic acquired resistance (SAR) to secondary infection by the same or by different pathogens. Similarly to what is observed in the macrophage action during the immune response, one of the earliest events in the HR is the rapid accumulation of reactive oxygen species (ROS) and nitric oxide (NO) through the activation of enzyme systems similar to neutrophil NADPH oxidase and nitric oxide synthase. Both NO and ROS are necessary to trigger host cell death, and several lines of evidence suggest that this cell death results from the activation of suicide processes. In fact, the HR is thought to be a form of programmed cell death, a genetically programmed process well known in animals, which is characterized by a distinct set of morphological and biochemical features.NO and ROS are also components of a highly amplified and integrated defense system that triggers the local expression of resistance genes. NO also functions independently of ROS in the induction of various defence genes including pathogenesis-related proteins and enzymes of phenylpropanoid metabolism involved in the production of lignin, antibiotics and the secondary signal salicylic acid. NO signaling functions depend on its reactivity and ROS are key modulators of NO in triggering cell death, although through mechanisms different from those commonly observed in animals. The establishment of SAR, an inducible plant defense response, involves the existence of a systemic signal that migrates from infected to systemic, non infected leaves and requires SA. Compelling evidence indicates that SA, although necessary both for local resistance and for SAR induction, is not the long-distance signal molecule that triggers systemic resistance. In mammals, NO circulates in the blood as S-nitroso adducts of proteins, or as low molecular weight S-nitroso thiols, such as nitroso glutathione (GSNO). This molecule, believed to act as both an intra- and intercellular NO carrier, is a powerful inducer of plant defense genes. Since glutathione is a major metabolite in the phloem, where the SAR signal is transmitted, it can be hypothesized that excess NO produced during the HR binds to glutathione and that in this form it may act as a long distance SAR signal. In summary, although several hypotheses still await experimental demonstration it is now clear that NO and ROS play a key role in disease resistance responses, and further studies of their regulation and mechanisms of action will offer the possibility to exploit new ways for improving plant resistance to a number of biotic and abiotic stresses.
34 Nitric oxide and auxin interactions in root formation
Lorenzo Lamattina*, Luciana Lanteri, Natalia Correa-Aragunde, Magdalena Graziano
Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Mar del Plata, Argentina *Email: [email protected]
Plants have developed a series of mechanisms for perception and transduction of environmental or endogenous stimuli and for integration and synchronization of physiological responses. Signal transduction, the way cells construct responses to a stimulus, is a recently defined focus in plant biology. Moreover, in addition to the accumulation of data generated by structural and functional genomic analysis, a revival of pharmacological, surgical and physiological experimental approaches assessed in the past years have led to a clear advancement in the comprehension of molecular mechanisms involved in plant physiological responses. As well as it was demonstrated in animal systems that nitric oxide (NO), cGMP and Ca2+ were interconnected to play a central role in vascular cell physiology, the same results are forthcoming from plant systems. Currently, the molecular basis that are responsible of transducing plant hormone messages into physiological responses are being deciphered. The auxin indole acetic acid (IAA) is the main hormone involved in determining the root architecture. Recently, we have demonstrated that NO is required in both the IAA-induced adventitious root formation (ARF) [Plant Physiol. (2002) 129: 954; (2003) 132: 1241; (2004) 135: 279] and lateral root development (LRD) [Planta (2004) 218: 900]. During ARF, the NO action is accomplished by at least two parallel mechanisms: i) the increase in cGMP level and ii) the activation of a MAPK pathway. The induction of LRD by NO is accomplished through a NO-mediated induction of cell cycle regulatory genes (cyclins and cyclin-dependent kinases) that leads to the activation of pericycle cells and their progress through G1-S phase. These and other recent findings concerning NO-related mechanisms that control root development will be presented.
Supported by Conicet, ANPCyT, UNMdP and Fundación Antorchas, Argentina.
35 Oxygen/Auxin influx and NO efflux: neural-like features of the transtion zone of the root apex
Stefano Mancuso1,*, Sergio Mugnai1, Anna Maria Marras2, Dieter Volkmann3, František Baluška3
1 Dept. Horticulture, University of Firenze, Viale delle idee 30, 50019 Sesto F.no (FI), Italy 2 Dept. of Pharmaceutical Science, University of Firenze, via Ugo Schiff 6, 50019 Sesto Fiorentino (FI), Italy 3 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany *Email: [email protected]
Root apices are composed of three distinct regions: an apical meristem where all cell divisions are taking place, a basal region of rapid cell elongation where cells extend parallel to the apical-basal axis of the root, and an interpolated transition zone (TZ). In contrast, shoot apices do not show such a clear zonation since cell divisions and rapid cell elongation occur side-by- side. In the root apex, the transition zone cells maintain an active cytoarchitecture, having central nuclei from which radiate both microtubules and actin filaments towards the peripheral plasma membrane. This specific cytoarchitecture is proposed to be optimally suited for the effective signal transmission between the plasma membrane and nucleus (Baluška et al. 2003a). Cells of the distal part of the transition zone, occupying approximately the region of 1,0 – 1,5 mm from the root tip, but not those of the meristem (approx. 0,0 – 1,0 mm from the root tip) and elongation region (approx. 2,0 – 7,0 mm from the tip), are characterized by the maximal degree of oxygen and IAA influx (Mancuso et al. 2005) into the root apex. Moreover, cells of the TZ are also specifically involved in the production of NO in response to environmental stimuli such as hypoxia. Immediately after the onset of hypoxia, NO is emitted specifically from the TZ of the root apex with a mean peak efflux of 925 ± 102 fmol NO cm-2s- 1. Approximately 230 pmol NO cm-2 are produced at the TZ. Interestingly, dramatic NO efflux was recorded only in the TZ of the root apex. Outside of the TZ, only small effluxes of NO (1/10 lower than those produced in the TZ) were detectable. To further support the idea of the TZ as a kind of sensory zone, w e will present, simultaneous measurements of tissue oxygenation and single-cell electrical activity in the TZ, showing that action potentials were accompanied by immediate decreases in tissue oxygenation. This behaviour surprisingly resembles results recently published on the neural activity of cats (Thompson et al. 2003). Obviously, cells of the transition zone are accomplishing processes requiring very large amounts of oxygen. All these advancements converge towards a concept proposing that the transition zone of the root apex represents a highly specialized sensory and information processing region which encompasses neuronal features (Baluška et al. 2003b). An attractive scenario would be that this root apex region acts as some kind of ‘brain-like’ command centre in higher plants (Baluška et al. 2004). Future studies should answer this important question of plant biology.
Baluška F, Wojtaszek P, Volkmann D, Barlow PW (2003a) The architecture of polarized cell growth: the unique status of elongating plant cells. BioEssays 25, 569-576 Baluška, F, Šamaj, J, Menzel, D (2003b). Polar transport of auxin: carrier-mediated flux across the plasma membrane or neurotransmitter-like secretion? Trends in Cell Biology 13: 282- 285 Baluška F, Mancuso S, Volkmann D, Barlow PW (2004) Root apices as plant command centres: the unique ‘brain-like’ status of the root apex transition zone. Biologia 59 (Suppl. 13): 7- 19 Mancuso S, Marras AM, Volker M, Baluska F (2005) Non-invasive and continuous recordings of auxin fluxes in intact root apex with a carbon-nanotube-modified and self-referencing microelectrode. Anal Biochem, In press Thompson JK, Peterson MR, Freeman RD (2003) Single-neuron activity and tissue oxygenation in the cerebral cortex. Science 299, 1070-1072
36 Polar signals in plant vascular tissue development
Enrico Scarpella1,Wenzislava Ckurshumova, Naden Krogan, Danielle Marcos, Philip Francis, Thomas Berleth*
Dept. of Botany, University of Toronto, 25 Willcocks Street, Toronto M5S 3B2, Canada 1 Present address: Department of Biological Sciences, University of Alberta, Edmonton, AB Canada *Email: [email protected]
Polar signals have long been implicated in vascular tissue pattern formation (summarized in 1). To better understand the molecular cues directing the patterned differentiation of vascular tissues we have adopted a number of strategies for the identification and functional characterization of genes preferentially expressed at early stages of vascular development. Several of these approaches are based on the observations that auxin signaling through the Auxin Response Factor (ARF) MP is critical for vascular differentiation in Arabidopsis and that vascular patterning depends on proper auxin transport at critical stages of organ development (1, 2). At least one other ARF acts redundantly in auxin signaling in vascular development and at least one Aux/IAA co-regulator functions as an in planta antagonist in this process (3). Auxin response maxima foreshadow sites of procambial differentiation (4) and other early markers indicate a polar mode of preprocambial cell fate acquisition (5). We report experiments addressing the acquisition of cell polarity during early stages of procambium formation and strategies towards the identification of signal transduction genes expressed at these early stages . Finally, further early vascular genes are identified in a collection of indirect enhancer-trap lines, which also served as genetic backgrounds in the search for mutants with abnormal preprocambial patterns.
Sachs, Adv. Bot. Res. 9, 152-262 (1981) Hardtke & Berleth. Embo J 17, 1405-1411 (1998). Mattsson et al. Development 126, 2979-2991 (1999). Hardtke et al., Development 131, 1089-1100 (2004) Mattsson et al. Plant Phys. 131, 1327-1339 (2003). Scarpella et al, Development 131, 3445-3455 (2004)
37 Leaf vascular patterning in monocots and dicots
Enrico Scarpella1,2,3,*, Danielle Marcos1, Philip Francis1, Annemarie H. Meijer2, Thomas Berleth1
1 Department. of Botany, University of Toronto, 25 Willcocks Street, Toronto ON, Canada M5S 3B2 2 Institute of Biology, Leiden University , Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands 3 Present address: Department of Biological Sciences, University of Alberta, CW-405 Biological Sciences Building, Edmonton AB, Canada T6G 2E9 *Email: [email protected]
In leaves, vascular strands of distinct hierarchical orders are organized in closed (i.e. connected) networks. Vascular networks are generated de novo during the development of each leaf primordium. Procambial cells, the precursors of all vascular cells, differentiate from preprocambial cells, which represent a molecularly defined subset of ground meristem cells in the leaf (1). Mechanisms integrating aligned cell differentiation during vascular strand formation were probably not reinvented in the evolution of leaves, but recruited and revised by leaf-specific controls. In addition, species-specific cues are likely to be involved in leaf vascular patterning, as shown by the successful use of species-specific leaf vascular patterns as taxonomic diagnostic features (2). Whereas most dicot leaves show a ramified pattern of progressively branched veins, most monocot leaves have striate venation patterns in which major veins lie parallel along the proximodistal axis of the leaf and are connected transversely by minor transverse veins (3). Auxin signals have a profound impact on vascular patterns of dicot leaves. Local auxin application induces the formation of a new vascular strand (4). Further, both defective auxin signal transduction and impaired auxin transport alter leaf vascular patterns in characteristic ways (5-8), and positions of leaf procambium formation are foreshadowed by sites of elevated auxin response (9). The reduced auxin sensitivity of monocot leaves (10), and their highly reproducible distribution and arrangement of veins (3) suggest that vascular patterns in monocot leaves may be rigidly specified. Recent pharmacological and genetic evidence suggest, however, a role for auxin transport and signal transduction in vascular patterning of monocot leaves (11,12).
Scarpella E, Francis P, Berleth T. 2004. Stage-specific markers define early steps of procambium development in Arabidopsis leaves and correlate termination of vein formation with mesophyll differentiation. Development 131:3445-55. Klucking EP. 1995. Leaf venation patterns. Berlin , Germany : J Cramer. Nelson T, Dengler N. 1997. Leaf vascular pattern formation. Plant Cell 9:1121-35. Sachs T. 1989. The development of vascular networks during leaf development. Curr Top Plant Biochem Physiol 8:168-83. Przemeck GKH, Mattsson J, Hardtke CS, Sung ZR, Berleth T. 1996. Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta 200:229-37. Hardtke CS, Berleth T. 1998. The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO J 17:1405-11. Mattsson J, Sung ZR, Berleth T. 1999. Responses of plant vascular systems to auxin transport inhibition. Development 126:2979-91. Sieburth LE. 1999. Auxin is required for leaf vein pattern in Arabidopsis. Plant Physiol 121:1179-90. Mattsson J, Ckurshumova W, Berleth T. 2003. Auxin signaling in Arabidopsis leaf Vascular development. Plant Physiol 131:1327-39. Wenicke W, Brettell R, Wakizuka T, Potrykus I. 1981. Adventitious embryoid and root formation from rice leaves. Z Pflanzenphysiol 103:361-5. Scarpella E, Boot KJM, Rueb S, Meijer AH. 2002. The procambium specification gene Oshox1 promotes polar auxin transport capacity and reduces its sensitivity towards inhibition. Plant Physiol 130:1349-60. Scarpella E, Rueb S, Meijer AH. 2003. The RADICLELESS1 gene is required for vascular pattern formation in rice. Development 130:645-58.
38 Signals and targets triggered by self-incompatibility: recognition of "self" can be deadly!
Noni Franklin-Tong
School of Biosciences, University of Birmingham, Edgbaston, Birmingham. B15 2TT, UK Email: [email protected]
Self incompatibility (SI) is one of the most important mechanisms for promoting outbreeding in flowering plants. It prevents self-fertilization through a genetically controlled cell-cell recognition system [1,2]. My lab studies SI in Papaver rhoeas (the field poppy), where SI is mediated by an interaction between the pistil S-gene product when it meets “self” (incompatible) pollen. This triggers a Ca2+-dependent signalling cascade in incompatible pollen, resulting in inhibition of pollen tube growth. The SI-specific signals involve activation of several protein kinases, including a MAP kinase, p56 [3]. Another phosphoprotein, p26, which is hyper-phosphorylated rapidly in response to SI, has been identified as a soluble inorganic pyrophosphatase [4], which we postulate may play an important role in inhibition of incompatible pollen. Another target of the SI response is the pollen actin cytoskeleton, which undergoes rapid reorganization and depolymerization [5,6]. We have recently demonstrated that programmed cell death (PCD) is triggered by the SI response and that a caspase-like/DEVDase activity is activated by SI in incompatible pollen [7]. Compelling evidence suggests that the caspase-like activity is triggered very early in the signalling cascade, and also that it plays an active role in processes leading to inhibition. SI-induced PCD appears to involve a number of signalling cascades and targets which recent data suggest may be involved in cross-talk. I will discuss progress on identifying the signals that mediate SI, the involvement of the actin cytoskeleton and PCD.
Franklin-Tong, V.E., Franklin, F.C.H. (2003) Trends in Plant Sciences 8, 598-605. Franklin-Tong, V.E. and Franklin, F.C.H. (2003) Phil. Trans Roy. Soc. Lond. B, 358, 1025-32.. Rudd, J.J., Osman, K., Franklin, F. C. H., Franklin-Tong V. E. (2003) FEBS Letters 547, 223- 227. Rudd, J.J. and Franklin-Tong, V.E. (2003) Journal of Experimental Botany 54, 141-148. Geitmann, A., Snowman, B.N., Emons, A.M.C. and Franklin-Tong, V.E. (2000) Plant Cell 12, 1239-1251. Snowman, B.N., Kovar, D.R., Shevchenko, G., Franklin-Tong, V.E., and Staiger, C.J. (2002) Plant Cell 14, 2613-2626. Thomas, S.G., Franklin-Tong, V.E. (2004) Nature 429, 305-309.
39 Signal perception and transduction in plant innate immunity
Thorsten Nürnberger*, Frederic Brunner, Stefan Engelhardt, Yvonne Gäbler, Birgit Kemmerling
Center for Plant Molecular Biology, Research Group Plant Biochemistry, University of Tübingen , Auf der Morgenstelle 5, D-72076 Tübingen , Germany *Email: [email protected]
Immunity of an entire plant species to microbial infection (non-host resistance) is determined by intertwined layers of defense including both constitutive barriers and inducible reactions. Activation of non plant cultivar-specific inducible responses is likely based upon recognition of pathogen-associated molecular patterns, which bind to plant receptors. We have identified a cell wall transglutaminase (TGase) from phytopathogenic Phytophthora spp. that triggers defense responses in parsley and potato. A surface-exposed fragment within this TGase (Pep- 13) was shown to be indispensable for both elicitor and TGase activity of the protein, suggesting a crucial role of this domain for protein stability and/or enzymatic activity. Ligand- induced receptor activation gives rise to elevated levels of cytoplasmic calcium, subsequent posttranslational activation of MAPK, production of reactive oxygen species and antimicrobial phytoalexins. NPP1, another Phytophthora-associated surface structure was shown to trigger a similar cascade of events through a receptor system distinct of the Pep-13 receptor. Currently, conditional expression of NPP1-induced cell death in Arabidopsis thaliana is employed to isolate mutants impaired in NPP1 perception. In addition, two molecular patterns associated with phytopathogenic pseudomonads, lipopolysaccharide and the typeIII effector, HrpZ, were shown to synergistically activate plant defense in parsley. Moreover, LPS alone as well as type III secretion-deficient pseudomonads were shown to trigger systemic acquired resistance (SAR) in Arabidopsis, suggesting that the plant defense-inducing capacity of microbial surface structures contributes to physiologically relevant resistance responses, such as SAR. Microarray experiments using the Affymetrix ATH1 chip and RNA prepared from pathogen- infected as well as elicitor-infiltrated A. thaliana plants were performed to identify genes whose expression pattern and predicted molecular function implied a role in signal perception and transduction. Plants carrying homozygous T-DNA insertions in a gene encoding a leucine-rich repeat receptor kinase were identified that showed an altered disease resistance. A molecular characterization of the protein will be presented.
40 TMV as a model for the analysis of RNA transport via plasmodesmata
Jamie Ashby1,2, Vitaly Boyko1,2, Emmanuel Boutant3, Anna Groner2, Monika Fasler1,2, Mark Seemanpillai3, Christophe Ritzenthaler3, Manfred Heinlein1,2,3,*
1 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 2 Department of Plant Physiology, Botanical Institute, University of Basel, Basel, Switzerland 3 Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Strasbourg, France *Email: [email protected]; [email protected]
Studies in higher plants have revealed the existence of proteins and RNA species that travel cell-to-cell and through the vasculature to serve as signaling molecules in plant development and gene silencing, thus confirming the role of plasmodesmata (Pd) in the mediation and control of intercellular and systemic communication via macromolecules. Compelling evidence for macromolecular trafficking through Pd comes from RNA viruses, which encode movement proteins (MP) to interact with Pd and other components of the RNA transport machinery in order to spread their genomes from cell-to-cell. The MP of Tobacco mosaic virus (TMV) is believed to form a ribonucleoprotein complex (RNP) with viral RNA (vRNA) and to represent the core of the infectious particle that spreads between cells. This hypothesis is supported by the ability of MP to bind single-stranded nucleic acids in vitro, by its localization to plasmodesmata (Pd), its interaction with cell wall proteins, as well as by its ability to modify the size exclusion limit (SEL) of Pd. Our research is aimed at elucidating whether the MP indeed forms an RNP in vivo, and also at understanding the cellular mechanism that targets the viral RNA genome (e.g. the RNP) and potentially other RNA molecules to Pd.
Boyko V, Ferralli J, Ashby J, Schellenbaum P, Heinlein M (2000) Function of microtubules in intercellular transport of plant virus RNA. Nat. Cell Biol. 2: 826-832 Heinlein M (2002) Plasmodesmata: dynamic regulation and role in macromolecular cell-to-cell signalling. Curr. Opin. Plant Biol. 5: 543-552 Heinlein M (2002) The spread of Tobacco mosaic virus infection: insights into the cellular mechanism of RNA transport. Cell. Mol. Life Sci. 59: 58-82 Heinlein M, Epel BL (2004) Macromolecular transport and signaling through plasmodesmata. Int. Rev. Cytol. 235: 93-164 Kovalchuk I, Kovalchuk O, Kalck V, Boyko V, Heinlein M, Hohn B (2003) Pathogen-induced systemic signal triggers genome instability. Nature 423: 760-762
41 Hydrotropism: root growth responses to water
Gladys Cassab
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, PO Box 510-3, Cuernavaca, Morelos 62250, México Email: [email protected]
Survival of terrestrial plants depends upon the capacity of roots to obtain water and nutrients from the soil. Directed growth of roots in relation to a gradient in moisture is called hydrotropism and begins in the root cap (RC) with the sensing of the moisture gradient. Even though the lack of sufficient water remains the single-most important factor affecting world agriculture, there are surprisingly few studies on hydrotropism. Using a screening system with a water potential gradient, we isolated a no hydrotropic response (nhr1) semi-dominant mutant of Arabidopsis that continued to grow downwardly into the medium with the lowest water potential contrary to the positive hydrotropic and negative gravitropic response seen in wild type (wt) roots. The lack of hydrotropic response of nhr1 roots was confirmed in a system with a gradient in air moisture. The hydrotropic response of wt roots in the screening system occurred with a concurrent drop in both starch content in columella cells and in gravitropic downward growth response. However, hydrotropicaly stimulated nhr1 roots in the screening system contained unusually large amyloplasts that persisted throughout the 8-day treatment. Since nhr1 roots maintained their large amyloplasts during hydrostimulation, their perception and gravitropic response in the screening system was not affected as in wt roots. Furthermore, nhr1 roots had abnormal RC morphogenesis and displayed decreases in auxin maximum. The genetic analysis of hydrotropism has provided new insights about the mechanisms that the RC uses to perceive and respond simultaneously to moisture and gravity signals. This knowledge might allow us to understand how RC processes environmental signals that are capable of regulating whole plant growth.
42 Cell wall integrity signalling in Arabidopsis thaliana
Thorsten Hamann*, Chris Somerville
Dept. of Plant Biology, Carnegie Institution of Washington, 260 Panama Street, Stanford 94305, CA, USA *Email: [email protected]
Signalling mechanisms coordinating interaction between the cell wall and the plant cell are crucial in several different biological contexts such as (a)biotic stress response, cell wall polysaccharide biosynthesis and cell morphogenesis. Over the last years evidence has surfaced that hints at the existence of a mechanism monitoring cell wall integrity. Both ethylene and jasmonic acid signalling mechanisms have been implicated while lignin and pectin biosynthetic processes are apparently downstream targets. We have performed expression profiling experiments monitoring the transcriptional response to cell wall stress caused by inhibition of cellulose biosynthesis through the herbicide isoxaben. One of the genes responding very strongly on the transcriptional level encodes a putative glutamate receptor protein (GLR). Homozygous mutant seedlings for this GLR exhibit cell wall defects and reduced lignification upon treatment with isoxaben. We will present our results regarding the biological function of this receptor.
43 N-Acylethanolamines: emerging Lipid Mediators of Seedling Development
Elison B. Blancaflor1,*, Kent D. Chapman2
1 Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma , USA 2 Department of Biological Sciences, University of North Texas, Denton, Texas, USA * Email: [email protected]
N-Acylethanolamines (NAEs) are lipid mediators derived from the hydrolysis of the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE). In animal systems, this reaction constitutes part of the endocannabinoid signaling pathway, which regulates a variety of physiological processes, including cell proliferation, immune cell signaling, neurotransmission, and embryo development. NAEs have been identified and quantified in a variety of plant tissues, and reports of biological activities of these lipids in plants are rapidly emerging. For example, NAEs have been implicated in the signal-mediated activation of gene expression, the inhibition of phospholipase D (PLD) alpha activity, and the regulation of ABA-induced stomatal closure. NAEs are prevalent in desiccated seeds and are metabolized rapidly during imbibition. We have shown previously that sustained levels of NAE12:0 have profound effects on Arabidopsis seedling growth and development, which includes a reduction in primary root and cotyledon expansion, increased radial swelling of root tips, and an inhibition of root hair initiation. Light and electron microscopic analysis of Arabidopsis roots reveal that the effects of NAE12:0 also are apparent at the cellular level as defects in cell division, cytoskeletal organization, membrane dynamics, and cell wall organization. We have developed a battery of tools that we expect will help in extending our understanding of NAE function in seedling development in Arabidopsis thaliana. These tools will be supported by sensitive quantitative procedures (gas chromatography/mass spectrometry) for determining endogenous levels of NAEs in plant extracts. Characterizing the effects of NAEs on seedling growth through these interdisciplinary approaches should provide new insights into the regulation of plant development by this endogenous group of bioactive lipids.
44 A Phytosynapse? The plant’s use of glutamate receptors to respond to aluminium
Tobias Baskin
Biology Department, University of Massachusetts, Amherst, MA 01003, USA Email: [email protected]
The synapse is a hallmark of the animal nervous system, sited literally at the crossroads between electrical and chemical information. Plants don’t have synapses, so it provokes surprise to discover that plants do have proteins that are homologues of ionotropic glutamate receptors. In animals, these receptors are synaptic cation channels, whose opening is gated by the neurotransmitter glutamate. In plants, we have found that glutamate receptors appear to be key players in signal transduction. Our data suggest that in the presence of aluminum, plant roots secrete glutamate, which binds to its receptor, allowing a calcium influx. The calcium transient leads to plasma-membrane depolarization as well as depolymerization of the cortical microtubules, both actions that will spread the message throughout the cell. Our original work was done with Arabidopsis roots and we are now finding a similar response network in wheat. This work suggests that in plants the concentrated synapse and brain of animals are replaced by a delocalized intelligence.
45 Glutamate signalling and root development in Arabidopsis
P. Walch-Liu*, T. Remans, B. G. Forde
Dept. of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK *Email: [email protected]
Like the rest of the plant, the root system has no pre-defined body plan, rather its development is continuously modified by interactions with environmental factors, including nutrients. Plants are known to forage for localised supplies of nitrate by proliferating their lateral roots within nitrate-rich patches. Recent evidence suggests that roots are also capable of sensing and responding in a highly specific manner to the presence of organic N in the soil. Arabidopsis root growth is very sensitive to the presence of external glutamate, but not to most other amino acids. The ability to sense glutamate appears to reside in the root tip itself and is common to both primary and lateral roots. Different ecotypes of Arabidopsis differ markedly in their glutamate sensitivity and we have used recombinant inbred lines to map a major QTL for glutamate sensitivity to chromosome 5. We will discuss our current understanding of the genetic and physiological basis of this phenomenon and its possible relationship to the existence of a family of glutamate receptor genes in plants.
46 Genetic and pharmacological evidence for a role of the GABA shunt in maintaining the levels of reactive oxygen intermediates in plant cells
Ayelet Yellin, Aaron Fait, Hillel Fromm*
Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel *Email: [email protected]
Disruption of the Arabidopsis gene encoding the last enzyme of the GABA shunt (SSADH) results in high levels of reactive oxygen intermediates (ROIs), cell death, and dwarfism, associated with hypersensitivity to stresses such as UV and heat. The ssadh mutants also contain high levels of gamma-hydroxybutyrate (GHB) a known neurotransmitters in mammals, which is derived from the GABA shunt. Currently we are undertaking genetic approaches aimed at isolating suppressor mutants of GABAshunt deficient mutants (ssadh in particular) with the hope of identifying novel components involved in GABA metabolism and signaling. A few suppressor mutants, which resemble wt plants in spite of the fact that they are homozygous for the disrupted SSADH gene, have been isolated and the genes with the suppressor mutations are being cloned. By taking a pharmacological approach, we found that vigabatrin (VGB), a specific inhibitor of mammalian GABA-transaminase, reduces the levels of GHB in ssadh mutants, similar to the effect of the drug in mammals deficient in SSADH. Moreover, VGB substantially reduces the levels of ROI, and improves plant growth. Thus both genetic and pharmacological evidence suggest the involvement of the GABA shunt in maintaining the levels of ROI in plant cells. Moreover, our studies reveal novel similarities between plants and animals in relation to two neurotransmitters: GABA and GHB.
47 Glutamate receptors and GABA in plant responses to environmental stress
Frank Turano*, Jiman Kang, Sivasubramani Balasubramanian
The George Washington University, Department of Biological Sciences, Washington, DC, USA *Email: [email protected]
The non-protein amino acid γ-aminobutyric acid (GABA) has been shown to rapidly increase in plants upon exposure to a variety of environmental stresses. However, little is known about the physiological role of GABA in higher plants or how alterations in the balance between Glu and GABA affect normal plant growth and development. Both molecules have been proposed to be signaling molecules in plants, we have taken two approaches to address this possibility. One approach focuses on the biosynthetic relationship between the putative ligands, Glu and GABA, while the other approach focuses on the putative receptors in the signaling pathway(s). GABA is synthesized from glutamic acid by the enzyme glutamic acid decarboxylase (GAD). The completion of the Arabidopsis genome has revealed that there are five AtGAD genes. AtGAD1 and AtGAD2 are expressed in roots and throughout the plant, respectively. However, the expression of the other three genes (AtGAD3-5) has not been reported. In order to gain a better understanding of the temporal and spatial expression of all the members of the AtGAD gene family, we made promoter and uidA gene fusion (AtGAD::GUS) constructs and determined the expression of each gene throughout development and upon exposure to distinct stresses. Our findings suggest that the AtGADs have non-redundant roles. Results from parsimony and incongruence length difference analyses demonstrate that regions of the N-termini of nearly a third of the twenty members of the putative glutamate receptors in Arabidospis thaliana (AtGLRs) are related to animal GABA B receptors. Immunoblot analyses and immunolocalization of one of these AtGLR gene products, AtGLR3.2, show that it is an integral membrane protein that accumulates in rapidly dividing cells and vascular tissue. These findings suggest that AtGLR3.2 may be associated with calcium distribution and allocation. We used an antisense approach to develop Arabidopsis lines deficient in AtGLR1.1 (antiAtGLR1.1). Our results suggest that AtGLR1.1 has functional characteristics similar to animal ionotropic glutamate receptors (iGLRs). Using a combination of targeted- proteomics and - metabolomics, genome-wide microarray and physiological analyses, we show that there is a strong interaction between carbon (C), nitrogen (N), sulfur (S) and abscisic acid (ABA) metabolic pathways, ABA sensing, and/or H2O utilization. In conclusion, our results show that the AtGLRs (i) regulate distinct pathways associated with N and S acquisition, distribution and metabolism, (ii) alter the expression of distinct carbon (C) metabolic pathways, (iii) alter the synthesis of the phytohormone ABA and sensitivity of the plant to ABA, and (iv) ultimately coordinate components of the C/N/S and ABA signaling pathways which change stomatal aperture to affect plant responses to H2O-related stresses. These findings suggest that the AtGLRs may coordinate the control of distinct components of C, N and S metabolism as well as ABA biosynthesis and sensing.
48 Arabidopsis knock out mutants of GABA metabolism and their response to different growth conditions
Frank Ludewig
Botanisches Institut, University of Cologne, Gyrhofstr. 15, D-50931 Cologne, Germany Email: [email protected]
Arabidopsis mutants defective in genes of the g-aminobutyric acid (GABA) pathway were isolated and analysed regarding their growth behaviour on agar plates containing different nitrogen sources. Several carbonic and amino acids including GABA were monitored under these conditions using GC/MS. GABA-transaminase knock out plants were hypersensitive against GABA when grown on agar plates.In contrast to WT plants, mutants did not grow on plates containing high concentrations of GABA as sole nitrogen source. Strikingly, growth of both, WT and mutant plants was much less inhibited without any nitrogen in the medium indicating that GABA sensing differs between Arabidopsis WT and mutant plants, i.e. a situation where GABA either can or cannot be used.
49 Does GABA act as a long-distance signal in the regulation of nitrate uptake in plants?
Erwan Le Deunff1,*, Nicolas Beuve1, Nicolas Rispail2, Cliquet J-B1, Philippe Lainé1, Alain Ourry1
1 UMR INRA -UCBN 950 EVA, Laboratoire d'Ecophysiologie Végétale, Agronomie & nutrition N,C,S; Esplanade de la Paix, F-14032 Caen Cedex, France 2 Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigfion SY 23 3EB, UK *Email: ledeunff@ ibfa.unicaen.fr
Nitrate uptake is the most limiting step of nitrate assimilation and consequently of higher plants growth (1). Nitrate uptake processes involve the functioning of several transporters with low-affinity (NRT1) and high-affinity (NRT2) to nitrate. These transporters are now well characterized at molecular level in many species and are regulated by complex local and long- distance signalling pathways (2). Until now, long-distance signals identified (such as glutamine) were supposed to mediate a feedback repression of nitrate uptake according to the modifications of internal N status varying with N demand of plants for growth. In order to identify others transported amino acids implicated in long-distance regulation of nitrate uptake, we have analysed in Brassica napus plants, composition of phloem exudates during N-deprivation (short-term experiment) and over a growth cycle (long-term experiment) in relation with variations of nitrate influx and BnNRT2 nitrate transporter gene expression. Our results show a positive correlation between GABA in phloem exudates and nitrate uptake in short and long-term experiments. The hypothesis that GABA could act as long-distance signal in up-regulation of nitrate uptake was tested by providing an exogenous GABA supply to the roots. By contrast to Glutamine treatment which inhibits both nitrate uptake and BnNRT2 gene expression, GABA treatment induces a significant increase of BnNRT2 mRNA expression but has less effect on nitrate influx. Despite the fact that this study provides the first evidence that GABA may act as putative long-distance inter-organ signal in plants, our results raise question of whether GABA could mediate a root-specific response in mineral acquisition via the modifications of root absorbing surfaces or increase conduction of ions transport (3). We are now investigating the role of GABA in root development, by analysing the effects of GABA treatments on exploratory root system: primary and lateral roots and absorbing root system: root hair development.
Malagoli P, Lainé P, Le Deunff E, Rossato L & Ourry A (2004) Modeling N uptake in Brassica napus L. Cv Capitol during a growth cycle using influx kinetics of root nitrate transport systems and field experimental data. Plant physiology 134, 388-400. Lainé P, Ourry A & Boucaud J (1995) Shoot control of nitrate uptake rates by root of Brassica napus L. effects of localiszed nitrate supply. Planta 196,77-83. Beuve N, Rispail N, Lainé P, Cliquet J-B, Ourry A & Le Deunff E (2004) Putative role of g - aminobutyric acid (GABA) as a long distance signal in up-regulation of nitrate uptake in Brassica napus L. Plant Cell and Environment 27, 1035-1046.
50 Is ATP a signalling agent in plants?
Vadim Demidchik1,*, Sergey N. Shabala2, Chris Nichols1, Adeeba Dark1, Julia M. Davies1
1 Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA , United Kingdom 2 School of Agricultural Sciences, University of Tasmania, Hobart, Tasmania 7001, Australia *Email: [email protected]
Very low concentrations of extracellular purines (ATP, ADP and AMP) can function as neurotransmitters in animal cells (1). At the cell surface they interact with specialised receptors (“purinoceptors”) which form transmembrane cation-selective channels (P2X receptors) or activate G-proteins (P2Y-receptors). Low concentrations of extracellular ATP (eATP) have recently been demonstrated to modify auxin distribution, gene expression and gravitropic responses in Arabidopsis roots (2). Since these effects have not been related to phosphorylation, a possible explanation is that eATP can act in plants in a neurotransmitter- like mode (2). Here, we test the hypothesis that ATP is a signalling agent in plants that functions in a similar way to animal neurotransmitters (activating ion fluxes at very low concentrations). We have found that [eATP] as low as 300 nM induced transient elevations of root ([Ca+2] cyt) in plants constitutively expressing cytosolic aequorin (3). ADP and non- hydrolysable analogues of ATP also caused [Ca+2] cyt increases, showing that the effect was not induced by phosphorylation. UTP was far less potent than all tested purines. Purine- induced [Ca+2] cyt increases were inhibited by classic purinoceptor blockers (suramin and PPADS) and by Gd+3 (3). These results have now been confirmed by Jeter et al. (2004) (4). Using the MIFE ® technique we have measured the effect of extracellular purines on net Ca+2 and K + fluxes in intact Arabidopsis root epidermis. ATP, ADP and the non-hydrolysable ATP analogue ab meATP (up to 100 m M) simultaneously caused transient Ca+2 influx and K+ efflux. Purine-induced fluxes were far larger in the elongation zone than in the mature epidermis. Using patch-clamp we have identified p urine- induced whole-cell cation conductances and single cation channels in root epidermis-derived protoplasts. Activity resembled animal ionotropic purinoceptors. We propose that extracellular ATP and ADP are important signalling agents in plants acting via plasma membrane cation channels.
Khakh BS (2001) Nat Rev Neurosci 2: 165. Tang et al. (2003) Plant Physiol 131: 147. Demidchik et al. (2003) Plant Physiol 133: 456. Jeter et al. (2004) Plant Cell 16: 2652.
51 Signaling role of extracellular ATP in growth control and in wound responses
Yu Sun, Iris Steinebrunner, Charlotte Song, Jian Wu, Timothy Butterfield, Xuanzhi Wang, Elizabeth Henaff, Stanley Roux*
Molecular Cell & Developmental Biology, University of Texas, Austin, TX 78712, USA *Email: [email protected]
Externally applied ATP (xATP) can induce increased [Ca2+] cyt in plant cells (Demidchik et al., 2003; Jeter et al., 2004), just as it does in animal cells. To evaluate the physiological significance of this signaling response, we have tested the effects of xATP on growth and development in Arabidopsis. Lower µM levels of xATP enhance and higher µM levels inhibit the hypocotyl growth of seedlings. Sub-µM xATP can induce in leaves an enhanced production of superoxide and downstream gene expression changes associated with wounding. Neither the growth nor superoxide responses can be induced by equivalent concentrations of phosphate or AMP. The inhibitory growth effects of xATP can be blocked by ethylene inhibitors, and the ability of xATP to induce superoxide production can be blocked by antagonists of animal purinoceptors and by inhibitors of calcium uptake into cells. Ectoapyrases (ecto-NTPDases) are ATP-hydrolyzing enzymes that play a key role in controlling the [xATP]. Two highly similar apyrases in Arabidopsis, Atapy1 and Atapy2, have putative signal peptides and have been postulated to function as ectoapyrases. They are important for pollen germination and other aspects of plant cell growth. Pollen from plants not expressing Atapy1 and Atapy2 (= double knock-out (DKO) plants) cannot germinate. DKO pollen from plants that are complemented with a wild-type gene under the control of a steroid-inducible promoter can be induced to germinate by dexamethasone. Fertilization by this pollen yields seeds that germinate normally, but in the absence of further dexamethasone treatment, the resulting DKO plants are stunted, typically < 10 cm tall at maturity. As evaluated by promoter:GUS constructs and other methods, the highest mRNA level for both apyrases was found in tissue poised for rapid growth (mature pollen), or tissue actually undergoing rapid growth (elongation zone of the root, hypocotyls of etiolated seedlings), consistent with the postulate that apyrases play a key role in growth control.
Supported by NSF IBN0344221 to S.J.R.
Demidchik V. et al. (2003) Plant Physiol. 133: 456-461 Jeter C.R. et al. (2004) Plant Cell 16: 2652-2664
52 Ion channels in plants: from DNA sequence to integrative biology
Benoît Lacombe
Biochimie et Physiologie Moléculaire des Plantes, UMR5004 Agro-M/CNRS/INRA/UMII, Place Viala, 34060 Montpellier Cedex1, France Email: [email protected]
Processes involved in plant growth, development and adaptation to changing environmental conditions are frequently accompanied by mass flow of ions or even electrical signals. Therefore signal perception, transduction or processing in higher plants significantly relies on the concerted action of receptors, ion channels and pumps. The functional properties of most ion channels cloned in Arabidopsis (and other plants) remain speculative and rely essentially upon sequence homologies with animal channels. The plant Shaker-like K+ channel family is a noticeable exception as this ion channel family has been well characterized at the molecular and physiological level. As their animals homologues, the so-called Shaker-like plant K+ channels are made of four a - subunits. Nine genes in Arabidopsis encode such polypeptides, which display the typical structural scheme with 6 transmembrane segments and one P-domain. In heterologous expression systems (Xenopus oocytes, Sf9 insect cells, COS cells, yeast) these genes yield K+ selective channels featuring both absence of inactivation and voltage-gating and are therefore capable of contributing as well to sustained K+ transport as to excitability. These channels can be sorted in 4 functional groups: (i) inwardly-rectifying (KAT1, KAT2, AKT1, SPIK), (ii) outwardly-rectifying (SKOR and GORK), (iii) weakly inwardly-rectifying channels (AKT2), and (iv) regulatory subunit (AtKC1). An overview of the current knowledge of these different subunits will be presented. A special emphasis will be given on the approaches that have allowed to decipher their role in planta: cloning, heterologous expression, expression pattern, regulation at the transcriptional/post transcriptional level and characterisation of knock-out mutants. Finally, the role of other proteins in the macromolecular assembly and regulation of plant potassium channels will be discussed.
53 Developmental effects of electric current in thermo- and photoperiodic plants
Ivana Macháčková1,*, Jan Krekule1, Maria Filek2, Jolanta Biesaga- Koscielniak2, Izabela Marcinska2
1 Institute of Experimental Botany AS CR, Rozvojová 135, 16502 Praha 6, Czech Republic 2 The Francisek Górski Institute of Plant Physiology, PAN, Ul. Niezapominajek 21,31- 016 Kraków, Poland *Email: [email protected]
The effect of electric current (EC) was tested on flowering induced by vernalization and by photoperiod in winter wheat, stem cuttings of spring and winter rape grown in vitro and to rape grafted plants (non-vernalized scion of the apical part grafted on vernalized stock of winter rape). Different time/voltage combinations were tested as well as both polarities. EC of positive polarity (anode close to apex, cathode in the medium) increased significantly percentage of flowering plants in partially vernalized winter wheat and of non-vernalized scions grafted on vernalized winter rape stocks, both under long and under short days. Transition to flowering of isolated apical stem segments of spring and winter rape was also stimulated by EC of positive polarity. EC of negative polarity inhibited flowering of the grafted scions in winter rape under long days. Thus, negative polarity seems to suppress inductive signals, while the positive polarity either mimics them or restrains the effects of non-inductive conditions.
54 Action potentials - from mechanism to function
Kazimierz Trebacz
Department of Biophysics, Institute of Biology, Maria Curie-Sklodowska University, Akademicka 19, PL-20-033 Lublin, Poland email: [email protected]
Action potentials (APs) can be evoked in plants by damaging and non damaging stimuli. After application of damaging stimuli, such as wounding or burning either series of APs or APs superimposed with variation potentials are registered. Non damaging stimuli, like illumination, cooling or direct current lead usually to generation of single AP. An important feature of APs is their ability to spread from the site of stimulation throughout the whole plant, specialized tissue or plant organ. Local circuits consisting of ion fluxes play important role in AP transmission and synchronization of plant response to remote stimuli. It is thus important to determine the ion mechanism of APs. It was first elaborated for giant characean cells. Later on, basic aspects of the mechanism were confirmed and modified using another model plant - the liverwort Conocephalum conicum. C. conicum belongs to phylogenically oldest terrestrial plants. Its thallus forms nearly homogenous network of cells connected with plasmodesmata. All cells of the thallus (including rhizoids) are excitable. Basing on experiments with application of ion-selective microelectrodes, and ion channel inhibitors one can conclude that the ion mechanism of APs in Conocephalum consists of the following steps. AP is initiated by Ca2+ influx into the cytosol. Increasing [Ca2+] cyt activates anion channels, and Cl - efflux leads to cell depolarization. Repolarization occurs as a result of K+ efflux and enhanced H+ extrusion through the electrogenic proton pump. Simultaneous application of anion- (A-9-C) and potassium channel inhibitors (TEA) allowed to separate a calcium phase of AP, called VT, and address the question concerning the source of calcium ions entering the cytosol. Substances responsible for Ca2+ liberation from internal stores (Sr2+) caused an increase, whereas chemicals blocking internal Ca2+ channels (neomycin) suppressed the calcium phase of AP. Partial inhibition of VT was also observed after application of impermeable Ca2+ channel inhibitors affecting Ca2+ influx through the plasmalemma. It was concluded that APs in Conocephalum are initiated by Ca2+ influx through the plasma membrane which then causes liberation of additional portion of Ca2+ ions according to the process known as calcium induced calcium release. The extent of [Ca2+] cyt increase determines the physiological response. It was demonstrated that in Conocephalum the rate of respiration increases up to twice after passing of AP evoked by non damaging electrical stimuli. Severe stimuli, like cutting of the thallus edge, evoked series of APs followed by shifted in phase oscillations in the rate of respiration. After blocking of AP with ion channel inhibitors, no significant increase in the respiration rate was observed irrespectively of the kind of stimulus. Significant increase in peroxidase activity is another consequence of excitation in C. conicum. It was registered only if the threshold of excitation was exceeded. Here again Ca2+ ions seem to play a role of coupling factors between AP and the physiological response. Different physiological responses evoked as a consequence of plant excitation are consistent with the concept of “calcium signature”.
55 Oscillations in plants
Sergey Shabala
School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, Tas 7001, Australia Email: [email protected]
Rhythmical behaviour is a quintessential pattern of life itself. Biological oscillations kept many generations of scientists fascinated, from such diverse areas of knowledge as physiology, biochemistry, biophysics, mathematics, and biological cybernetics. An avalanche-like increase in publication number led to foundation of several specialised journals on biological rhythms. However, plants have always been treated as “Cinderellas” in studies on biological rhythms. In contrast to quite obvious circadian rhythms (with 24 h period), ultradian oscillations (with periods of minutes) are not as widespread in plants as they are in animal organisms, at least at first glance. With a possible exception of plant movements (such as leaves or plant axial organs) and oscillations in stomatal aperture, many plant physiologists treat oscillations as some unwanted “noise”. More recently, a breakthrough in understanding of the signalling role of Ca2+ in cell metabolism caused a vivid interest in calcium oscillations in stomatal guard cells, as reflected by a large number of excellent reviews [1-3]. The physiological role of ultradian oscillations in other plant tissues and organs is still underestimated. In this review, an attempt is made to summarise the recent progress in this area and highlight the paramount role of oscillatory processes in plant life. First, advantages and principles of oscillatory control are considered in the context of plant physiology, with a major emphasis on feedback control and self-sustained oscillations, as well as on determenistic chaos and “strange” behaviour in plants. Next, a possible role for ultradian rhythms in timekeeping and the link between ultradian and circadian oscillators is discussed. Several models of circadian oscillators are analysed, and various pros - and contras - for each of them are discussed. The major emphasis is made on possible role of cellular membranes as an important component of the feedback loop in circadian clock mechanism. The importance of membrane- related oscillations is further illustrated by their crucial role as a part of the encoding mechanism, mediating plant- environmental interaction. Practical examples include cell differentiation and morphogenesis, growth, development and adaptive responses to various abiotic and biotic stresses.
Trewavas A (1999) Plant Physiology 120: 1-6 Blatt MR (2000) Current Opinion in Plant Biology3: 196-204 Webb AAR (2003) New Phytologist160: 281-303
56 Slow wave potentials – higher plants’ very own propagating electrical signals
Rainer Stahlberg
Department of Biology, University of Washington, Seattle, WA 98195, USA Email: [email protected]
Unlike action potentials (APs) slow wave potentials (SWPs, sometimes called variation potentials) are a particular creation of higher plants with no parallel occurrence in animals. The generation of SWPs occurs under different conditions than that of action potentials (APs). APs are generated and can be studied in excised stems and leaves. SWPs depend on the pressure difference between the atmosphere and an intact plant interior. Excision of leaves, anti- transpirants, air of high humidity any other condition that compromises the tension of the plant interior will severely reduce the range of SWP signals or suppress them altogether. SWPs and APs both involve transient depolarizations of plant cells that spread from their place of generation to distant parts of the plants. However, when compared in the same plant the depolarization of APs is always shorter than that of an SWP. The available data suggest that this feature reflects different ionic mechanisms. While opening anion channels mediate most of the depolarization in APs, the depolarization of SWPs probably involves both opening of calcium channels and a transient shutdown of stretch-sensitive H+-ATPases at the plasma membrane. A third defining particularity of SWPs consists in their mode of propagation. SWPs appear to involve a sequence of local depolarizations that arise in the wake of a rapid wave of positive pressure spreading through the xylem and finally resulting in rapid water uptake and turgor increase by peripheral cells. Since poisoned or killed stem sections do not interrupt SWP propagation, it follows a hydraulic rather than electric mechanism. Studying Mimosa responses, Haberlandt suggested as early as 1890 (when only APs were known) to look for a hydraulically propagating signal. It took a long time to realize that such a signal exists in the form of SWPs.
57 Hydro-electrochemical integration of the higher plant - basis for electrogenic flower initiation
Edgar Wagner*, Lars Lehner, Johannes Normann, Justyna Veit, Jolana Albrechtova
Albert-Ludwigs University Institute of Biology Schanzlerstr. 1 D-79104 Freiburg Germany * Email: [email protected]
The integration of activity of Chenopodium plants on hydraulic-electrochemical levels is expressed by a diurnal rhythm in resting membrane potential measured with contact electrodes. Membrane state could be gated by the energy state of cells. From earlier studies we compiled evidence in favour of a circadian rhythm in overall energy transduction producing a circadian rhythm in energy charge and redox state (NADP/NADPH). The ratio of metabolic coupling nucleotides would be relatively temperature independent and thus could fulfill the requirements for precise temperature-compensated time keeping. The photoreceptor phytochrome, involved in photoperiodic control of development, could via changes in pyridine nucleotide pool sizes and changes in nucleotide ratios regulate transcription via redox controlled transcription factors. Spontaneous action potentials (APs) have been shown to correlate with turgor controlled growth movements. The accumulation of spontaneous APs at specific times during daily light- dark spans were recorded giving specific electrophysiograms (EPGs). There was a switch in predominant propagation direction of APs along the stem axis (acropetal-basipetal) in the transition from vegetative to reproductive growth; opposite in long- and short-day Chenopodium plants. The information from EPGs showing the frequency distribution and polarity changes of spontaneous APs in response to flower inducing and non inducing photoperiods have been used to induce flowering in non inductive photoperiods by specific timing and polarity of direct current pulses via contact electrodes. It is anticipated that hydraulic changes at the apex leading to flower initiation are mediated by a specific hydro- electrochemical communication between leaves and the shoot ape
58 Forisomes as sensors and aphids as reporters of Ca2+-influx and efflux during depolarization waves along sieve tubes
Aart J.E. van Bel*, Jens B. Hafke, Torsten Will
Plant Cell Biology Research Group, Institute of General Botany, Justus-Liebig- Universität, Senckenbergstrasse 17, D-35390 Giessen, Germany *Email: [email protected]
Experiments on electrical propagation are often lacking of a precise definition of the cell types and the ion species involved. We developed an in vivo system that unequivocally shows calcium involvement in electrical long-distance signalling via the sieve tubes in intact plants. Shallow windows paradermally cut in the cortex of major leaf veins of Vicia faba allowed observation of events in sieve tubes and the insertion of microcapillaries into sieve elements. Exclusively in sieve elements of Fabaceae, protein bodies (forisomes) occur which reversibly disperse and contract in dependence of calcium. Forisome dispersion enabled to determine the in vivo Ca2+ concentration in the sieve tubes. Burning of the leaf tip triggered a depolarization wave along the sieve tubes which concurred with an increase of intracellular calcium in the sieve elements as shown by Oregon Green. Upon arrival of the depolarization wave, forisomes often readily dispersed. The forisomes re-contracted several minutes after passage of the wave. The behaviour of forisomes and the depolarization profiles indicate the involvement of calcium channels and calcium pumps in electrical propagation along the sieve elements. As forisomes temporarily plug sieve plates, the reaction of aphids to burning-induced plugging of the sieve plates - and thus food deprivation - was investigated. Behaviour of aphids and the position of the stylet insertion can be monitored by the so-called EPG-technique. Microelectrodes attached onto the aphid's abdomen record electrical patterns produced by the aphid during foraging. Each specific wave pattern is related to foraging of a defined cell type. After puncturing of a sieve tube, aphids produce watery saliva which is reflected by a E1-wave pattern. As soon as the aphids are settled, an E2-wave pattern establishes which can hold for many hours. The hypothesis that the aphids would react to sudden food deprivation or turgor drop was tested in the plant/aphid combinations Vicia faba/Megoura viciae and Hordeum vulgare/Schizaphis graminae by burning the leaf tips. It appeared that plugging of sieve plates causes an immediate transition from the E2- back to the E1-wave pattern. Aphids were then used to mimick a row of microelectrodes along the phloem. The behaviour of aphids at various distances from the site of burning enabled us to record speed and magnitude of the depolarization wave. The degree of the aphid's reaction was supposed to be related to the degree of sieve plate plugging which in turn is related to the degree of local depolarization and Ca2+ influx. The EPG-data seem to indicate that the propagation speed slows down with the distance and that the depolarization wave becomes extinct after 10 to 20 cm.
59 Heat-induced electrical signals change photosynthesis in poplar
Silke Lautner, Thorsten E.E. Grams, Rainer Matyssek, Jörg Fromm*
Fachgebiet Holzbiologie,TU München, Winzererstr. 45, 80797 München, Germany *Email: [email protected]
Plants respond to various abiotic stimuli by generation and propagation of electrical signals. To get more information on the response of photosystem II (PSII) in higher plants, we investigated heat-induced long and short-distance signalling in poplar trees (Populus trichocarpa) by means of chlorophyll fluorescence imaging. In both types of signalling, PSII quantum yield of electron transport is reduced significantly (from c. 0.55 to 0.35). Two- dimensional imaging analysis of the fluorescence signal manifests the yield reduction which spreads via the veins through the leaves. The different types of signal propagation were shown by microelectrode measurements in leaf-vein phloem via the aphid technique; basipetal signal transduction leads to rapid membrane hyperpolarisation within the same leaf, whereas acropetal long-distance signalling causes depolarisation of the membrane potential in leaf phloem. Moreover, gas exchange measurements revealed that the depolarising signals travel distances across the stem to neighbouring leaves where the net CO2 uptake rate is temporarily depressed towards compensation. Controls show that after cooling of the stem to +4°C electrical signal transmission via the phloem is disrupted so that leaf gas exchange stays unchanged. By measuring calcium-deficient poplar plants, a much lower amplitude of the electrical signal was detected, and no significant response in gas exchange was observed after heat-induced wounding of leaves. We therefore conclude that electrical signals significantly affect the photosynthetic performance of poplar trees.
60 Electrophysiology and Phototropism
Alexander G. Volkov
Department of Chemistry, Oakwood College, Huntsville, Alabama 35896, USA Email: [email protected]
Plants generate different types of electrical events in connection to environmental stress. Cells, tissues, and organs transmit electrochemical signals over short and long distances. Action potentials in higher plants may be the information carriers in intercellular and intracellular communication in response to environmental changes. Here, we show the generation of bioelectrochemical responses induced by blue photosensory system in soybean plants. A phototropic response is a sequence of the four following processes: reception of the directional light signal, signal transduction, transformation of the signal to a physiological response, and the production of directional growth response. The irradiation of soybean plants at 450 ± 50 nm induces action potentials with duration times of about 0.3 ms and amplitudes about 60 mV. Action potentials play an active role in the expedient character of the response reactions of plants as a reply to external stimuli. Blockers of ionic channels inhibit phototropism in soybean plants. The role of the electrified interface of the plasma membrane in signal transduction is discussed.
O. S. Ksenzhek and A. G. Volkov, Plant Energetics, Academic Press , San Diego , New York , 1998 A. G. Volkov, Interfacial Catalysis, Marcel Dekker, New York , 2003. A. G. Volkov, T. C. Dunkley, S. A. Morgan, D. Ruff, J. L. Boyce, A. J. Labady, Bioelectrochemistry, 63, 91-94, 2004.
61 Signals and signalling pathways in plant wound responses
Jeremy Rhodes, John Thain, David Wildon*
School of Biological Sciences, University of East Anglia, Norwich, UK *Email: [email protected]
A widely studied system is the wound induction of proteinase inhibitors (PIs), and other proteins, that are produced locally by the wounded organ, and systemically in organs distant from the wound site. PIs are inhibitors of a variety of proteases, including those from pathogens and herbivorous insects, and are important components of plant resistance to herbivores. There are several possibilities for the signal(s) that link a local wound to the systemic responses, including various chemical elicitors, a pressure wave caused by release of tension in the xylem, and an electrical signal that could be propagated in the phloem. In tomato seedlings, following a severe wound, evidence favours the hydraulic dispersal of chemical elicitors by reversal of flow in the xylem, a mechanism proposed by Malone and co- workers, whereas minor wounds seem to be associated with the transport of chemical elicitors in the phloem. These various possibilities for signals and signalling pathways will be discussed.
62 Effects of high frequency electromagnetic stimulation on plants
Alain Vian
Université Blaise Pascal, Campus universitaire des Cézeaux, 24 avenue des Landais, 63177 AUBIERE Cedex, FRANCE Email: [email protected]
Studies on the effects of high frequency electromagnetic stimulation on living beings have grown in number over the last few years, particularly in relation to the exponential increase in use of cell phones among the general population. This telecommunication device uses high frequency, non thermic electromagnetic waves (900–1800 MHz) coupled with a lower frequency carrier (230 Hz). Shortly after the advent of cell phones, the question arose as to whether this kind of radiation could constitute an environmental threat for living organisms, particularly in relation to possible health effects. We chose to use tomato plants as our model biological system along with a Mode Stirring Reverberation Chamber (MSRC) as the stimulating device. Plants offer numerous advantages compared with animals (ease of growth, lack of mobility, absence of psychological side-effects), while the MSRC allows us to generate a highly controlled and reproducible isotropic, homogeneous electromagnetic field. Our investigation was focused at the molecular level, where we measured rapid changes in mRNA accumulation (using qRT-PCR) of stress-related genes as molecular markers. We found that stress-related mRNAs such as calmodulin, bZIP transcription factor and protein kinase strongly accumulate within minutes after a short (10 min), low amplitude (5 V/m), 900 MHz EMF stimulation. This accumulation does not occur if the plants are placed into an EMF-proof container. Futhermore, exposure of a single leaf (while masking the rest of the plant) shows that a traumatic signal rapidly moves from the stimulated leaf to the rest of the plant and elicits accumulation of these same stress-related transcripts. In conclusion, we demonstrated that low amplitude EMF stimulation constitutes an environmental stimulus able to activate stress-related genes in tomato plants.
63 Shade avoidance without photoreceptors
Thijs L. Pons*, Ronald Pierik, Niels P.R. Anten, Rens (L.) A.C.J. Voesenek
Dept. Plant Ecophysiology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands *Email: [email protected]
One of the best-known examples of adaptive plasticity in plants is the shade avoidance response of plants to canopy density perceived by phytochrome. However, it is now evident that proximity detection by means of this family of photoreceptors is not the only signalling pathway available to plants for this critical information for survival in a competitive situation. Other photoreceptors are involved as well, and ethylene is important also. We found that the response of plants to a mechanical stimulus works in a manner that is consistent with shade avoidance. The examples mentioned so far are concerned with avoidance of shading of leaves, which thus maximizes photosynthetic carbon gain. Another form of shade avoidance is the reallocation of photosynthetic capacity from lower shaded leaves to upper leaves in more favourable light conditions, which has a similar function. Photoreceptors do not play an important role in this process. The distribution of the transpiration stream parallel to the light gradient appeared to be involved in an alternative signalling pathway.
64 Root Exudation and Rhizosphere Biology: Allelochemicals and Cell Death
Laura G. Perry*, Mark W. Paschke, Jorge M. Vivanco
Center for Rhizosphere Biology, Colorado State University, Fort Collins CO 80523, USA *Email: [email protected]
Plant roots release an enormous variety of secondary metabolites into the soil. These root exudates can play important roles in mediating positive and antagonistic root-root and root- microbe interactions. We will focus our discussion on the role of root exudates in negative and positive communication between roots of Centaurea maculosa and competing plant species. Centaurea maculosa , a native species in western Europe, is an invasive weed in North America . Our work on C. maculosa root exudates indicates that C. maculosa invasions in North America are partly mediated by root-secretion of a potent phytotoxin, (±)-catechin. Roots of susceptible plants such as Arabidopsis thaliana exposed to (±)-catechin exhibit waves of ROS and Ca 2+ signals that begin at the meristematic and elongation zones, and lead to genome-wide changes in expression and cell death. We investigated the genes involved in early signal transduction triggered by (±)-catechin using T-DNA mutants. Our studies have shown that some T-DNA mutants exhibit resistance to (±)-catechin phytotoxicity and at least one gene mutation increases (±)-catechin-sensitivity in Arabidopsis. These results indicate a complex network of genes working simultaneously to cause cell death and to counteract cell death, possibly by affecting levels of ROS. Soil (±)-catechin concentrations in C. maculosa populations can be very high (mean = 1.55 mg g -1), and tend to be higher in North American populations than in European populations. Further, North American grassland species tend to be more sensitive to (±)-catechin than the European species with which C. maculosa naturally coexists, suggesting that (1) European species may have co-evolved (±)-catechin-resistance and (2) the (±)-catechin-naïvité of North American species may account for C. maculosa invasiveness in North America. Our research also suggests that high concentrations of (±)-catechin are autotoxic to C. maculosa seedlings and inhibit C. maculosa recruitment in well-established stands, indicating chemical control of population density. In addition, we have found that low catechin concentrations can induce growth and defense responses in (±)-catechin-susceptible species, indicating the potential for chemical facilitation by C. maculosa under some conditions. Finally, our work on mechanisms of (±)-catechin-resistance has shown that at least two North American species (Lupinus sericeus and Gaillardia grandiflora) also rely on root exudates to resist (±)-catechin toxicity. Lupinus and Gaillardia root-secrete oxalic acid in response to exposure to (±)-catechin. Oxalic acid, by working as an antioxidant, protects the resistant species and their (±)-catechin- susceptible neighbors from (±)-catechin toxicity under in vitro conditions and in the field. The presence of the resistant species or exogenous oxalic acid reduces C. maculosa (±)-catechin production, revealing the potential for chemical cross-talk between allelopathic and allelochemical-resistant species, and the coordination of defense responses between plants.
65 The transmogrification of plant invaders: biogeographic differences in allelopathic effects and native evolutionary responses
Ragan M. Callaway1,*, Jorge M. Vivanco2
1 The Division of Biological Sciences, The University of Montana, Missoula, MT 59812, USA 2 Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, CO 80523, USA *Email: [email protected]
Many exotic plant species undergo astounding increases in dominance when introduced to new communities by humans. This is primarily attributed to escape from specialist consumers. However, strong allelopathic effects (a form of plant-plant communication) by some powerful invaders and much stronger allelopathic effects in invaded than in native ranges suggest a new theory for invasive success – the novel weapons hypothesis. We discuss the evidence for allelopathic effects of Centaurea maculosa and C. diffusa, evidence for belowground cross-talk, and propose that some invaders transmogrify because they possess novel biochemical weapons that function as unusually powerful allelopathic agents or as mediators of new plant- soil microbial interactions. Novel biochemical weapons possessed by some plant invaders may provide an advantage based on differences in historical coevolutionary trajectories. Furthermore, exotic plant invasions often cause high mortality in native populations and therefore have the potential to be a selective force on natives. We found that individuals of some North American species that survived Centaurea maculosa invasion have higher tolerances to the European invader than individuals from communities that had not experienced invasion. These results provide initial evidence that native plants species may evolve to tolerate the effects of an exotic invader, and in particular an invader’s novel allelochemistry.
66 Interplant communication: From induced volatiles to signal transduction pathways
Jürgen Engelberth*, Irmgard Seidl-Adams, Jack C. Shultz, James H. Tumlinson
Penn State University, Dept. of Entomology, 117 A Chemical Ecology Lab, University Park, PA 16802, USA *Email: [email protected]
Plants under insect herbivore attack have evolved various mechanisms to counteract this threat. Among the measures plants undertake to survive with the least damage are the recognition of insect-derived elicitors, production of proteins that block digestion or disrupt intestinal tissue, and the production of defense-related secondary metabolites, which directly or indirectly affect the herbivore performance. Volatile organic compounds (VOC), a mixture of volatile secondary metabolites from various pathways, serve as signals not only to attract predators and parasites of attacking herbivores, but also can be recognized by neighboring plants resulting in defense-related gene expression. Jasmonic acid (JA) and other lipid-derived compounds (oxylipins), which are activated by wounding and insect elicitors, represent important signals in this process. In corn (Zea mays), insect-derived elicitors not only increase the production of oxylipins locally, but also induce JA distal from the application site within 5- 10 min. Green leafy volatiles (GLV), which are rapidly emitted during herbivory, serve as potent volatile signals for neighboring receiver plants. By inducing specific sections of the octadecanoid signaling pathway GLV can act as priming signals preparing those plants against impending herbivory. Structure/function analysis of natural GLV as well as synthetic analogs clearly showed certain structural requirements, but excluded α,β-unsaturated carbonyls as active centers. A comparison of gene expression after wounding, wounding with application of crude regurgitant elicitors (CRE), and exposure to Z-3-hexenyl acetate (Z-3-HAC) further demonstrated the specificity of the GLV signal in plant-plant communication through selective activation of genes involved in JA biosynthesis. In conclusion, inter-plant communication via GLV results in an enhanced preparedness specifically directed against insect herbivore attack mediated by specific activation of distinct parts of the octadecanoid signaling pathway.
67 Communication between undamaged plants by volatiles: the role of allelobiosis
V. Ninkovic*, R. Glinwood, J. Pettersson
Swedish University of Agricultural Sciences, Department of Entomology, Box 7044, 75007 Uppsala, Sweden * Email: [email protected]
Plant/plant communication via volatiles, allelopathy, may have profound effects on development of exposed plant individuals (1). Hypothetically these responses affect insects adapted to living on and/or around these plants. To identify effects of plant-plant communication via volatiles on different trophical levels we have used a model system consisting of different barley cultivars and two common weeds, an aphid pest, Rhopalosiphum padi (L.), and an aphid predator, ladybird, Coccinella septempunctata (L.). Plant-plant communication mediated by volatile messenger substances significantly affect plant leaf temperature (2) and biomass allocation (3) but not relative growth rate and total biomass. Significant changes in leaf temperature and biomass allocation of exposed plants to allelobiotic volatilise show that the allelopathic effect was systemic. Barley plants exposed to volatiles from neighbouring plants were less readily accepted by aphids when specific cultivars of barley were combined (intra-species) (2; 4) and between cereals and a number of aggressive weeds (inter-species) (5). Exposure to volatiles from creeping thistle, Cirsium arvense (L.), also causes barley plants to become more attractive to ladybird (L.) (6). For this tritrophic effect of plant/plant communication we suggest the term allelobiosis defined as the effects of chemical interactions between plants across trophic leaves (7). Our results support the hypothesise that plant responses to allelobiosis is an adaptation for coexistence with other which is a basis for follow up effects in higher trophic levels. The positive effect on polyphagous predators may increase the ecological success of listening” plant stand and, also have negative effects on herbivore plant acceptance.
Rice, E. L. 1984. Allelopathy. Academic press, INC, Oriando, Florida, USA. Pettersson, J., Ninkovic, V. & Ahmed, E. (1999) Acta Agriculture Scandinavica Section B, Plant and Soil, 49, 12-157. Ninkovic, V. (2003) Journal of Experimental Botany, 54, 1931-1939. Ninkovic, V., Olsson, U. & Pettersson, J. (2002) Entomologia Experimentalis at Applicata. 102, 177-182. Glinwood, R., Ninkovic, V., Pettersson, J. & Ahmed, E. (2004) Ecological Entomology, 29, 188- 195. Ninkovic, V. & Pettersson. J. (2003) OIKOS, 100, 65-70. Pettersson, J., Ninkovic, V & Glinwood, R. (2003) BCPC Crop Science and Technology 2003, volume 2, 1135-1144
68 Self/non-self recognition in mycorrhizal networks
Manuela Giovannetti
Department of Chemistry and Agricultural Biotechnology, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy Email: [email protected]
Most terrestrial plant species establish mutualistic symbioses with arbuscular mycorrhizal (AM) fungi, which develop extensive, belowground extraradical hyphae fundamental for the uptake of nutrients from soil and their transfer to the host plant (1) . Since AM fungi have a wide host range, they are able to colonize and interconnect plants of different species, genera and families, by means of hyphae extending from one root system to another . Such mycorrhizal networks, visualized and quantified in vivo by using a two-dimensional experimental system, extend at a mean growth rate ranging from 738 to 1067 mm per day, depending on the host plant, and reach hyphal densities of 10-40 mm per mm of root length. Moreover, AM extraradical networks are highly interconnected due to the widespread occurrence of hyphal fusions (anastomoses) (2). Successful anastomoses , first described during the pre-symbiotic growth of AM fungi, occur between hyphae belonging to the same individual and to different individuals of the same strain. They are characterized by self compatibility, consisting in complete fusion of hyphal walls, cytoplasmic flow and migration of nuclei through hyphal bridges, as revealed by time- lapse, video-enhanced and epifluorescence microscopy (3) . By contrast, hyphae of individuals belonging to different genera and species, and even to geographic isolates of the same species, do not fuse, and show rejection responses, either before or after anastomosis , revealing AM hyphal ability to discriminate self from non-self. Incompatibility responses consist of protoplasm retraction from hyphal tips and septum formation in approaching hyphae, even before physical contact, suggesting that specific recognition signals are involved in hyphal fusion (4). Extraradical mycorrhizal networks maintain the capacity of self recognition, evidenced by the high frequency of anastomosis between hyphae originating from the same root system (2). Recent results show that the root systems of plants belonging to different species, genera and families become interconnected by means of anastomosis formation between mycorrhizal mycelia, which can potentially create indefinitely large continuous fungal network linking together plants in a community (5). The emerging picture of mycorrhizal networks is one of previously unimagined dynamism and provides further support to the view that AM fungal symbionts play a fundamental role in the distribution of resources by the establishment of functional guilds in plant communities.
Smith and Read (1997). Mycorrhizal symbiosis. London , UK . Academic Press. Giovannetti et al. (2001). New Phytologist, 151, 717-724. Giovannetti et al. (1999). Applied and Environmental Microbiology, 65, 5571-5575. Giovannetti et al. (2003). Applied and Environmental Microbiology, 69, 616-624. Giovannetti et al. (2004). New Phytologist,164, 175-181.
69 Poster Presentations
Charzewska A, Zawadzki T, Krupa M, Stolarz M: The free-running rhythm of circumnutation activity in sunflower (Helianthus annuus L.)
Tamás Visnovitz, Ildikó Világi, Zoltán Kristóf: Mechanoreceptor cells on the tercier pulvinus of Mimosa pudica L.
Elzbieta Krol, Halina Dziubinska, Kazimierz Trębacz, Maria Stolarz, Maria Filek: Different Responses to cold recorded in spring - and winter - varieties of rape (Brassica napus L. var. oleifera)
Maria Stolarz, Halina Dziubińska, Elżbieta Król, Maciej Krupa, Agnieszka Charzewska, Tadeusz Zawadzki, Kazimierz Trębacz: Light-induced changes in stem growth and circumnutations in Helianthus annuus L.
Viktor E. Tsyganov, Alexander I. Zhernakov, Anna V. Khodorenko, Zlata B. Pavlova, Pavel Y. Kisutin, Andrei A. Belimov, Vera I. Safronova, Tatyana S. Naumkina, Alexey Y. Borisov, Peter Lindblad, Karl-Josef Dietz, Igor A. Tikhonovich: Genetic approach to study pea (Pisum sativum L.) adaptations to mechanical and cadmium stresses during development its symbioses with Rhizobium and arbuscular mycorrhizal fungi
Kiril N. Demchenko, N.P. Demchenko, Katharina Pawlowski: Myosin VIII, actin and tubulin in the development of symbiotic contacts by actinorhizal and rhizobial root nodules
Miroslav Ovecka, Irene Lichtscheidl, František Baluška: Plant synapses in plant root apex are enriched with lipid rafts
Boris Voigt, Molly Craxton, Bazbek Davletov, Patrick J. Hussey, Diedrik Menzel, František Baluška: Expression and localization of Arabidopsis synaptotagmins
Tomohiro Uemura, Takashi Ueda, Akihiko Nakano, Masa H. Sato: Plant SNARE molecules involved in endocytosis and exocytosis
Pilar Gil Montenegro: Root–to–leaf electrical signaling in Avocado (Persea americana Mill.)
Markus Schlicht, Boris Voigt, Alina Schick, Miroslav Strnad, Klaus Palme, Dieter Volkmann, Diedrik Menzel, František Baluška: End-Poles of Root Cells as Auxin Transporting Plant Synapses
Susanna M. Messinger, Keith Mott, David Peak: Noise Enhanced Distributed Emergent Computation in Plants
Maja Kovač, Darja Milovanovič Jarh, Axel Műller, Anita Purnat, Špela Baebler, Hana Krečič, Mojca Milavec, Maruša Pompe-Novak, Kristina Gruden, Maja Ravnikar: Jasmonic acid, salicylic acid and gene expression in early response of potato plants to virus infection
Jolana Albrechtova, Edgar Wagner: Hydraulic signals and plant development
Ying-Lang Wan, Halina Gabrys, František Baluška, Diedrik Menzel: Blue Light-Regulated Vesicular Recycling of Phototropin1 in the Root Transition Zone
Jevin West, Keith Mott, David Peak: Why decentralized computation in plants?
Weronika Krzeszowiec, Halina Gabrys: What do myosins do in blue light?
70
Vladimíra Hlaváčková, Pavel Krchňák, Petr Ilík, Jan Nauš, Ondřej Novák, Radek Kaňa, Martina Špundová: The first moving signal inducing systemic changes in photosynthesis after local burning of tobacco plants
Wasser K, Müller J, Preuss ML, Holstein S, Böhm N, Nielsen E, Hirt H, Menzel D, Šamaj J: Signalling endosomes in plants: activated MAPKs associate with CCVs and endosomes
Jens Müller, Nils Böhm, Heribert Hirt, Diedrik Menzel, Jozef Šamaj: Dynamic behaviour of SIMKK, a plant stress-induced mitogen-activated protein kinase kinase and its downstream target SIMK
71 The free-running rhythm of circumnutation activity in sunflower (Helianthus annuus L.)
Charzewska A*, Zawadzki T, Krupa M, Stolarz M
Department of Biophysics, Institute of Biology, M. Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland
Introduction: Circumnutation is a revolving movement of elongating cylindrical plant organs such as stems, hypocotyls, tendrils and roots. It is the consequence of helical growth and reversible volume variations occurring in the cells of the moving part of the organ (the bending zone below the apex). The bending is caused by the difference in the water content and cell volume between the convex and concave sides of the bending zone, associated with turgor and ion concentration differences between opposite sides of the stem. It is proposed that turgor changes are generated by endogenous, spontaneous oscillations. As a consequence oscillatory growth and movement are generated. We aimed to compare the rhythmicity of circumnutation in sunflower with regard to the trajectory length, period, shape and surface area of individual nutations in different photoperiod conditions. Material and Methods: Measurements lasting about 7 weeks were executing using a picture analysis system. Variable photoperiods – a 24h photoperiod (LD 16:8 followed by LL), a 30h photoperiod (LD 20:10 followed by LL and again by LD 20:10) and a 16h photoperiod (LD 8:8 followed by LL) were applied. In order to determine whether a periodicity of circumnutation parameters existed or not, the data were processed by the Fourier spectral analysis and the wavelet analysis. Results and conclusions: We found that all parameters revealed a beautiful daily modulation in each LD condition applied. After LD-LL transition, the parameters were gradually loosing their modulation. In the transient phase (about 7 days after LD-LL transition) the rhythms of the circumnutation parameters turned out to be close to 24h in the 24h photoperiod as well as in the 30h and 16h photoperiod. These findings suggest the possibility for circumnutation of being circadian regulated. After the LL-LD transition the rhythms regained their daily modulation after one night. These findings strongly support the view that circumnutation in sunflower, widely known as an ultradian rhythm, also possesses daily modulations of its intensity and it can tune in a wide range to the frequency of environment.
Buda A, Zawadzki T, Krupa M, Stolarz M, Okulski W. 2003. Daily and infradian rhythms of circumnutation intensity in Helianthus annuus L. Physiol Plant 119: 582-589 Caré AF, Nefed’ev L, Bonnet B, Millet B, Badot PM. 1998. Cell elongation and revolving movement in Phaseoulus vulgaris L. twining shoots. Plant Cell Physiol 39: 914-921.
72 Mechanoreceptor cells on the tercier pulvinus of Mimosa pudica L.
Tamás Visnovitz1,*, Ildikó Világi2, Zoltán Kristóf1
Department of Plant Anatomy 1 and Department of Physiology and Neurobiology 2, Eötvös Lorand University of Sciences, Faculty of Natural Sciences, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary *Email: [email protected]
Mimosa pudica is a famous plant, grown for its curious movement; the leaves close up and drop when touched, and re-opening within a few minutes. It has become a subject of many studies (even Charles Darwin dealt with the seizmonastic movement of Mimosa), “but the receptor cells have not yet been identified” (Shimen, 2001). We described a special cell type on the adaxial part of the tercier pulvini. Stimulating these cells, the leaf that contains the pulvinus closed. A very gentle mechanical stimulation, -carried out with a micromanipulator needle - was effective in these cells, but ineffective for any other cells of the tercier pulvinus. Light and electron microscopy were carried out to reveal the connections between cells and the motor cells. The receptor cells and the motor organ have connections through plasmodesmata. Samples were taken from different stages of developing leaves to study the differentiation of the receptor cell. The sensitivity of the receptor cells for mechanical stimulation requires a special developmental stage. The electrophysiological studies supported our receptor cell theory. We measured electrochemical potential in these cells. After mechanical stimulation (about 0,3 sec) hyperpolarisation and then a depolarisation peak was recorded. Repolarisation developed about 1 sec later. Leaves in refracter stadium could not produce those potential changes. We make efforts to understand the function of that cells also at molecular level.
73 Different responses to cold recorded in spring and winter varieties of rape (Brassica napus L. var. oleifera)
Elzbieta Krol1,*, Halina Dziubinska1, Kazimierz Trębacz1, Maria Stolarz1, Maria Filek2
1 Department of Biophysics, Institute of Biology, Maria Curie-Skłodowska University, Akademicka 19, PL-20-033 Lublin, Poland 2 Department of Plant Physiology, Polish Academy of Science, Podłużna 3, PL-30-239 Kraków, Poland
In our study we used intracellular microelectorde technique to demonstrate that the spring and winter varieties of Brassica napus L. var. oleifera respond to cold in different ways. The transmembrane potentials were measured in the mesophyll cells of leaves in both varieties. Cold stimulation was obtained by local application of 0.5 ml of cold solution (1oC) to the leaf surface and a local drop of temperature was approx. to 8o – 10oC. The transmembrane potentials and the durations of cold-induced potential changes depended on a kind of a leaf. The mesophyll cells of a spring variety displayed low transmembrane difference (-100 mV) and its cold-induced potential changes lasted on average 8 s (measured in a half of amplitude). The cells from a winter variety, which had been vernalized, had almost two fold larger transmembrane difference (-185 mV) and their cold-induced potential changes lasted significantly longer – approx. 18 s . The results are consistent with the general assumption that plants have a kind of memory remembering previous environmental conditions.
74 Light-induced changes in stem growth and circumnutations in Helianthus annuus L.
Maria Stolarz*, Halina Dziubińska, Elżbieta Król, Maciej Krupa, Agnieszka Charzewska, Tadeusz Zawadzki, Kazimierz Trębacz
Department of Biophysics, Institute of Biology, Maria Curie-Skłodowska University, Akademicka 19, PL-20-033 Lublin, Poland
Many studies including applications of external stimuli like different gravitational forces, touch, high and low temperature and chemicals have been carried out to understand the mechanisms underling circumnutations. Circumnutations are defined as helical movements of a growing plant organ. We investigated the effects of light/dark 16/8h period on stem growth and circumnutations in three-week-old sunflowers. The growth kinetics was being monitored by an angular position-sensing transducer for a three days and for circumnutation characteristic the time-lapse images from top view camera were registered and analyzed. Under 24 h light/dark cycle both the stem growth and circumnutation parameters displayed diurnal rhythm . Besides long-lasting fluctuation in a range of many hours, the light- induced short sunflower stem response in a range of a few minutes were observed. After switching on the light a rapid decrease of the growth rate and even a stem contraction was observed. The kinetics of stem reaction had a characteristic wave-like shape and all in all persisted about 60 minutes. These drastic changes in stem length were accompanied by phase dependent circumnutation disturbances. These results suggest that after non-injured light stimuli hydro-electrochemical signals which might interfere with circumnutation mechanism appeared in sunflower stem.
75 Genetic approach to study pea (Pisum sativum L.) adaptations to mechanical and cadmium stresses during development its symbioses with Rhizobium and arbuscular mycorrhizal fungi
Viktor E.Tsyganov1,*, Alexander I. Zhernakov1, Anna V. Khodorenko1, Zlata B. Pavlova1, Pavel Y. Kisutin1, Andrei A. Belimov1, Vera I. Safronova1, Tatyana S. Naumkina2, Alexey Y. Borisov1, Peter Lindblad2, Karl-Josef Dietz3, Igor A. Tikhonovich1
1 All-Russia Research Institute for Agricultural Microbiology, Podbelsky ch. 3, Pushkin 8, 196608, St.-Petersburg, Russia 2 Institute of Grain Legumes and Groat Crops, Orel, p/b Streletskoe, Russia 3 Department of Physiology and Biochemistry of Plants, University of Bielefeld, Germany *Email: [email protected]
Legume endosymbioses with nodule bacteria and arbuscular mycorrhizal fungi is sensitive to stresses and, at the same time, is able to decrease the stress effects on plants. Using of genetic approach allows to identify plant genes involved in the control over adaptations of legume symbioses to stresses. In this study, two mutants with changed reactions to stresses have been analysed by their ability to interact with Rhizobium and mycorhhizal fungi: SGEcrt (crt) with hypersensitivity of roots to touch stimuli [1] and SGECd t (cdt) with increased tolerance to cadmium (Cd) [2]. It was demonstrated that mutant SGEcrt (crt) has decreased nodulation ability which can be restored by addition of ethylene action inhibitors. In contrast, the mutant SGEcrt (crt) is characterized with accelerated mycorrhizal colonization. The mutant SGECd t (cdt) was characterized with increased tolerance of nodulation to toxic Cd concentration in comparison with wild type.
Supported by RFBR (04-04-48462-a), St. Petersburg Governement (2004), INTAS (01-270) and for VET by St. Petersburg Governement fellowship (PD04-1/4-230) .
Tsyganov et al., Ann. Bot. 2000. 86:975-981; Tsyganov et al., Biology of Plant-Microbe Interactions, Vol. 4, 2004. pp: 506-509
76 Myosin VIII, actin and tubulin in the development of symbiotic contacts by actinorhizal and rhizobial root nodules
K.N. Demchenko1,*, N.P. Demchenko1, K. Pawlowski2
1 Komarov Botanical Institute, Russian Academy of Sciences, St.-Petersburg, Russian Federation 2 Albrecht von Haller Institute for Plant Sciences, Plant Biochemistry, University of Göttingen, Göttingen, Germany *Email: [email protected]
Two groups of plants can enter root nodule symbioses with nitrogen-fixing soil bacteria. Gram- negative, unicellular rhizobia induce nodules on legume roots, while Gram-positive mycelial actinomycetes of the genus Frankia induce nodules on the roots of plants from eight angiosperm families, mostly woody shrubs, collectively called actinorhizal plants. Based on molecular phylogenetic studies, both types of root nodule symbioses are supposed to go back to a common ancestor. We compared infection thread growth in actinorhizal and legume nodules by immunolocalization of components of the cytoskeleton. We used myosin VIII- specific antibodies and antibodies against actin and tubulin to analyse the intracellular accommodation of microsymbionts in legume (Medicago truncatula, pea) and actinorhizal symbioses (Casuarina glauca, Datisca glomerata) on light microscopy and TEM level. Myosin VIII accumulated around infection threads in actinorhizal systems, but not around legume infection threads.
This study was supported by the Russian Foundation for Basic Research (04-04-48282) and by the German Research Council.
77 Plant synapses in plant root apex are enriched with lipid rafts
Miroslav Ovecka1,2,*, Irene Lichtscheidl1, František Baluška2,3
1 Institution of Cell imaging and Ultrastructure Research, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria 2 Institute of Botany, Slovak Academy of Sciences, Dubravska cesta 14, SK-84523 Bratislava, Slovak Republic 3 Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany *Email: [email protected]
Structural sterols are integral components of the plasma membrane. They are enriched in spatially differentiated microdomains, “lipid rafts”. We visualized structural sterols in root apex of Arabidopsis thaliana by fluorescence microscopy. In our study, we compared the distribution of structural sterols in the plasma membrane in two types of root cells with distinct mode of elongation: diffusely expanding meristematic cells arranged in cell files and tip-growing tubular root hairs. Our results show that structural sterols were abundant at the plasma membrane of root hair apices and they were internalized into endosomal compartments. In root meristematic cells they were present in the plasma membrane and endosomal compartments, but in certain developmental zones of the root apex they were enriched in non-growing cross cell poles representing plant synapses. Both actin-enriched root hair apices and non-growing end poles of the cell surface enriched in actin and myosin VIII are cell periphery domains known to be active sites of intense endocytosis and vesicular trafficking. Enrichment of structural sterols in these domains indicates that they can be involved in the modulation of the physiological properties of the plasma membrane responsible for the maintenance of actin- dependent rapid membrane trafficking and recycling.
Supported by EU, project TIPNET (HPRN-CT-200200265) and by the Grant Agency VEGA (grant No. 2/5085/25)
78 Expression and localization of Arabidopsis synaptotagmins
Boris Voigt1,*, Molly Craxton2, Bazbek Davletov2, Patrick J. Hussey3, Diedrik Menzel1, František Baluška1
1 Institute of Molecular and Cellular Biology, University of Bonn , Bonn , Germany 2 MRC Laboratory of Molecular Biology, Cambridge, UK 3 Integrative Cell Biology Laboratory, University of Durham, Durham, UK *Email: [email protected]
Synaptotagmins are known Ca2+ dependent triggers of exocytosis and endocytosis in animals with nervous system. Sequence analysis of various animal and plant genomes revealed the presence of synaptotagmin genes in all animals and land plants, but there is no evidence of synaptotagmin genes in unicellular organisms or those with simple forms of multicellularity. Synaptotagmins have a common structure, a N-terminal transmembrane sequence is followed by a linker and two distinct C2 domains, C2A and C2B. These two domains are known Ca2+ binding domains, but there is nothing more known about the function of the proteins in plants. To get more information about the 6 members of the Arabidopsis synaptotagmin gene family, we are investigating expression patterns by promotor-GUS fusions and cellular localizations by GFP-fusions as well as antibody labelings. Preliminary data suggest that the members of the synaptotagmin gene family are differently expressed, but show similar cellular localization.
79 Plant SNARE molecules involved in endocytosis and exocytosis
Tomohiro Uemura1,2, Takashi Ueda3, Akihiko Nakano3,4, Masa H. Sato2
1 Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 2 Laboratory of Developmental Cell Biology, Department of Biological Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 3 Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 4 Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan *Email: [email protected] ([email protected])
In all eucaryotic cells, specific vesicle fusion during vesicular transport is mediated by membrane-associated proteins called SNAREs ( soluble N-ethyl-maleimide sensitive factor attachment protein receptors). Sequence analysis identified a total of 54 SNARE genes (18 Qa- SNAREs/Syntaxins, 11 Qb-SNAREs, 8 Qc-SNAREs, 14 R-SNAREs/VAMPs and 3 SNAP-25) in the Arabidopsis genome. Almost all of them were ubiquitously expressed through all tissues examined. A series of transient expression assays using green fluorescent protein (GFP) fused proteins revealed that most of SNARE proteins were located on specific intracellular compartments. As the results , we identified 18 SNARE molecules on the plasma membrane; 9 Qa-SNAREs (SYP111, SYP112, SYP121, SYP122, SYP123, SYP124, SYP125, SYP131 and SYP132), 1 Qb-SNAREs (AtVTI12), 3 Qc-SNAREs (N`PSN11, NPSN12 and NPSN13) and 5 R- SNAREs (AtVAMP721, AtVAMP722, AtVAMP724, AtVAMP725 and AtVAMP726). These SNARE proteins also localized endosomal compartment, suggesting that these SNAREs shuttle between the plasma and endosome. We propose possible combinations of SNARE proteins on plasma membrane, and discuss the SNARE proteins involved in endocytosis or exocytosis
80 Root–to–leaf electrical signaling in Avocado (Persea americana Mill.)
Pilar Gil Montenegro
Pontificia Universidad Católica de Chile, Chile Email: [email protected]
Our work using phytomonitoring techniques for Avocado (Persea americana Mill) tree irrigation indicate that plant response to changed soil water availability is a very fast process. Root–to– leaf ABA transport or hydraulic processes cannot fully explain the almost immediate stomata physiological response (less than 15 minutes) to either water application and/or sudden ETp increments or reductions. Some studies in Avocado physiology report that root–to–leaf ABA transport is a transpirative mass-flow process. In other tree species a decline in stomatal conductance (gs) was detected when volumetric soil water content declined below 0.12, but the decline in gs, could not have been mediated by increasing [ABA(xyl)] because stomatal closure appeared to precede any increase in [ABA(xyl)]. Sap flow velocity in the range of 30 to 35 cm/h has been reported in avocado, indicating that hydraulic forces cannot either fully explain the fast stomatal response to soil water availability. This work is aimed to study the eventual existence of an electrical signaling process regulating stomata behavior. Two year old avocado trees were subjected to several drying and re- watering cycles, as well as to modifications on some ETp parameters, as incident radiation and air flux conditions. Extracellular electrical potential was continuously recorded between trunk and leaf petiole; leaf stomata conductance was also registered. Drying the root system with a continuous air flow at room temperature generated the arrival of an electrical signal to the leaf petiole; the same signal was detected when the root system was re-watered. Our results indicate that a sudden change in soil water availability creates a significant electrical signal, which reaches leaf petiole in 10 to 50 min. Other measurement were made on girdled plants, in several cycles, re-watering plants after 4 drying days, indicating that the electrical signal detected is possibly conducted by the xylem, and not by the phloem tissue. The eventual existence of root–to–leaf electric information exchange mechanisms opens interesting possibilities to artificially modify plant response to environmental or agronomic management strategies, aimed to increment water use efficiency.
81 End-Poles of Root Cells as Auxin Transporting Plant Synapses
Markus Schlicht1, Boris Voigt1, Alina Schick1 Miroslav Strnad2, Klaus Palme3, Dieter Volkmann1, Diedrik Menzel1, František Baluška1,*
1 Institute of Molecular and Cellular Biology, University of Bonn , Bonn , Germany 2 Laboratory of Growth Regulators, Palacký University & Institute of Experimental Botany AS CR, CZ-78371 Olomouc, Czech Republic 3 Institut für Biologie II, Universität Freiburg, Freiburg, Germany *Email: [email protected]
Putative auxin influx and efflux carriers (PIN/AUX proteins) show polar localizations in root cells which is in a good agreement with predictions of the chemiosmotic theory as related of the polar auxin transport (PAT). In this classical model, the role of these carriers have been interpreted to act as plasma membrane transporters. However several published data indicate that the exocytosis inhibitor Brefeldin A stops the PAT within few minutes, while the putative transport proteins of the PIN and AUX families are still polarly localised at the plasma membrane of the end-poles. Furthermore, it became clear that PAT inhibitors like TIBA and NPA are, in fact, general inhibitors of the endocytosis in plant cells (1). These data support an alternative model proposing that auxin is secreted in a neurotransmitter-like mode via BFA- sensitive and F-actin dependent vesicular exocytosis (2). This new view of the PAT is in a good accordance with the preferential localization of polarized secretion machinery at the end-poles of maize root cells transporting auxin (3). These end-poles have several other properties allowing to define them as plant synapses (4). Besides abundant F-actin, myosin VIII, dynamins, Rho-GTPases (5); the auxin secreting end-poles are equipped also with plant homologues of mammalian neuronal molecules synaptotagmins. Taken together, all these data enable us to propose that the end-poles of root cells, especially those of the root transition zone which are particularly active in the PAT, resemble neuronal synapses.
Geldner N, Friml J, Stierhof YD, Jurgens G, Palme K (2001) Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 413: 425-428 Baluška F, Šamaj J, Menzel D (2003) Polar transport of auxin: carrier-mediated flux across the plasma membrane or neurotransmitter-like secretion? Trends Cell Biol 13: 282-285 Baluška F, Hlavačka A, Šamaj J, Palme K, Robinson DG, Matoh T, McCurdy DW, Menzel D Volkmann D (2002) F-actin-dependent endocytosis of cell wall pectins in meristematic root cells: insights from BFA-induced compartments. Plant Physiol 130: 422-431 Baluška F, Volkmann D, Menzel D (2005) Plant synapses: actin-based domains for cell-to-cell communication. Trends Plant Sci 10: 106-111 Baluška F, Wojtaszek P, Volkmann D, Barlow PW (2003) The architecture of polarized cell growth: the unique status of elongating plant cells. BioEssays 25: 569-576
82 Noise Enhanced Distributed Emergent Computation in Plants
Susanna M. Messinger1,*, Keith Mott1, David Peak2
1 Biology Department, Utah State University, Logan, UT 84322-5305, USA 2 Physics Department, Utah State University, Logan, UT 84322-4415, USA *Email: [email protected]
The idea of biological organisms as natural computers has been tantalizing researchers for decades, but substantial, quantitative proof of computation is elusive and difficult to obtain because of the inherent complexity of biological systems. A recent study has, however, presented evidence supporting the existence of distributed emergent computation in plants (Proceedings of the National Academy of Science 101(4), 918-922). Plants daily solve a constrained optimization problem, maximizing CO2 uptake while minimizing water loss, through the dynamic adjustment of stomatal conductance. Stomata form a locally connected network that collectively processes environmental stimuli to perform the optimization task. Like all biological systems, plants are subject to noise, i.e., random variability in both space and time. Biological noise has traditionally been viewed as a nuisance that organisms cope with by developing sometimes elaborate noise-attenuating mechanisms. Although this view is substantiated by biological processes like DNA replication and some cellular signaling cascades, recent research has revealed that some biological processes are actually improved by, or even driven by, the presence of noise. Motivated by the possibility that noise enhances some biological processes, this study examined the possible effects of noise on distributed emergent computation in plants by introducing similar noise into a standard artificial cellular computing system that also performs distributed emergent computation. We found that small amounts of spatial and temporal noise added to the system independently improve computational efficiency. Added together, spatial and temporal noise improves the computational ability of the system more than either independently. Thus, it may be that biology not only coexists with noise, but actually uses it to enhance adaptive, information processing ability.
83 Jasmonic acid, salicylic acid and gene expression in early response of potato plants to virus infection
Maja Kovač1, Darja Milovanovič Jarh1, Axel Műller2, Anita Purnat1, Špela Baebler1, Hana Krečič1, Mojca Milavec1, Maruša Pompe-Novak1, Kristina Gruden1, and Maja Ravnikar1
1 Department of Plant Physiology and Biotechnology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia 2 Lehrstuhl fűr Pflanzenphysiologie, Ruhr-Universität Bochum, Univeristätsstr. 150, 44801 Bochum, Germany *Email: [email protected]
Jasmonic acid (JA) and salicylic acid (SA) are central players in mediating responses to pathogens and wounding in plants. These molecules induce expression of several defence- related genes. The involvement of JA and SA in early response of plants to virus infection was investigated in potato plants (Solanum tuberosum L.) infected with potato virus Y NTN. Various potato cultivars are differently sensitive to the virus. In susceptible variety the symptoms of infection are ring shaped necrosis of the tubers, chlorosis and curling of leaves, accelerated senescence, and sever reduction in crop yield. In our study, two potato cultivars were chosen: sensitive cv. Désirée and resistant cv. Santé. JA and SA were measured 1 and 3 hours after inoculation using a multiplex GC-MS/MS and HPLC, respectively. The study showed the involvement of JA in very early response of potato to PVY NTN infection, as significant increase of JA, was detected in inoculated leaves 1 hour post-inoculation. The increase was most pronounced in resistant variety indicating the correlation of JA metabolism with the expression of the resistance. The role of SA in very early defence response is less clear. Gene expression profile of virus-potato plant interaction was studied by cDNA microarrays. Most prominent changes were observed in genes associated with defence response.
84 Hydraulic signals and plant development
Jolana Albrechtova*, Edgar Wagner
Albert-Ludwigs University Institute of Biology Schanzlestr. 1 D-79104 Freiburg Germany *Email: [email protected]
Systemic responses by definition involve a whole plant body, though they are usually evoked by local signal(s). The signals spread through the plant body via chemical, electrical, hydraulic and molecular pathways. Hydraulic signals are especially important in drought stress and in wound reactions. However, turgor changes also drive rhythmic growth, leaf movements as well as circumnutations and therefore seem to co-ordinate plant development. Hydraulic signals are probably perceived as pressure changes both by mechano-transductive ion channels and as local deformations in cytoskeleton and nucleoskeleton, with the possibility of influencing gene expression. Physical strain at the surface of the apical meristem was previously suggested to play a key role in the patterning of organogenesis. We have studied the influence of local water transport and turgor changes on organogenesis at the apical meristem of Chenopodium plants. Specific changes in shape and size of the apical meristem were found to precede reorganisation of organogenesis under photoperiodic flower induction. Optical properties of cell walls at the surface of the apical meristem were changing during flower induction. Expression of the aquaporin CrAQP increased at the apex during an early phase of flower induction and the application of an inhibitor of aquaporin activity partially inhibited flowering. Changes in ion balance and carbohydrate levels in the cells seem also to be involved in the process. Altogether, the results support a hypothesis about the involvement of hydraulic signals in organogenesis at the apical meristem. It is anticipated that hydraulic changes at the apex leading to flower initiation are mediated by a specific hydro-electrochemical communication between (roots), leaves and the shoot apex.
85 Blue light-regulated vesicular recycling of phototropin1 in the root transition zone
Ying-Lang Wan1, Halina Gabrys2, František Baluška1, Diedrik Menzel1,*
1 Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany 2 The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, Krakow, Poland *Email: [email protected]
Phototropin is essential for phototropism of plants. Phototropin 1 is a 120kD ser/thr kinase, which can receive photons through the LOV domain at the N-terminus. The changes in conformation of the LOV domain activate the C-terminal kinase domain, leading to the autophosphorylation of PHOT1. But how the signal is transduced into the cell and leads to the bending response is still unknown. Our studies use Arabidopsis seedlings transformed by a GFP-PHOT1 construct expressed under the control of endogenous promoter. We have found that PHOT1 recycles between the plasma membrane at cross-poles and endosomes in cells of the root apex transition zone. The recycling pathway is active at a basal level even in darkness and under low light-condition, but can be stimulated by blue light. Stimulation is dependent on the light intensity, and therefore could be the mechanism for sensing the light gradients when the plants are illuminated unilaterally. PHOT1 recycling requires an intact actin cytoskeleton and is sensitive to Brefeldin A (BFA), an exocytosis blocker. BFA induces formation of PHOT1- enriched endocytic compartments. The amount of PHOT1 within these BFA-induced compartments is enhanced by blue light in correlation of the light intensity. This recycling pathway can be affected by ROS, suggesting that PHOT1 could participate in stress response pathways.
86 Why decentralized computation in plants?
Jevin West1,*, Keith Mott1, David Peak2
1 Biology Department, Utah State University, Logan, UT 84322-5305, USA 2 Physics Department, Utah State University, Logan, UT 84322-4415, USA *Email: [email protected]
The what part of information processing in leaves has been addressed by the plant physiology community. Plants on a daily basis must solve a constrained optimization problem: how should stomatal aperture should vary for the plant to take up the most amount of CO2 for a given amount of H2O loss as environmental conditions change throughout the day? The how part of information processing remains an open question. Traditionally, stomata were assumed to operate independently, with each stoma processing signals from the environment and the rest of plant in isolation. However, recent studies have shown that the plant may be solving this constrained optimization task using emergent distributed computation. The purpose of this study is to address the why part of the story. Why do plants resort to decentralized computation when there clearly are disadvantages to this type of information processing (e.g., redundancy, pathological deadlocks and increased processing time and resource costs)? To address this question, a theoretical analysis has been done addressing the topics of robustness, metabolic investment costs and evolutionary happenstance.
87 What do myosins do in blue light?
Weronika Krzeszowiec, Halina Gabrys*
Department of Plant Physiology and Biochemistry, Jagiellonian University Gronostajowa 7, 30-387 Krakow, Poland *Email: [email protected]
Myosins are members of an actin-activated family of ATPases. The Arabidopsis genome contains 17 myosin-like genes that belong to classes VIII and XI. Myosins transport various cargoes along microfilaments and are essential in many physiological processes. They are also components of motor systems responsible for chloroplast photo-orientation in the cell. It has been established that actin cytoskeleton responds to light conditions (e.g. in Vallisneria, Mougeotia, Ceratodon). Photoreceptors mediating chloroplast movements in Arabidopsis thaliana are two phototropins, PHOT1 and PHOT2, members of the blue light photoreceptor family. They are involved in phototropism, chloroplast movements and stomatal opening. So far, no evidence has been presented that myosins might be controlled by light absorbed by a photoreceptor. Myosins of both classes are present on chloroplast surface [1,2] and are believed to participate in chloroplast translocations in Arabidopsis. The aim of this work was to obtain evidence that the surface-associated myosins are involved in the mechanism of chloroplast movements, by establishing whether they undergo changes under the influence of light. Blue light intensities were applied that induce saturated avoidance and accumulation responses of chloroplasts. The experiments were carried out on mature lives of Arabidopsis thaliana wild type, first irradiated and then fixed with paraformaldehyde solution. Myosins were visualized with animal-antibodies: anti-myosin (smooth & skeletal) and secondary FITC-labeled antibodies. The fluorescence was observed in a confocal microscope. Localization of myosins was different in the cells irradiated with strong and weak blue light. Myosins were found on the chloroplast surface in almost all cells irradiated with weak blue light. Strong blue light displaced them from that surface. The effect was blue light-specific and did not occur in strong red light. We suggest that the light-induced reorganization of myosins is essential in the mechanism of chloroplast movements and that it is the final step in the phototropin signal transduction.
Wojtaszek P, Anielska-Mazur A, Gabryś H, Baluška F, Volkmann D: Recruitment of myosin VIII towards plastid surfaces is root cap-specific and provides the evidence for actomyosin involvement in root osmosensing. Funct. Plant Biol. in press. Wang Z, Pesacreta TC: A subclass of myosin XI is associated with mitochondria, plastids, and the molecular chaperone TCP-1α in maize. Cell Motility and Cytoskeleton57, 218-232, 2004.
88 The first moving signal inducing systemic changes in photosynthesis after local burning of tobacco plants
Vladimíra Hlaváčková1,*, Pavel Krchňák1, Petr Ilík1, Jan Nauš1, Ondřej Novák2, Radek Kaňa1, Martina Špundová1
1 Department of Experimental Physics, Palacký University , tř. Svobody 26, 771 46 Olomouc, Czech Republic 2 Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71 Olomouc, Czech Republic *Email: [email protected]
Plant species developed some systemic self-protecting mechanisms inducible by local stress. Some questions about the nature of moving signals and their connection to systemic changes in the energetic processes including photosynthesis are still unresolved. A short-term systemic response of tobacco plants (Nicotiana tabacum cv. Samsun ) to local burning was investigated. A basipetal spreading of electrical signal along the plant, evoked by the local burning of the upper leaf, was detected by changes in the extracellular electrical potential differences (EEPD) between Ag/AgCl electrodes placed on three untreated leaves and a reference electrode placed in the root nutrient solution. Simultaneously, the photosynthetic parameters (gas exchange and chlorophyll fluorescence) were measured on the selected leaves. Concentrations of potential chemical signal compounds - jasmonic (JA) and abscisic acid ( ABA ) - in measured leaves were measured by the LC-MS method. Changes in EEPD were observed in several seconds after burning depending on the distance from the site of burning. The extent of EEPD changes was mostly within 10 - 70 mV. Main changes lasted several minutes and a steady state value was achieved in about 1 hour. In most cases the amplitude of changes decreased with increasing distance of the measured leaf from the burned one. The propagation velocity was about 1 cm s-1. The EEPD changes were followed in several minutes by a pronounced decrease in the rate of CO2 assimilation, transpiration and stomatal conductance. Nearly no change was detectable in the sub-stomatal concentration of CO2. Similarly, negligible short-term changes in chlorophyll fluorescence induction and photochemical quenching were detectable. In the measured leaves, also changes in JA and ABA concentrations were observed. We suggest that the short-term changes in photosynthesis evoked by local burning are stimulated (directly or indirectly) by the moving electrical signal. Interestingly, these photosynthesis changes do not include changes of electron transport in PSII.
Acknowledgements. The project has been supported by grant from Ministry of Education of the Czech Republic , No. MSM 6198959215.
89 Signalling endosomes in plants: activated MAPKs associate with clathrin-coated vesicles and endosomes
Wasser K, Müller J, Preuss ML, Holstein S, Böhm N, Nielsen E, Hirt H, Menzel D, Šamaj J*
Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D – 53115 Bonn, Germany *Email: [email protected]
Mitogen-activated protein kinase (MAPK) pathways including ERK and p38 associate with signalling endosomes upon activation of growth factor receptors in mammalian cells. It is believed that this scaffolding and compartmentalization of MAPK pathways is essential for achieving signal specificity and more efficient transduction. In plants, almost nothing is known about targeting of MAPKs to the endomembranes and to the cytoskeleton. We provide biochemical and cell biological evidence that two Arabidopsis MAPKs, MPK4 and MPK6 are weakly associated with endosomes (later one also with CCVs) in control cells. Additionally, three MAPKs, MPK3, 4 and 6 are associated in their activated state with endosomes following diverse abiotic stresses. Active MPK4 and MPK6, but not MPK3, associate also with isolated clathrin-coated vesicles (CCVs) upon biotic stress induced by elicitor (flagellin) treatment. These data indicate a constitutive signalling from the endocytic compartment in plants.
90 Dynamic behaviour of SIMKK, a plant stress-induced mitogen- activated protein kinase kinase and its downstream target SIMK
Jens Müller, Nils Böhm, Heribert Hirt, Diedrik Menzel, Jozef Šamaj
Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D – 53115 Bonn, Germany *Email: [email protected]
SIMKK, a stress-induced mitogen-activated protein kinase kinase (MAPKK) is a specific activator of SIMK, but nothing is known about the dynamic behaviour of these two signalling enzymes in plants. Therefore, we aimed to study dynamic localization changes of SIMKK and SIMK upon diverse abiotic stresses. Both, transiently or stably expressed CFP/YFP-tagged SIMKK and SIMK have shown predominantly nuclear localization under unstressed conditions. By contrast, both kinases partially relocated to the cytoplasm and co-localized on spot-like vesicular structures upon salt and oxidative stress. Other abiotic stresses, such as heat and wounding, but not the exposure to elicitors (flagellin, chitin, β-glucan and ergosterol) also caused relocation of SIMKK to cytoplasmic vesicular compartments. Detailed time-lapse observations revealed that SIMKK was actually recruited to motile endomembraneous organelles (most likely representing endosomes) upon stress.
91
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Poster session sponsored by
92 Abstracts for Oral Presentation Session 1
The Plant Vascular System: A Macromolecular Information Superhighway
William J. Lucas
Section of Plant Biology, College of Biological Sciences, University of California-Davis, USA
A new paradigm is emerging in which plants utilize proteins and RNA as non-cell-autonomously acting signaling macromolecules to mediate local and long-distance regulation over physiological and developmental processes. The cell-to-cell pathway for the trafficking of these non-cell-autonomous proteins (NCAPs) and RNA, in the form of ribonucleoprotein (RNP) complexes, is established by plasmodesmata (PD), the intercellular organelles unique to plants. The interconnection of local tissues to the phloem sieve tube system, through PD, establishes an integrated supracellular organism. Regulation of these local and long-distance macromolecular trafficking networks is likely essential for the coordinated exchange of information between distantly located plant organs to orchestrate events at the whole plant level. In angiosperms, the sieve tube system is comprised of two main cell types, the sieve elements (SEs) and their associated companion cells (CCs). At maturity, the enucleate SEs are highly modified to create a low-resistance pathway, the sieve tube, for the translocation of photoassimilates; CCs function in the maintenance of the associated SEs. Specialized, branched PD interconnect these two cell types, thereby forming the CC-SE complex. Analysis of the phloem sap collected from a number of species has demonstrated that the translocation stream contains a complex set of proteins (~ 1000 in number). Detailed analyses of many of these phloem proteins have demonstrated their capacity for cell-to-cell movement through PD: thus, the entry and exit of these phloem proteins appears to be regulated by the CC-SE PD. Interestingly, a number of these phloem proteins can bind to RNA and one, CmPP16, has been shown to mediate the Thecell-to-cell andSecond long-distance translocation Symposium of RNA, in a non-sequence-specific manner. These studies provide support for the concept that the CC-SE complex has the machineryon necessary Plant to mediate long-dist Neurobiologyance delivery of NCAPs and RNPs. This notion is consistent with the discovery that the phloem translocation stream contains a specific population of RNA molecules (>1,500 mRNA and many 1000s of si/miRNA species). Grafting experiments have proven that many of these RNA molecules are translocated within the phloem and, in some situations, delivery of these RNA have 1 Abstracts for Oral Presentation Session 1
been correlated with development of specific phenotypes within the shoot apex. Systemic spread of RNA interference (RNAi), a sequence-specific RNA degradation process, is also consistent with the concept that RNA can be delivered to distant organs, through the phloem. Thus, PD and the sieve tube system act, in concert, to establish an information superhighway in plants. To advance our understanding of the roles played by PD and the phloem, in the trafficking of NCAPs and RNPs, we are currently developing proteomic-based strategies to (a) elucidate the supramolecular structure of PD, (b) establish the function of the phloem-mobile proteins, and (c) develop a phloem transcriptome for the RNA molecules (both large and small) that function within the context of the enucleate SEs. The impact of these findings on studies in plant biology will be discussed in terms of RNA as a long-distance information macromolecule within the plant kingdom.
The Second Symposium
on Plant Neurobiology
2 Abstracts for Oral Presentation Session 1
Plant Neurobiology: Why Now, and What For?
František Baluška1,4, Dieter Volkmann1, Peter Barlow2, Stefano Mancuso3,4
1 University of Bonn, Germany 2 University of Bristol, UK 3 University of Florence, Italy 4 Plant Neurobiology Laboratory (Florence & Bonn)
Recent advances in plant cell biology, molecular biology, and ecology have accumulated a critical mass of data which are not ‘digestible’ within the framework of these, now classical, disciplines of plant sciences. New approaches are required, and these should be characterized by system-like analysis of information acquisition, storage, processing, and the making of decisions. Plants retrieve from the abiotic environment information critical for their survival, especially relating to light and gravity, two physical forces pervading the universe. Intriguingly, the translation of these physical forces into plant activities − typically differential growth responses − is based on the transcellular transport of auxin, which help to bring about the final shape of the plant body. Thus, this information-bearing molecule is central to our call for plant neurobiology. Although the history of auxin can be traced back to the Darwin’s early experiments with phototropism of coleoptiles, we still know almost nothing about its peculiar features. Let us examine the mystery of this unique molecule. Although auxin can be synthesized probably in each plant cell, it is tediously transported from cell to cell throughout the plant body. Similarly puzzling is the well-known phenomenon that, although the auxin molecule is sufficiently small to pass easily through plasmodesmatal channels, plants cells somehow manage to prevent this direct cell-to-cell means of auxin transport. Rather, plants maintain an energetically costly system based on vesicle trafficking, closely resembling neuronal and immunological cell-cell communication, in order to drive transcellular auxin transport. The next peculiarity is that when extracellular auxin hits the Theplasma membrane, Second it induces electric reSymposiumsponses based on the ABP1 auxin-binding protein. All this suggests that auxin, besides hormone- and morphogen-like properties, also possesses neurotransmitter-like properties. As the cell-to-cell transport of auxin is also involvedon in plantPlant response the light Neurobiology and gravity, as well as to vesicular trafficking, plant neurobiology is needed to explain this great mystery of plant nature. In a recent Nature article, DeWeese and Zador (2006) loosely defined neurobiology as having three basic characteristics: 1) all biological systems (organisms)
3 Abstracts for Oral Presentation Session 1
are embedded within a physical environment that shapes their organization and behaviour; 2) in order to survive, all biological systems need to effectively retrieve of information from their physical environment; 3) neuronal activity is essential to translate information sensed concerning the environment into electrical impulses which are then capable of rapid transformation into biological signals that induce motoric responses. Importantly, also communication from the biotic environment is physically mediated through the senses: hearing, seeing, feeling, or smelling; all this is based on the laws of physics. In short, the neurobiological apparatus translates sensory information first into electrical impulses and only then into biological information inducing organismal actions. Human perception of the outside world relies on so-called ‘neural code’ which links together sensory signals and neural responses. Similarly in plants, numerous parameters of the physical environment, especially light and gravity, are monitored. Specialized cells (e.g., root cap statocytes and root transition zone cells) are evolutionarily optimised to translate sensory information obtained from this environment into motoric responses (e.g. gravibending of root apices). Moreover, physical forces, influences, and insults, all induce immediate electrical responses in plants. Obviously, one task is to make a connection betwee all these events with the molecules and cellular processes which are known from neurobiology, and for which there is firm or emerging evidence from plants. Finally, we need to understand those processes which transform physical information (e.g. light, gravity, temperature, mechanical and osmotic forces etc.) into biological information. Particularly, we need to know if it is possible to convert physical information directly into biological information without inducing any bioelectrical responses, or if physical information needs to be first transformed into bioelectrical information before it can be translated into purely biological information. For this, we need a merging of classical electrophysiology with cell biology and molecular biology. Obviously, plant neurobiology as a new branch of plant sciences is not only justified but also is very competent to solve the new and urgent questions of contemporary plant biology.
TheBaluška F, Šamaj Second J, Menzel D (2003) Polar Symposium transport of auxin: carrier-mediated flux across the plasma membrane or neurotransmitter-like secretion? Trends Cell Biol 13: 282-285 Baluška F,on Mancuso Plant S, Volkmann D, Barlow Neurobiology PW (2004) Root apices as plant command centres: the unique ‘brain-like’ status of the root apex transition zone. Biologia 59: 9-17 Baluška F, Volkmann D, Menzel D (2005) Plant synapses: actin-based adhesion domains for cell-to-cell communication. Trends Plant Sci 10: 106-111
4 Abstracts for Oral Presentation Session 1
Baluška F, Mancuso S, Volkmann D (2005) Communication in Plants: Neuronal Aspects of Plant Life. Springer-Verlag Baluška F, Hlavacka A, Mancuso S, Volkmann D, Barlow PW (2005) Neurobiological view of plants and their body plan. In Communication in Plants: Neuronal Aspects of Plant Life, F Baluška, S Mancuso, D Volkmann (eds), Springer-Verlag DeWeese MR, Zador A (2006) Efficiency measures. Nature 439: 920-921 Felle H, Peters W, Palme K (1991) The electrical response of maize to auxin. Biochim Biophys Acta 1064: 199-204 Friml J (2003) Auxin transport – shaping the plant. Curr Opin Plant Biol 6: 7-12 Friml J, Wiśniewska J (2005) Auxin as an intercellular signal. In: Intercellular Communication in Plants, Flemming A. (ed), Annual Plant Reviews 16, Blackwell Publishing Mancuso S, Barlow PW, Volkmann D, Baluška F (2006) Actin turnover-mediated gravity response in maize root apices: gravitropism of decapped roots implicates gravisensing outside of the root cap. Plant Signal Behav 1: 52-58 Šamaj J, Read ND, Volkmann D, Menzel D, Baluška F (2005) The endocytic network in plants. Trends Cell Biol 15: 425-433 Steffens B, Feckler C, Palme K, Christian M, Böttger M, Lüthen H (2001) The auxin signal for protoplast swelling is perceived by extracellular ABP1. Plant J 27: 591-599
The Second Symposium
on Plant Neurobiology
5 Abstracts for Oral Presentation Session 1
How and Why do Plants Support Electrical Long-distance Signals?
Rainer Stahlberg, Robert E Cleland, and Elizabeth Van Volkenburgh
University of Washington, POB 355325, Seattle WA 98195, USA
L. Hermann, W. Nernst and J. Bernstein determined our modern understanding of transmission of action potentials (APs) by demonstrating that excitation travels not as a direct electrical current but in the form of an area of altered ion currents along a frequently myelin-coated plasma membrane 1. After characterizing APs in animal nerves, finding similar propagating electrical signals in nerveless plants was unexpected 2. When neurobiologists later discovered that maintenance of large amplitudes of APs over long distances was achieved by the intermittent renewal of the signal by electrogenic (mostly depolarizing) chemicals (neurotransmitters) in synapses, the degree of astonishment and curiosity about plant signals should have increased even more 1,2. After all, (i) plant cells are separated by thick cell walls that leave no space for synaptic structures and (ii) their sessile life style leaves most plants in no need for fast signals. In spite of such consideration we know today that plants have not only one but two types of propagating, electrical long-distance signals; (i) action potentials (APs), which they share with animals and (ii) slow wave potentials (SWP, also called “variation” potentials), which are unique to vascular plants 2, 3. The mechanisms of how plants can support APs and SWPs over long distances of hundreds of cells is one of the great challenges in the emerging field of plant neurobiology together with the exploration of their roles in signaling internal and external situations from one plant part to another.
1. Stahlberg R (2006) Historical overview on plant neurobiology. Plant Signal. Behav. 1: 6-8 2. Trebacz K (2006) Electrical signals in long-distance communication in plants. In Communications in Plants. Neuronal Aspects of Plant Life (Baluška F, Mancuso S, TheVolkmann Second D, eds). Berlin, Springer VerlagSymposium , pp. 277-290 3. Stahlberg R, Cleland RE, Van Volkenburgh E (2006) Slow wave potentials – a propagating electrical signal unique to higher plants. In Communications in Plants. Neuronalon Aspects Plant of Plant Life (Baluška Neurobiology F, Mancuso S, Volkmann D, eds). Berlin, Springer Verlag , pp. 291-308 4. Stahlberg R, Cleland RE, Van Volkenburgh E. (2006) Shade-induced action potentials in Helianthus annuus L. originate primarily from the epicotyl. Plant Signal. Behav. 1: 15-22 6 Abstracts for Oral Presentation Session 1
Gravity as Physical Force Shaping Plants
Dieter Volkamnn
IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany
Gravity is the most constant environmental factor which influences both ontogeny as well as phylogeny, and in particular the diversity, of land living organisms. As prominent mechanical signal, gravity controls a large number of plant genes belonging to different categories, including signalling, metabolism, cytoskeleton, vesicle trafficking, and cell wall. Several signal transduction cascades have been proposed for the gravity controlled processes like gravitropism and gravimorphogenesis. Experiments under temporarily reduced gravity conditions (parabolic flights) suggest that extremely fast electrical signals, like action potenials (APs), as well as very rapid changes in oxygen and nitric oxide (NO) concentrations, play a crucial role in these plant morphogenesis processes. The cell wall-plasma membrane-cytoskeleton continuum, known as the Ingber’s tensegrity concept, in connection with vesicle trafficking and cytoskeleton dynamics, will be discussed as structural basis of a common mechanism enabling gravity to shape the plants.
The Second Symposium
on Plant Neurobiology
7 Abstracts for Oral Presentation Session 1
Potential Calcium Sensor Proteins Function in Perception and/or Response to Environmental Stimuli
Yu-Chang Tsai, Nikkí A. Delk, Elizabeth McCormack, Naweed I. Chowdhury, Roque Sanchez and Janet Braam
Biochemistry and Cell Biology, Rice University, Houston TX 77005-1892 USA
Plants are highly responsive to diverse stimuli enabling them to acclimate and thrive under diverse environmental conditions. Fluctuations in free cytosolic calcium (Ca2+) serve as second messengers in transducing perceived stimuli into cellular responses. The quintessential Ca2+ sensor is calmodulin (CaM), which upon binding Ca2+ undergoes a profound conformational change that affects the activity of interacting proteins. In this way, Ca2+ signals are converted to changes in target protein activity and consequent physiological responses. Arabidopsis has a large gene family encoding CaM and CaM-like (CML) proteins (McCormack and Braam, 2003; McCormack et al., 2005). To reveal functions of Arabidopsis CaMs and CMLs, we are characterizing phenotypes of plants harboring mutations in a subset of the gene family members. CML11 is an unusual CaM isoform that has a polymorphic glutamine-rich domain at the amino terminus. CML11 insertional mutants show defects in root hydrotropism. CML23 and CML24, closely related paralogs, regulate flowering time through the photoperiod and autonomous pathways, affecting both CONSTANS and FLOWERING LOCUS C expression. The altered transition to flowering is a consequence, at least in part, of altered nitric oxide accumulation in the mutants. Thus, these potential Ca2+ sensors have specialized and specific roles in plant physiology enabling diverse behaviors, including response to water stress and seasonal regulation of the transition to flowering. This material is based upon work supported by the National Science Foundation under grant no. IBN0321532 and the Department of Energy under grant no. FG02-03ER15394. The Second Symposium Delk, N., Johnson, K.A., Chowdhury, N.I., and Braam, J. (2005) "CML24, regulated in expression by diverse stimuli, encodes a potential Ca2+ sensor that functions in responseson to ABA, Plant day length and ionNeurobiology stress." Plant Physiol., 139: 240-253. McCormack, E., Tsai,Y.-C., and Braam, J. (2005)"Handling calcium signaling: Arabidopsis CaMs and CMLs." Trends Plant Sci., 10: 383-389. McCormack, E., and Braam, J. (2003)"Calmodulins and Related Potential Calcium Sensors of Arabidopsis." New Phytologist, 159: 585-598.
8 Abstracts for Oral Presentation Session 2
Cellular Signals Governing Vascular Tissue Organization in Plants
Hiroo Fukuda
Department of Biological Sciences, Graduate School of Science, the University of Tokyo, Hongo, Tokyo 113-0033, Japan
The body plan of plants is governed by a combination of clonal fate and positional information that is provided by local signals, as is commonly seen in multicellular organisms. Recent advances in molecular genetics and cell biology have uncovered unique mechanisms of plant body formation. We have focused on the vascular system, because the vascular system becomes a good model system for understanding molecular mechanism of plant body formation and also becomes a good target for improving crops and trees. The vascular system is composed of various types of vascular cells, which usually differentiate at predicted position and at predicted timing according to genetic programs and cell-cell communications (1). Here I report our recent findings about genetic program and cell-cell communication in association with vascular organization.
1） Vesicle transport plays a role in continuous formation of vascular strands We isolated Arabidopsis van1-van7 mutants with discontinuous vascular strands (2). Of them, the van3 mutant has been analyzed in detailed, revealing that the VAN3 gene encodes an ARF-GAP, which regulates vesicle transport from the trans Golgi network (3). This protein seems to be involved in auxin perception but not closely in auxin transport which is regulated by GNOM ARF-GEF, resulting in fragmented vascular formation. We also found that a dynamin protein is bound to the VAN3 protein specifically and may function in continuous formation of the vascular system together with the VAN3 protein (4).
2） Various extracellular factors regulate vascular cell differentiation We have searched an extracellular factor(s) promoting tracheary element Thedifferentiation Second from medium of Zinnia xylogenic Symposium culture, in which isolated mesophyll cells transdifferentiate into xylem cells (5). Isolation of the factor, called xylogen and its gene indicated that xylogen is a non-classical arabinogalactan protein with non-specificon lipid Planttransfer protein-like Neurobiologysequence. Two similar genes are found in Arabidopsis genome sequence. Introduction of the genes into tobacco BY-2 culture cells induced a xylogen activity in culture medium, demonstrating that the genes encode xylogen. Double mutants of these genes exhibited a distinctive phenotype defective in vascular continuity. Because the xylogen gene is expressed in
9 Abstracts for Oral Presentation Session 2
procambium or xylem precursor cells, xylogen may function as an apoplastic signal molecule directing xylem cell specification continuously (5). We also have found other factors promoting tracheary element differentiation, brassinosteroids (7,8) and phytosulfokine in Zinnia xylogenic culture. Recently we identified a novel peptide that inhibits tracheary element differentiation from medium of Zinnia non-xylogenic culture. These findings clearly indicated that spatial and temporal expression of these intercellular factors.
3） Systematic analysis of gene expression with DNA arrays for Zinnia and Arabidopsis xylogenic cultures revealed master genes directing xylem cell differentiation Using a Zinnia xylogenic culture we have revealed comprehensive gene expression profiles (9). Recently we newly established Arabidopsis cell culture in which cultured cells differentiate into xylem cells at high frequency. Using the culture system, we also performed a comprehensive analysis of gene expression during xylem cell differentiation. The two comprehensive analyses revealed that similar gene sets are expressed in association with xylem cell differentiation. Detailed analysis of gene function revealed a new class of transcription factors, VND6 and VND7, which can induce xylem cell differentiation (10). Interestingly, VND6 and VND7 induce different types of xylem cells, that is, VND6 induces metaxylem vessel cells and VND7 does protoxylem vessel cells. On the other hand. the suppression of function of VND6 and VND7, results in the inhibition of metaxylem and protoxylem formation, respectively. These results indicate that VND6 and VND7 are master genes for inducing xylem cell differentiation.
1. Fukuda, H. (2004) Signals that govern plant vascular cell differentiation. Nature Rev. Mol. Cell Biol. 5, 379-391 2. Koizumi, K., Sugiyama, M. and Fukuda, H. (2000) A series of novel mutants of Arabidopsis thaliana that are defective in the formation of continuous vascular network: Calling the auxin signal flow canalization hypothesis into question. TheDevelopment, Second 217, 3197-3204 Symposium
3. Koizumi, K., Naramoto, S., Sawa, S., Yahara, N., Ueda, T., Nakano, A., Sugiyama, M. and Fukuda, H. (2005) VAN3 ARF–GAP-mediated vesicle transport is involved in leaf vascularon networkPlant formation. Development Neurobiology 132, 1699-1711
4. Sawa, S., Koizumi, K., Naramoto, S., Demura, T., Kubo M., Ueda, T., Nakano A., and Fukuda, H. (2005) DRP1A is responsible for vascular continuity synergistically working with VAN3 in Arabidopsis. Plant Physiol. 138, 819-826 10 Abstracts for Oral Presentation Session 2
5. Motose, H., Sugiyama, M. and Fukuda, H. (2001) An arabinogalactan protein(s) is a key component of a fraction that mediates local intercellular communication involved in tracheary element differentiation of Zinnia mesophyll cells. Plant Cell Physiol., 42, 129-137 6. Motose, H., Sugiyama, M. and Fukuda, H. (2004) A proteoglycan mediates inductive interaction during plant vascular development. Nature 429, 873-878 7. Yamamoto, R., Demura, T., and Fukuda, H. (1997) Brassinosteroids induce entry into the final stage of tracheary element differentiation in cultured Zinnia cells. Plant Cell Physiol. 38, 980-983 8. Yamamoto, R., Fujioka, s., Demura, T., Takatsuto, S. Yoshida, S. and Fukuda. H. (2001) Brassinosteroid levels increase drastically prior to morphogenesis of tracheary elements. Plant Physiol., 125, 556-563 9. Demura, T., Tashiro, G., Horiguchi, G., Kishimoto, N., Kubo, M., Matsuoka, N., Minami, A., Nagata-Hiwatashi, M., Nakamura, K., Okamura, Y., Sassa, N., Suzuki, S., Yazaki, J., Kikuchi, S., Fukuda, H. (2002) Visualization by comprehensive microarray analysis of gene expression programs during transdifferentition of mesophyll cells into xylem cells. Proc. Natl. Acad. Sci. USA. 99, 15794-15799 10. Kubo, M., Udagawa, M., Nishikubo N., Horiguchi, G., Yamaguchi, M., Ito, J., Mimura, T., Fukuda, H. and Demura T.(2005) Transcriptional switches for protoxylem and metaxylem vessel formation. Genes Dev. 19, 1855-1860
The Second Symposium
on Plant Neurobiology
11 Abstracts for Oral Presentation Session 2
Rice ARF-GAP Mediates Auxin Influx and Auxin-dependent Root Growth
Xiaolei Zhuang1, Jiafu Jiang1, Junhua Li1, Qibin Ma1, Yunyuan Xu1, Yongbiao Xue2, Zhihong Xu1, Kang Chong1
1Research Center for Molecular Developmental Biology, Key Laboratory of Photosynthesis and Molecular Environmental Physiology, Institute of Botany; Chinese Academy of Sciences, Beijing 100093, China; 2Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100086, China
Development and organogenesis in plants are highly dependent on polar auxin transport, which requires the proper asymmetric localization of both auxin influx and efflux carriers. Auxin efflux carriers such as PIN1 are regulated by GNOM ADP ribosylation factor (ARF)-GEF in Arabidopsis, a GDP/GTP exchange factor for a small G protein of ARF-GTPase. However, little is known about the regulation of auxin influx carriers. Here we identified OsAGAP gene in rice which encoded a protein with predicted structure similar to ARFGAP. It was able to stimulate the GTPase activity of rice ARF. Furthermore, OsAGAP can rescue the defect of vesicular transport in the yeast gcs1 Δ glo3 Δ double-mutant cells. Transgenic Arabidopsis with OsAGAP constitutively expression showed reduced apical dominance, shorter primary roots, increasing number of longer adventitious roots. Overexpression of OsAGAP in rice, an activating protein for ARF-GTPase, impaired polar auxin transport and reduced lateral root development and growth. OsAGAP was expressed mainly in the cortex, vascular tissues and meristematic cells under the root cap. The transgenic plant phenotype of reduced number of lateral roots was rescued by treatment with 1-naphthyl acetic acid (NAA), which can enter the cells via diffusion independent of auxin influx carriers, but not by indole 3-acetic acid (IAA), which requires influx carriers to enter the cells. Total [3H]IAA transported in roots of transgenic plants was significantly reduced as compared with that in the wild type. TheThe auxin influx Second carrier AUX1 is asymmetri Symposiumcally localized in the plasma membrane in wild-type plants but in the cytoplasm in transgenic Arabidopsis plants overexpressing OsAGAP. OsAGAP-overexpressed rice plants also showed altered vesicle trafficking.on PlantOur data and other Neurobiologyevidences support a previously undescribed model of PAT regulation: a loop mechanism mediated by ARF-GAP and GEF is involved in regulating polar auxin transport at influx and efflux carriers, which controls root development in plants.
12 Abstracts for Oral Presentation Session 2
Gamma Aminobutyric Acid (GABA) Metabolism in Plants: Analysis of knock-out Mutants
Anke Hüser1, Rainer Waadt2, Dennis Fink1, Iris Schmitz1, Ulf-Ingo Flügge1, and Frank Ludewig1
1Botanical Institute II, University of Cologne, Gyrhofstr. 15, 50931 Cologne, Germany 2Botanical Institute and Botanical Garden, University of Münster, Schlossplatz 4, 48149 Münster, Germany
GABA metabolism is compartmentalized. Anabolism takes place in the cytosol and catabolism occurs in mitochondria. The GABA catabolic ssadh mutant is strongly impaired in growth, most likely due to the accumulation of a toxic compound. Two candidate metabolic intermediates (SSA and GHB) were analyzed for their responsibility to cause the phenotypic aberrances of mutant plants. The ssadh phenotype can be rescued by simultaneously knocking out the gaba-t gene, the gene upstream in GABA catabolism. This phenotype suppression can be explained by preventing the intermediate to accumulate in the double knock-out plants. Based on this finding and seeking for unknown genes being involved in GABA metabolism an ssadh suppressor screen has been performed, where ssadh mutants have been mutagenized using EMS. Suppressor mutants have been collected and analyzed from the M2 generation. Ultimately, EMS mutagenized genes should be mapped and cloned to assign a function for them in GABA metabolism or regulation of the pathway.
The Second Symposium
on Plant Neurobiology
13 Abstracts for Oral Presentation Session 2
Functional Characterization of Plant Steroid Binging Protein
Hong-Wei Xue
National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences; Partner Group of Max-Planck-Institute of Molecular Plant Physiology (MPI-MP) on “plant Molecular Physiology and Signal Transduction”
Steroid-binding proteins (SBPs) are essential for growth and development of both animals and higher plants. Most known animal SBPs not only regulate the concentrations of general available steroids but also function in steroid signal transduction through interacting with specific receptors on the plasma membrane of target cells, thus directly involve in regulation of cell growth and differentiation (Rosner et al., 1999; Breuner and Orchinik, 2002). Although some SBP coding genes have been annotated in higher plants, less studies on the isolation and functional characterization of plant SBP to date. Based on the homologous analysis, we identify one putative arabiopsis MSBP1. Full-length cDNA was isolated and recombinant expressed MSBP1 was used for biochemical characteristics analysis. MSBP1 could bind progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro, demonstrating the presence of a steroid-binding protein in higher plants. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, indicating that MSBP1 negatively regulates hypocotyl elongation and as a negative regulator of cell elongation in Arabidopsis thaliana. Altered expressions of genes involved in cell elongation, such as expansins and extensins, were detected, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in Thesteroid signaling. Second Further studies indi cateSymposium that MSBP1 involve in BR signaling through interaction with BAK1.
on Plant Neurobiology
14 Abstracts for Oral Presentation Session 2
Evidence that Leaf Attachment is Required for Auxin-Induced Inhibition of Leaf Expansion in Arabidopsis
Christopher P. Keller and Morgan L. Grundstad
Department of Biology, Minot State University, Minot, ND 58707
Previous work suggests auxin (indole-3-acetic acid) may function in leaf expansion. Increasing the auxin content of intact expanding leaves of Arabidopsis and Phaseolus, either through exogenous application or through trapping the endogenous hormone in leaves, results in inhibition of leaf expansion.1 Paradoxically, other work has clearly shown that treatment of excised leaf strips from tobacco (Nicotiana) with auxin stimulates rather than inhibits growth.2 Auxin treatment, whether of intact or of excised leaf tissue results in epinastic curvature due to relatively greater growth by the adaxial side of the tissue. The current, not yet complete, project reexamines the auxin growth sensitivity of attached leaves and excised leaves and leaf tissues of Arabidopsis attempting to determine if the reversed growth response to auxin of excised tissues is a wound response or a result of detachment from the plant. For our experiments, 10-14 day old soil-grown Arabidopsis with both the first two true leaves 2.7-3.3 mm in diameter were used - one randomly selected leaf to be the treatment leaf and the other a control. Depending on the experiment, digital images used to determination initial leaf or leaf strip area of intact attached leaves, detached leaves, excised leaf strips (0.7 mm wide cut transversely at midleaf), or wounded attached leaves (sliced transversely from leaf edge to near midvein in three places). Treatment solutions included: full strength Murashige and Skoog media, 10 mM KCl, 0.1 mM Mes/Btp (pH 6.0), +/- indole-3-acetic acid at various concentrations (between 1 µM and 1 mM). Attached leaves and wounded attached leaves received either a 5µl drop of the treatment or control solution. Detached leaves and leaf strips were incubated in 3 mL of the same solutions. After 24 hours, Thethe area of the Second variously treated leaves and Symposium strips was re-determined. The growth of intact attached leaves was found to be relatively insensitive to auxin. While lower concentrations of IAA were ineffective, 300 µM and 1 mM were inhibitory.on For example, Plant at 300 µM the Neurobiologyarea of IAA treated leaves increased 51.7 +/- 10.1(95% C.L.)% compared to 77.2 +/- 6.3 % for the controls (n=12). The growth of detached leaves floated on IAA was sensitive to the same high concentrations but here growth was increased. For example, at 300 µM the area of auxin treated leaves increased 52.3 +/- 3.1 % compared to 43.7 +/- 4.5 % for the controls (n=12). Leaf
15 Abstracts for Oral Presentation Session 2
strips incubated in auxin grew significantly more than control leaves across a range of concentrations 10 µM and higher. In limited tests, wounded attached leaves were significantly inhibited by auxin. Applied at 50 µM, the area of auxin treated leaves increased 14.9 +/- 6.9 % compared to 35.2 +/- 8.8 % for the controls (n=20). In the data so far collected the growth of attached leaves, whether intact or wounded, is inhibited by auxin treatment while detached leaves and strips grow more. Possible explanations will be discussed. (This project is supported by National Institutes of Health of the USA grant P20 RR016741 from the NCRR.)
1. Keller CP, Stahlberg R, Barkawi L, Cohen JD (2004) Plant Physiology 134: 1217-1226. 2. Keller CP, Van Volkenburgh E (1997) Plant Physiology 113: 603-610.
The Second Symposium
on Plant Neurobiology
16 Abstracts for Oral Presentation Session 3
Viroid to Probe Intracellular and Systemic RNA Trafficking in Plants
Biao Ding
Department of Plant Cell and Molecular Biology and Plant Biotechnology Center, 1060 Carmack Rd., Ohio State University, Columbus, OH 43210 USA
RNA trafficking plays important roles in plant growth and development and plant-pathogen interactions. How an RNA traffics within a cell and across specific cellular boundaries is a central mechanistic issue that is poorly understood. Viroid infection provides an excellent model system to address this issue. Viroids are small, noncoding and autonomously replicating RNAs that infect plants. Without encoding proteins, the viroid RNA genome must have evolved structural motifs to exploit an endogenous system for trafficking. We have developed methods that combine single cell replication and whole plant infection assays to investigate how Potato spindle tuber viroid (PSTVd) traffics within a cell and systemically throughout a plant. Our studies show that the strand polarity of PSTVd RNAs can dictate subnuclear trafficking and localization patterns. We have also obtained evidence that trafficking of the PSTVd RNA across different cellular boundaries is mediated by distinct RNA motifs. These findings have important implications in investigating the general regulatory mechanisms of RNA trafficking in plants, including characterizing cell-specific protein factors that recognize an RNA for trafficking between different cells and in particular directions.
The Second Symposium
on Plant Neurobiology
17 Abstracts for Oral Presentation Session 3
Genetic and Proteomic Analysis of Gravity Signal Transduction in Arabidopsis Roots
Patrick H. Masson, Li-Sen Young, Narayana Murthy UM, Gregory Sabat2, Benjamin R. Harrison, John Stanga, Carolyn Neal, Laura Vaughn
Laboratory of Genetics and UW Mass-Spec Facility2, University of Wisconsin-Madison, 425G Henry Mall, Madison, WI 53706, USA
Arabidopsis roots respond to gravistimulation by developing a curvature that is modulated by a lateral gradient of auxin. This gradient originates in the columella statocytes, and is associated with a lateral repositioning of the PIN3 auxin efflux facilitator in these cells. We used genetics to identify proteins that contribute to gravity signal transduction in the statocytes. ARG1 and ARL2 are needed for lateral auxin transport across the cap. ARG1 is associated with the vesicular trafficking pathway, suggesting it regulates PIN3 function or trafficking. Accordingly, immunolocalization studies confirm a lack of PIN3 relocalization in gravistimulated statocytes of arg1-2 and arl2-1 mutant root caps. Genetic modifiers of arg1-2 were obtained and shown to enhance the gravitropic defect of arg1-2. The corresponding proteins function in interpretation of the gravity signal. Furthermore, a proteomic approach allowed identification of root-tip proteins that are differentially represented early in response to gravistimulation. Subsequent reverse genetic studies demonstrated a role for adenosine kinase and the AdoMet pathway in gravity signal transduction (Support from NASA and NSF).
The Second Symposium
on Plant Neurobiology
18 Abstracts for Oral Presentation Session 3
Polarity Control of Auxin Transport
Rujin Chen, Ashverya Laxmi and Jianwei Pan
Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401 USA
Polar auxin transport plays an important role in many plant developmental processes and responses to the environment. It is mediated by plasma-membrane localized auxin influx and efflux carrier proteins of AUX1 and PIN families of proteins, respectively. In the Arabidopsis genome, there are 8 closely related and 7 distantly-related PIN sequences. Loss-of-function analysis and over-expression in heterologous systems of PINs suggested that PIN proteins are the facilitator/regulator/component of the auxin efflux carriers. PIN proteins are undergoing constitutive cycling between plasma membrane and endocytic compartments called endosomes. Experimental evidence suggests that this cycling is important for PIN function as the auxin efflux facilitator and is subject to feedback regulation by auxin. We are interested in understanding how the polarity of auxin transport is established at the molecular and cellular levels. Our inhibitor studies suggest that protein phosphorylation/dephosphorylation plays a role in the control of auxin transport.
The Second Symposium
on Plant Neurobiology
19 Abstracts for Oral Presentation Session 3
Signaling in Chloroplast Movement
Halina Gabrys, Anna Anielska-Mazur, Agnieszka Katarzyna Banas, Weronika Krzeszowiec
Department of Plant Physiology and Biochemistry, Faculty of Biotechnology, Jagiellonian University, Kraków, Poland
Chloroplasts change position in plant cells to optimize light conditions for photosynthesis. In leaves of higher plants these movements are activated by blue light absorbed by phototropins. Phototropin2 controls avoidance responses of chloroplasts in strong light whereas phototropin1 mediates both, accumulation responses in weak light and avoidance responses. Chloroplasts move along actin tracks using myosins as motor molecules. The signal transduction pathway downstream the photoreceptor remains largely unknown. Our study focused on the chloroplast end of the pathway. In particular, we asked the question whether information on the spectral range, intensity and direction of the actinic light is transmitted to the actomyosin system, and if so, in what way it is reflected in the operation of this system. Using transgenic tobacco plants we showed that wavelength differences are not sensed by actin filaments. The actin cytoskeleton was visualised due to expression of a human plastin (actin bundling protein) gene fused with GFP. Images of blue and red-irradiated actin network were similar. The filaments responded however to fluence rate: strong light caused their widening and diffusion, reversible in weak light. No blue specific effect was detected. A potential role of Ca2+ ions in light signal transduction was addressed in experiments with several compounds disturbing calcium homeostasis. The results emphasize the necessity of calcium for proper organization and dynamics of the actin network. Its role as a secondary messenger remains elusive. Immunofluorescence studies using antimyosin antibodies provide evidence that myosins residing on chloroplast surface may be a target of strong blue light signal. Only in this light were these myosins detached from the chloroplast surface. The strong blue light-activated separation of myosin did not occur in the phot2 mutant of Arabidopsis thaliana deficient in phototropin2. This finding sheds a new light on the mechanism operating at the chloroplast-myosin interface and on the mechanism of chloroplast movements in Thegeneral. Intracellular Second responses of the actinSymposium cytoskeleton and chloroplasts to blue light signals are modulated by the presence of sugars in the leaf apoplast. This modulation occurs at the level of gene expression and is pretty complex, depending on the sugar type, concentration and length of exposure. A hexokinase-dependent signalingon pathway Plant is partly involved Neurobiology in this effect. This has been shown in experiments using a non-metabolisable glucose analog, 3-O-methylglucose that did not affect the movement apparatus. On the other hand, Hexokinase1, the unique known Arabidopsis hexose sensor, effected only a reduction in the avoidance response amplitude. 20 Abstracts for Oral Presentation Session 4
A ROP GTPase Signaling Network in the Control of Plant Cell Morphogenesis
Ying Fu, Shundai Li, Tongda Xu, Ying Gu and Zhenbiao Yang
Department of Botany and Plant Sciences, Center of Plant Cell Biology, University of California at Riverside
Rho-family small GTPases are ubiquitous signaling switches in eukaryotic signal transduction. They are considered master regulators that control many important cellular, developmental, and physiological processes through their ability to coordinate multiple pathways and feedback loops. Plants possess a unique subfamily of RHO GTPases, known as ROP, that regulate a diverse array of processes ranging from polar cell growth through hormone responses to defense reactions. However, the molecular mechanisms of Rho GTPase signaling in plants remain poorly understood. We are investigating ROP GTPase signaling networks using two model systems: tip growth in pollen tubes and intercalary cell growth in pavement cells that form jigsaw puzzle appearance. In this talk, I will focus on our recent understanding of how the ROP2 GTPase signaling network coordinates the organization of actin microfilaments (MFs) and microtubules (MTs) to achieve the intercalary cell growth in pavement cells. Both MFs and MTs play an important role in cell morphogenesis, and earlier drug experiments suggest that these two cytoskeletal elements interact and coordinate to modulate cell shape formation. The intercalary growth in pavement cells of Arabidopsis leaves provide an exciting system to investigate how these cytoskeletal elements coordinate with each other at the molecular level to regulate cell growth and cell shape formation. Earlier studies suggest well-ordered cortical MTs, which are found in the indenting zone, promoting indentation, while more recent studies suggest that lobe formation and outgrowth requires cortical fine MFs that are localized to the tip of outgrowing lobes. We have shown that ROP2 GTPase controls intercellular growth to from interlocking lobes and Theindentations bySecond coordinating these two typesSymposium of cytoskeleton. ROP2 regulates these two cellular targets using two molecular targets, RIC4 and RIC1. ROP2 is activated at the site of lobe formation and growth and then activates RIC4, leading to the formationon of localized Plant fine cortical MFs. Neurobiology In the meantime, active ROP2 inhibits RIC1, which are associated with the indenting zone and promotes ordering of cortical MTs. Therefore, in lobe-forming zone, ROP2 promotes outgrowth by activating actin assembly and suppressing MT ordering. Interestingly, in the indenting zone, RIC1-dependent well-ordered MTs suppresses ROP2 activation, assuring that no
21 Abstracts for Oral Presentation Session 4
ROP2 activity is present in this region. Our ongoing research is aimed at understanding how ROP2 is activated in the lobe-forming zone, how RIC1 is activated in the indenting zone, how cortical MTs regulate ROP2 activity, and whether cell-cell communication is important for the coordination of lobe growth and indentation formation between neighboring cells. Progress in answering these questions will be discussed.
The Second Symposium
on Plant Neurobiology
22 Abstracts for Oral Presentation Session 4
Roles of Syntaxins in Disease Resistance
Hans Thordal-Christensen, Ziguo Zhang, Angela Feechan, Carsten Pedersen
Dept. of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark
Plant disease resistance is the result of the collective activity of several separate defence mechanisms. In genetic analyses, we have previously discovered that syntaxin SYP121 in Arabidopsis is required for penetration resistance (Collins et al., 2003; Assaad et al., 2004). SYP121 is probably necessary for vesicle trafficking leading to formation of papillae, which are local cell wall appositions functioning as barriers against fungal penetration. The closely related SYP122 is not required for penetration resistance. Other defence mechanisms are controlled by different signalling pathways, which are activated upon pathogen attack. The signalling compounds salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) each define such pathways. In addition, plant cells can undergo the “hypersensitive response” (HR), a program cell death (PCD) reaction, that confer resistance to biotrophic pathogens. In mutant studies, we have examined the involvement of SYP121 and SYP122 in these four signalling pathways, and found that the syntaxins act as negative regulators of all four. While SYP121 is the primary regulatory protein, SYP122 is partially able to take over it role. The release of this negative regulation in the syp121 syp122 double mutant results in strong PCD-mediated resistance to an otherwise virulent powdery mildew fungus.
Assaad FF, Qiu JL, Youngs H, Ehrhardt D, Zimmerli L, Kalde M, Wanner G, Peck SC, Edwards H, Ramonell K, Somerville CR, Thordal-Christensen H (2004) The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papilla. Molecular Biology of the Cell 15: The5118–5129. Second Symposium Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu JL, Hückelhoven R, Stein M, Freialdenhoven A, Somerville SC, Schulze-Lefert P (2003)on SNARE Plant protein mediated diseaseNeurobiology resistance at the plant cell wall. Nature 425: 973-977.
23 Abstracts for Oral Presentation Session 4
Regulation of pH Homeostasis in Arabidopsis by a Protein Kinase
Yan Guo
National Institute of Biological Sciences, Beijing 100093 China
Regulation of cellular pH is an important part of plant responses to several hormonal and environmental cues such auxin, blue light and fungal elicitors. However, little is known about the signaling components that mediate cellular pH homeostasis in plants. Here we report that an Arabidopsis serine/threonine protein kinase, PKS5, is a critical regulator of cellular pH homeostasis and plant responses to alkaline conditions. Loss-of-function pks5 mutant plants are more tolerant of high external pH. PKS5 negatively regulates the activity of plasma membrane H+-ATPase, and it can phosphorylate the H+-ATPase AHA2 at a novel site, Ser-931, in the C-terminal regulatory domain. We show that PKS5 interacts with an EF-hand calcium-binding protein SCaBP1, and that high external pH can trigger an increase in the concentration of cytosolic free Ca2+. Yeast reconstitution experiments show that the activity of AHA2 in vivo is repressed by SCaBP1 and PKS5, and this repression requires Ser-931. These results suggest that PKS5 is part of a calcium-signaling pathway mediating H+-ATPase regulation.
The Second Symposium
on Plant Neurobiology
24 Abstracts for Oral Presentation Session 4
Involvement of Vesicular Trafficking in Abiotic Stress Tolerance
Miguel A. Botella, Arnaldo Schapire, David Posé, Abel Rosado, and Victoriano Valpuesta
Laboratory of Plant Biochemistry and Biotechnology. Department of Biochemistry and Molecular Biology, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain
We have identified a number of Arabidopsis mutants that show hypersensitivity to several abiotic stresses. Two of these mutants suggest that membrane trafficking is important for abiotic stress tolerance. The 746 mutant was identified in a screening of plants that showed hypersensitivity to NaCl stress. Identification of the gene altered in this mutant encodes for a protein with homology to synaptotagmins. Synaptotagmins are proteins that share a common structure: an N-terminal transmembrane domain, a linker segment and two C2 domains. The dry2 mutant is hypersensitive to oxidative stress and the gene altered encodes a protein involved in sterol biosynthesis. Previous analysis of Arabidopsis mutants affected in sterol composition demonstrated the importance of sterol biosynthesis in cell polarity and auxin efflux. The importance of vesicular trafficking relative to abiotic stress tolerance will be discussed.
The Second Symposium on Plant Neurobiology
25 Abstracts for Oral Presentation Session 5
Lipid-Mediated Signaling in Plant Stress Responses
Xuemin Wang
Department of Biology, University of Missouri, St. Louis, MO 63121; Danforth Plant Science Center, St. Louis, MO 63121, USA
Cell membranes are the initial and focal points of stimulus perception and signaling messenger production. Phosphatidic acid (PA) is the simplest membrane phospholipid and also a central intermediate for the synthesis of membrane lipids and storage lipids. The metabolism of PA occupies a key position in glycerolipid metabolism and membrane biogenesis. It is the regulatory function of PA, however, that has attracted increasing attention in recent years. PA is involved in various cellular processes, such as signal transduction, membrane trafficking, secretion, and cytoskeletal rearrangement. The effects of PA have been linked to the survival, proliferation, reproduction, and response to abiotic and biotic stresses. Signaling PA can be produced by multiple enzymes, and the activation of specific enzymes regulates the timing, location, and molecular species of PA. The modes of PA action are multifaceted and include membrane tethering, direct modulation of enzymatic activity, and effects on membrane structures and metabolism. Phospholipase D (PLD) is one super-family of enzymes that produce PA. Recent results have provided insights into the molecular mechanism by which the PLD family and PA molecular species mediate stress signaling. The functions of specific PLDs and PA have been linked to programmed cell death and plant tolerance to water, temperature, and nutrient stresses. This talk will discuss the multifaceted roles and the mechanisms of action of PLDs and PA in mediating different plant stress responses. It will also describe lipidomics approaches used to systematically understanding membrane lipid functions.
The Second Symposium
on Plant Neurobiology
26 Abstracts for Oral Presentation Session 5
Plant Molecular Response to Al Toxicity in Acid Soil: Regulation of Organic Acid Exudation
Hideaki Matsumoto １ , ＊ , Hong Shen1, ２ ,Takayuki Sasaki １ , Yoko Yamamoto１, Mineo Yamaguchi１, 3, Hiroki Osawa１,4, Peter R. Ryan5, and Emmanuel Delhaize5
1Research Institute for Bioresources, Okayama University, Kurashiki 710-0046、 Japan 2College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, China 3Div0ision of Plant Science, 1-31 Agriculture Building University of Missouri Columbia, MO 65211 USA 4Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan 5CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia
Acid soils occupy 30% or 3950 billion ha of the world’s ice free land area and are the largest problem soils for the crop production. Thus attention has been paid to improving the crop production in acid soils. The adverse effect of acid soil on plant growth is strongly related to the toxicity of Al3+. Some plants acquires Al tolerance simply by exclusion of toxic Al3+ by chelation with exuded organic acids from root by Al. Malate exudation from wheat root: Activation of malate efflux occurred in 5 min after exposure of root apex of Al tolerant wheat to Al3+. The channel highly permeable to malate was demonstrated in Al-tolerant wheat by the whole patch clamp method but the gene encoding the malate transporter had not been discovered until recently. We cloned ALMT1 gene (aluminum-activated malate transporter) expressed dominantly in the root apices of the Al-tolerant wheat line. ALMT1 localized in the plasma membrane (PM) and constitutively expressed in the root apices of the Al-tolerant line at greater levels than in the Al-sensitive line (1). Transgenic barley expressing ALMT1 demonstrated that ALMT1 encoding Al-activated malate transporter is capable of conferring Al tolerance to plant cells (2). The Citrate exudationSecond from soybean root : SymposiumAl initiates citrate efflux from the soybean root apices 30min after the addition of Al. PM H+-ATPase regulated the efflux of citrate by Al. Vanadate and fusicoccin extended inhibitory and stimulatory effects on the Al-induced efflux of oncitrate. HigherPlant activity of PM NeurobiologyH+-ATPase coincided with more citrate efflux in Al-resistant than –sensitive soybean cultivars. The increase of PM H+-ATPase activity by Al was caused by transcriptional and translational regulation. PM H+-ATPase activity and expression were higher in an Al-resistant cultivar than in Al-sensitive cultivar. Al activated the 27 Abstracts for Oral Presentation Session 5
threonine-oriented phosphorylation of PM H+-ATPase. The up-regulation of PM H+-ATPase activity was associated with the secretion of citrate from soybean roots (3).
1. Sasaki T et al. (2004) A what gene encoding an aluminum-activated malate transporter. Plant J 37: 645-653 2. Delhaize E et al. (2004) Engineering high-level aluminum tolerance in barley with the ALMT1 gene. Proc. Natl Acad. Sci. USA. 101:15249-15254 3. Shen H et al. (2005) Citrate secretion coupled with the modulation of soybean root tip under aluminum stress. Up-regulation of transcription, translation, and threonine-oriented phosphorylation of plasma membrane H+-ATPase. Plant Physiol. 138: 287-296
The Second Symposium
on Plant Neurobiology
28 Abstracts for Oral Presentation Session 5
The E3 Ubiquitin Ligase Activity of Arabidopsis PLANT U-BOX17 and Its Functional Tobacco Homolog ACRE276 Are Required for Cell Death and Defense
Cheng-Wei Yanga, Rocio Gonza´ lez-Lamotheb, Richard A. Ewana, Owen Rowlandb, Hirofumi Yoshiokab, Matt Shentona, Heng Yea, Elizabeth O’Donnella, Jonathan D.G. Jonesb, and Ari Sadanandoma
a Plant Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom b Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom
Previous analysis of transcriptional changes after elicitation of Cf-9 transgenic tobacco (Nicotiana tabacum) by Avr9 peptide revealed a rapidly upregulated gene, ACRE276. We show that ACRE276 is transiently induced in wounded leaves within 15 min, but upon Avr9 elicitor treatment, this upregulation is enhanced and maintained until cell death onset in Cf-9 tobacco. ACRE276 RNA interference (RNAi) silencing in tobacco results in loss of hypersensitive response (HR) specified by Cf resistance genes. CRE276 RNAi plants are also compromised for HR mediated by the tobacco mosaic virus defense elicitor p50. Silencing tomato (Lycopersicon esculentum) ACRE276 leads to breakdown of Cf-9–specified resistance against Cladosporium fulvum leaf mold. We confirmed that tobacco ACRE276 is an E3 ubiquitin ligase requiring an intact U-box domain. Bioinformatic analyses revealed Arabidopsis thaliana PLANT U-BOX17 (PUB17) and Brassica napus ARC1 as the closest homologs of tobacco ACRE276. Transiently expressing PUB17 in Cf-9 tobacco silenced for ACRE276 restores HR, while mutant PUB17 lacking E3 ligase activity fails to do so, demonstrating that PUB17 ligase activity is crucial for defense signaling. Arabidopsis PUB17 knockout plants are compromised in RPM1- and RPS4-mediated resistance against Pseudomonas syringae pv tomato containing avirulence genes AvrB and AvrRPS4, respectively. We identify a conserved class of U-box ARMADILLO repeat E3 ligases that are positive regulators of cell death and Thedefense across Second the Solanaceae and Brassicaceae. Symposium
Cheng-Wei Yang, Rocio Gonzalez-Lamothe, Richard A. Ewan, Owen Rowland, Hirofumion Yoshioka, Plant Heng Ye, Elizabeth Neurobiology O’ donnell, Jonathan DG Jones, Ari Sadanandom(2006) The E3 Ubiquitin Ligase Activity of Arabidopsis PLANT U-BOX17 and Its Functional Tobacco Homolog ACRE276 Is Required for Cell Death and Defence. Plant Cell, 10.1105/tpc.105.039198
29 Abstracts for Oral Presentation Session 5
Plant Immunophilins Regulate ABC Transporter Activity and Cell Morphogenesis
Burkhard Schulz1, Markus Geisler2, Enrico Martinoia2, Wendy Peer1 and Angus Murphy1
1 Department of Horticulture, Purdue University, West Lafayette, IN 47907, USA 2 University of Zurich, Institute of Plant Biology, CH-8008 Zurich, Switzerland
E-mail: [email protected]
Polar transport of the phytohormone auxin is required for the plant polarity and coordinated development. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in AtPGP1 and AtPGP19 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from atpgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors such as NPA and Quercetin. Expression of AtPGP1 in yeast and in mammalian cell expression systems results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1- naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is again sensitive to auxin efflux and ABC transporter inhibitors. The plasma membrane bound FKBP-like immunophilin protein TWD1 (TWISTED DWARF1/FKBP42) from Arabidopsis physically interacts with ABC transporters AtPGP1 and AtPGP19. Disruption of the TWD1 gene in Arabidopsis results in dwarfed plants exhibiting a reduction in cell elongation as well as desorientation of cell growth. This leads to strong epinasty of leaves and size reduction of organs. Biochemical analysis of resembling dwarf phenotypes of the double mutants atpgp1/atpgp19 and of the single mutant twd1 suggested a positive regulatory role of TWD1 on AtPGP-mediated auxin export activities. We verified the Theregulatory effect Second of TWD1 on PGP-mediated Symposium auxin efflux by employing plant specific as well as heterologous auxin transport systems. Using an IAA-specific microelectrode we demonstrate that IAA influx in the root elongation zone is reduced and shiftedon apically Plant in atpgp and Neurobiologytwd1 mutant roots. As a consequence, atpgp1/atpgp19 and twd1 mutant roots reveal elevated levels of free IAA in the elongation zone and above. Yeast and plant transport data suggest that TWD1 defines not only transport activities but also substrate specificities.
30 Abstracts for Oral Presentation Session 6
Emerging Roles of Endocytosis in Plant Development
Akihiko Nakano1,2, Tatsuaki Go1,2, Wakana Uchida2, Satoko Arakawa-Kobatashi2 and Takashi Ueda1
1Dept. of Biol. Sciences, Grad, School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, 2RIKEN Discovery Research Institute, Wako, Saitama 351-0198, Japan
Endocytosis plays important roles in various functions of plants, such as polar transport of auxin, establishment of cell polarity, cell plate formation during cytokinesis, and cell wall morphogenesis. To understand molecular mechanisms of endocytosis in plants, we are focusing our attention on Rab5 GTPases. In mammalian cells, Rab5 is known to organize many events relating endocytosis, such as homotypic fusion between early endosomes, alteration of lipid composition of the endosomal membrane, and signal transduction through endosomes via specific interactions with effector proteins. Arabidopsis thaliana contains three Rab5-related GTPases (conventional Ara7 and Rha1, and plant-unique Ara6) in the genome. These three Rab5 GTPases are localized on differentiated plant endosomes and regulate endosomal membrane fusion. We have recently identified an Arabidopsis Rab5-specific guanine nucleotide exchange factor (GEF), AtVps9a, and shown that this is the sole activator of the all three Rab5 members. Two tagged lines are available for the AtVPS9a locus. In the atvps9a-1 mutant whose GEF activity is completely lost, embryogenesis is arrested at the torpedo stage. In the atvps9a-2, a leaky allele lacking the C-terminal regulatory domain, shoot appears to develop normally but elongation of the primary root was severely affected. These results indicate that endocytosis plays very basic roles in plant development.
The Second Symposium
on Plant Neurobiology
31 Abstracts for Oral Presentation Session 6
Cloning and Functional Characterization of Arsenate Reductase from Rice
Gui-Lan Duan1, Barry P Rosen2, Yi-Ping Tong3, and Yong-Guan Zhu1*
1 Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences; 2 Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA; 3 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences *Corresponding author
Rice (Oryza sativa) is the staple food for more than half the world's population. Arsenic ingestion through by rice consumption attracts more and more attention in the last few years. The genetic engineering techniques can be hopeful in improving the yield and quality of rice grown in arsenic-impacted soils. The rice genome contains two genes with significant homology to the CDC25 from Arabidopsis thaliana and ACR2 from S. cerevisiae: OsACR2.1 (137 aa, 14,963 Da; GenBankTM accession number AY860059) and OsACR2.2 (130 aa, 14,330 Da; GenBankTM accession number AY860058). The predicted proteins contain the
HC(X)5R catalytic motif, and exhibit high sequence identity with ScAcr2p (21.25% and 26.24%, respectively) and AtCDC25 (50.98% and 55.48%, respectively). Both genes complemented the E. coli mutant strains with ArsC deletion and yeast mutant strains with a disrupted ACR2 gene. Detailed molecular evidence will be presented.
The Second Symposium
on Plant Neurobiology
32 Abstracts for Oral Presentation Session 6
Sec14p-like Phosphatidylinositol Transfer Proteins in Arabidopsis
Patrick Vincent and Vytas A. Bankaitis
Department of Cell & Developmental Biology, University of North Carolina, Chapel Hill, NC 27599-7090 USA
Phosphatidylinositol transfer proteins (PITPs) represent novel regulators of specific signaling interfaces between lipid metabolism and membrane trafficking. The Sec14p-like PITPs major comprise a eukaryotic protein superfamily that is highly represented in plants. A particularly interesting group of plant Sec14p-like proteins is the large and uncharacterized Arabidopsis Sec14p-nodulin domain family. AtSfh1p, one such Sec14p-nodulin protein, is a focus of our work. AtSfh1p is a PITP that regulates a specific stage in root hair development by integrating phosphoinositide signaling with polarized membrane trafficking in developing Arabidopsis root hairs. Compromise of this signaling node results in deranged polarized root hair expansion in a manner that coincides with loss of tip-directed
PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, precocious Ca2+ entry into root hairs, and disorganization of the root hair MT cytoskeleton. We propose that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.
1. Sha, B., Phillips, S.E., Bankaitis, V.A. and Luo, M. (1998) Crystal structure of the Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p. Nature 391: 506-510. 2. Vincent, P., Chua, M., Nogue, F., Fairbrother, A., Mekheel, H., Xu, Y., Allen, N., Bibikova, T.N., Gilroy, S., and Bankaitis, V.A. (2005) A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis root hairs. Journal of Cell Biology 168: 801-812. The3. Phillips, S.E.,Second Vincent, P., Rizzieri, KSymposium., Schaaf, G., Gaucher, E.A., and Bankaitis, V.A. (2006) The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes. Crit. Rev. in Bioch. & Mol. Biol. 41: 1-28. on Plant Neurobiology
33 Abstracts for Oral Presentation Session 6
The Arabidosis thaliana SNARE Protein PEN1 Localizes to Endosomes and at Sites of Polarized Endosomal Secretion in Root Cells
Boris Voigt1,2,3, Hans Thordal-Christensen4, Stefano Mancuso2,3, Diedrik Menzel1, František Baluška1,3
1 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany 2 Dept. Horticulture, University of Firenze, Viale delle idee 30, 50019 Sesto F.no (FI), Italy 3 Plant Neurobiology Laboratory, Viale delle idee 30, Sesto F.no (FI), Italy; Kirschallee 1, Bonn, Germany 4 Plant and Soil Science Laboratory, Dept. of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40,1871 Frederiksberg C, Copenhagen, Denmark
The syntaxin SYP121/PEN1 was first isolated in a non-host penetration resistance screen in Arabidosis thaliana. SYP121/PEN1 shows a localization at the plasma membrane and endomembrane compartments in leaves. It is accumulating at papillae upon pathogen infection. In knock-out mutants, the papilla formation is delayed and the penetration resistance is decreased (Collins et al. 2003, Assaad et al. 2004). The syntaxin SYP121/PEN1 is well expressed in many root cells and has there probably different functions than penetration resistance. We show that it is localized, besides to the plasma membrane, also to numerous vesicular compartments of root cells. These are of endosomal nature and obviously participate in the polarized endosomal secretion in tip growing root hairs as well as in cytokinetic root cells. Both the root hair apices as well as cytokinetic cell plates are sites of the polarized endosomal secretion (Voigt et al. 2005, Ovecka et al. 2005, Dhonukshe et al. 2006, Dettmer et al. 2006). Importantly, PEN1 labelled endosomes exhibit a Ca2+ dependent behavior. All this suggests that the SYP121/PEN1 function in calcium triggered polarized secretion in root cells. As papilla also represents an example of polarized secretion, (Schmelzer 2002), our data suggest for the first time that the syntaxin SYP121/PEN1 is driving calcium-regulated polarized secretion based on secretory endosomes. The Second Symposium Assaad FF, Qiu JL, Youngs H, Ehrhardt D, Zimmerli L, Kalde M, Wanner G, Peck SC, Edwards H, Ramonell K, Somerville CR, Thordal-Christensen H (2004) The PEN1 syntaxinon defines Plant a novel cellular compar Neurobiologytment upon fungal attack and is required for the timely assembly of papillae. Mol Biol Cell 15: 5118-5129 Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu JL, Huckelhoven R, Stein M, Freialdenhoven A, Somerville SC, Schulze-Lefert P
34 Abstracts for Oral Presentation Session 6
(2003) SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425: 973-977 Dhonukshe P, Baluška F, Schlicht M, Hlavacka A, Šamaj J, Friml J, Gadella Jr TWJ (2006) Endocytosis of cell surface material mediates cell plate formation during plant cytokinesis. Dev Cell 10: 137-150 Ovecka M, Lang I, Baluška F, Ismail A, Illeš P, Lichtscheidl IK (2005) Endocytosis and vesicle trafficking during tip growth of root hairs. Protoplasma 226: 39-55 Schmelzer, E. (2002) Cell polarization, a crucial process in fungal defence. Trends Plant Sci 7: 411-415 Voigt B, Timmers A, Šamaj J, Hlavacka A, Ueda T, Preuss M, Nielsen E, Mathur J, Emans N, Stenmark H, Nakano A, Baluška F, Menzel D (2005) Actin-based motility of endosomes is linked to polar tip-growth of root hairs. Eur J Cell Biol 84, 609-621
The Second Symposium
on Plant Neurobiology
35 Abstracts for Oral Presentation Session 6
Hydrogen Peroxide-Based Signals Co-Ordinated the Response to Sodium Stress in Plants
Guoyong An, Chun-Peng Song
Lab of Plant Stress Biology, College of Life Sciences, Henan University, Kaifeng 475001, China
It is now clear that hydrogen peroxide (H2O2) acted as the second messenger
involved in abscisic acid (ABA) signaling in the regulation of stomatal behavior. H2O2 generation is induced in plants following exposure to a wide variety of abiotic and biotic stimuli, including salt, drought, UV irradiation and ABA treatment, etc. Roots are exposed in soil and always challenged by salt stress. It is not clear how plant root cells sense and transduce the stress signal to aerial parts of plants, e.g. stomata, to
adjust stress adaptation. Here we report that H2O2 generation in guard cells mediates stomatal behavior to control the water loss resulting in development of salt tolerance under salt stress. We used infrared camera, laser scanning confocal microscope and patch clamp to identify the long-distance signal from root cells as component of guard cell signaling. The stomata of Vicia faba leaves closed when plants were treated by 300 mM NaCl in the soil for 3 hours, thereby accompanying the increase of temperature of leaf surface. Monitoring Na+ changes in guard cells by using fluorescent probe Sodium-Green AM indicate that the concentration of Na+ of guard cells from the plant challenged with NaCl was higher than that of control. Exogenous
NaCl could induce both closure of stomata and enhancement of H2O2 production in guard cell. CAT and DPI partly reversed the stomatal closure induced by NaCl. The
pattern of stomatal behavior in response to H2O2 and NaCl is similar. Whole-cell recording showed that NaCl could inhibit inward K+ current of guard cell. These results suggested that stomata could probably act as sensor after NaCl treatment to co-ordinate whole plant to adapt to salt stress through transduction of long-distance signal.
The Second Symposium
on Plant Neurobiology
36 Abstracts for Oral Presentation Session 7
Extracellular ATP and signalling in Arabidopsis
A. R. Slabas1, W.J.Simon1, K.Lindsey1, S.Chivasa2, 1
1. The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, South Rd, Durham DH1 3LE UK. 2. Creative Gene Technology, School of Biological and Biomedical Sciences, University of Durham, South Rd, Durham DH1 3LE UK.
Following a proteomic investigation of the extracellular matrix of Arabidopsis, we observed that a number of proteins contained potential phosphorylation domains [1]. This prompted us to investigate if extracellular ATP existed in plants and if so, what the function of the ATP was. Using 32P-phosphate labelling of cell cultures, we have demonstrated that extracellular ATP exists. We have used ATP depletion systems to observe the physiological effects of extracellular ATP removal. Removal of ATP results in cell death – this is a phenomenon which is not only observed in suspension cultures, but also in whole plants. Addition of non-hydrolysable ATP also results in cell death –indicating that extracellular ATP is essential for cell viability. Fumonisin B1 [FB1] triggers depletion of extracellular ATP, which precedes cell death. Addition of exogenous ATP rescues the cells from FB1-mediated cell death, indicating that extracellular ATP may be an important component of sensing pathogen attack [2]. We are currently using both transcriptomic and proteomic approaches to try and identify the candidate genes in this cell death pathway.
1. Chivasa et al., (2002) Electrophoresis 23:1754-1765 2. Chivasa et al., (2005) Plant Cell 17:3019-3034
The Second Symposium
on Plant Neurobiology
37 Abstracts for Oral Presentation Session 7
Apoplastic Calmodulin: Existence, Functions, and Transmembrane Mechanism
Daye Sun, Ying Sun
Institute of Molecular Cell Biology, Hebei Normal University, Shijiazhuang 050016
Calmodulin (CaM), traditionally a well known intracellular calcium sensor that mediates many signaling pathway, has been found in the apoplast of many species of plants, and functions of apoplastic CaM in accelerating cell proliferation, protoplast cell wall regeneration, and in promoting pollen germination and tube growth, have also been described physiologically in our previous studies. To provide further genetic evidence for the existence and functions of apoplastic CaM to support our hypothesis that apoplastic CaM might be used as a polypeptide signal in plant development, we first verified cell wall localization of CaM in living cells by visualizing fluorescence of GFP fusion protein with soybean CaM(SCAM), and interestingly found that highly conserved isoform SCaM-1, -2, -3, but not divergent isoform SCaM-4,-5, all of which are stably expressed in tobacco cells are preferably secreted. Next we confirmed the promotion effect of apoplastic CaM on pollen germination and tube growth by phenotypic analyzing Arabidopsis wild type pollen, transgenic pollen over-expressing apoplastic AtCaM-2, a conserved isoform in Arabidopsis, and transgenic pollen with apoplastic CaM attenuated by over- expressing apoplastic CaM-binding peptide. To understanding transmembrane mechanism of apoplastic CaM function, we did radio-ligand binding assay with 35S-labeled AtCaM-2, and disclosed the specific, reversible, and saturable calmodulin binding sites on the surface of both A. thaliana suspension-cultured cells and its protoplasts. Chemical crosslinking of 35S-labeled AtCaM-2 further revealed 117- and 41-kDa plasma membrane proteins, which might be promising candidates for receptor-like protein specifically bound to apoplastic CaM. Besides, phospholipase C activity, cytosolic Ca2+ concentration were also Thefound to be increasedSecond specifically in responsSymposiume to exogenous CaM when applied outside of pollen protoplast. Furthermore, patch clump experiment showed that heterotrimeric Gα protein regulated pollen plasma membrane Ca2+ channel is involved onin mediating Plant apoplastic CaM signal.Neurobiology All our findings suggest that apoplastic CaM may be a polypeptide signal in plants development, and its signaling pathway is different from that of intracellular CaM.
38 Abstracts for Oral Presentation Session 7
Redox-Responsive Calcium Signaling in Plants
Tomonori Kawano1, Takuya Furuichi2, and Takashi Kadono1
1 Graduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu 808-0135, Japan 2 Graduate School of Medicine, Nagoya University, 65 Tsurumai, Nagoya 466-8550, Japan
Calcium ion (Ca2+), one of the major necessary elements in living cells, plays an essential role as an intracellular secondary messenger in plant cells. When plant cells are exposed to environmental stresses or perceive signaling molecules, calcium channels can be transiently activated to convert these stimuli into intracellular events. In the present talk, our recent studies on the redox-responsive calcium signaling mechanism in tobacco (Nicotiana tabacum L.) cells will be described. Three cell lines of tobacco cell suspension cultures expressing aequorin genes 2+ were used to study the changes in cytosolic calcium concentration ([Ca ]c) in 2+ responses to extracellular redox changes. The [Ca ]c elevation-stimulating oxidative stimuli related to reactive oxygen species (ROS) studied here include hydrogen peroxide, salicylic acid, aluminum ion and ozone. Salicylic acid and aluminum added to the cells rapidly stimulate the generation of superoxide anion via peroxidase- and NADPH oxidase-dependent mechanisms, respectively, and in turn superoxide induces the influx of Ca2+ into the cells. In tobacco cell suspension cultures, exposure to ozonized air can result in generation of a variety of ROS such as singlet oxygen, hydrogen peroxide and hydroxyl radicals and these ROS members eventually stimulate the influx of Ca2+ into the cells. When the entry of Ca2+ into the cells were prevented by Ca2+ chelators such as BAPTA and EGTA, the ozone-induced cell death was significantly prevented, supporting our view that Ca2+ plays a key role in oxidative cell death. Our data suggested that the recently characterized TPC1-type Ca2+-permeable channels behave as the key oxidative stress-responsive 2+ 2+ Ca -permeable channels involved in transient increase in [Ca ]c. In addition to oxidative stresses, effect of reducing environments in the 2+ extracellular space on [Ca ]c was tested by adding some low molecule thiols such as cysteine, N-acetylcysteine, glutathione, dithiothreitol and 2-mercaptoethanol. TheSimilarly to Secondthe responses to oxidativ Symposiume stresses, tobacco cells responded to 2+ extracellularly added thiols by rapidly elevating [Ca ]c. Such thiol-induced increases 2+ 2+ 2+ in [Ca ]c were shown to be sensitive to Ca chelators and Ca channel blockers thus suggestingon the involvement Plant of Ca2+ channels Neurobiology as the targets of extracellular thiols. We are presently trying to propose a conceivable model that explains the plant calcium responses to both oxidative and reductive environments surrounding the plant cells.
39 Abstracts for Oral Presentation Session 7
Histone Modification: A Link Between Signals and Programs
Shunong Bai
College of Life Sciences, Peking University, Beijing, 100871
It is widely accepted that plant development is heavily affected by environmental signals. During previous two decades, through analyzing Arabidopsis mutants, people were enabled to dissect the developmental events those are responded to environmental signals. It is interesting that the investigations were concentrated at the two ends of an integrated process: the dissection of the signaling system at the one end, centered with the signaling receptors; and the identification of the components involved in the developmental program at the other. Taking the cellular patterning of root epidermis as an example, genes such as CPC, GL2 and WER have been isolated and identified to play roles in determining the patterning process. The highly positional dependence to the cortical cells and the identification of SCM gene suggested a “positional cue” is involved in directing the patterning. But little is known how the signals linked with the patterning genes. Such a situation is not a solo case. How to link signals and programs in developmental events is an obvious challenge to our understanding of regulatory mechanism in plant development. Our work on histone acetylation affecting the cellular patterning of root epidermis suggested that it is possible to approach the link not from the both ends inward, but from the center outward by deciphering the mechanism of the chromatin regulation of gene expression.
The Second Symposium
on Plant Neurobiology
40 Abstracts for Oral Presentation Session 8
Signalling to the Actin Cytoskeleton
Patrick. J. Hussey
The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, South Rd., Durham DH1 3LE, UK
One of the most intriguing issues in plant cytoskeleton biology is that whereas the fundamental components of the cytoskeleton appear similar to their animal counterparts (actin and microtubules) the actin regulatory proteins in particular are utilised (e.g. SCAR/WAVE ) and regulated (e.g ADF) differently. This is perhaps not surprising because the modes of development in plants and in animals are different. For example embryogenesis in animals is dependent on cell migrations whereas plant cells cannot move. In plants the simple body plan is established during embryo development but most organ development is established after embryogenesis and after seed germination. Plant cells have to respond to different environmental cues in order to maximise energy production, to take up nutrients from the soil, to reproduce and to protect from pathogen invasion. In all these cases the cytoskeleton has to respond to signals and reorganise to generate organelle movement and cell expansion, polarise cell growth and thicken the cell wall. My lab is involved in elucidating the plant signalling pathways that control actin reorganisations that govern plant cell morphogenesis and this presentation will give an overview of the key stimulus responsive molecules of the actin cytoskeleton.
The Second Symposium
on Plant Neurobiology
41 Abstracts for Oral Presentation Session 8
How to Stop a Pollen Tube? Signaling to the Targets of Self-incompatibility in Papaver Pollen
Steven G. Thomas, Shutian Li, Shanjin Huang1, Christopher J. Staiger1 and Vernonica Franklin-Tong
School of Biosciences, University of Birmingham, Edgbaston, Birmingham. B15 2TT, UK 1Dept. Biological Sciences & The Bindley Bioscience Center, Purdue University, West Lafayette IN 47907-1392, USA
Sexual reproduction in higher plants involves interactions between pollen and pistil. Self incompatibility (SI) prevents self-fertilization and is an important mechanism for promoting outbreeding. SI is controlled by the S-locus; discrimination occurs between incompatible (“self”) pollen, which is rejected, while compatible (“non-self”) pollen can achieve fertilization. In Papaver rhoeas, S proteins encoded by the pistil part of the S-locus interact with incompatible pollen. The “self” SI interaction triggers a Ca2+-dependent signalling cascade, resulting in rapid depolymerization of the actin cytoskeleton and inhibition tip growth in incompatible pollen. A MAP kinase, p56, is also activated specifically in incompatible pollen. We recently showed that programmed cell death (PCD) involving a caspase-3-like activity is triggered by SI. This provides a precise mechanism for the specific destruction of “self” pollen. We are currently investigating a possible role for actin depolymerization in signaling to PCD and also whether the p56-MAPK might signal to PCD. Recent data providing evidence for their involvement in SI-mediated PCD will be discussed. As the SI-induced F-actin depolymerization was far in excess of that required to inhibit growth, this suggested an additional function. We have used drugs that affect actin dynamics, latrunculin B and jasplakinolide, to explore a possible role for actin depolymerization in signaling to PCD. Our data show that alterations to actin dynamics play a functional role in the early stages of the PCD signaling cascade in Papaver pollen. Furthermore, a significant alleviation of SI-induced PCD in incompatible pollen was achieved by adding jasplakinolide, showing that actin Thedepolymerization Second plays a functional roleSymposium in SI-induced PCD. We believe this represents the first demonstration of signaling between the actin cytoskeleton and PCD in plants. Because MAPKs are known to play a role in PCD in both animal and plant cells, we have oninvestigated Plant a possible link betw Neurobiologyeen the p56-MAPK and PCD triggered by SI. Using the MAPK inhibitor, U0126, we have obtained evidence that this blocks both p56-MAPK activation and DNA fragmentation induced by SI. This provides the first evidence that the p56-MAPK is involved in signaling to PCD in the SI response.
42 Abstracts for Oral Presentation Session 8
The Cytoskeleton and Intercellular Communication
Christine Faulkner1,2, Leila Blackman3, David Collings3, Robyn Overall1
1School of Biological Sciences, University of Sydney, NSW, Australia 2Present address: Institute of Molecular Plant Sciences, University of Edinburgh, Scotland 3Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, ACT, Australia
As conduits for electrical signals, developmental messages, water and metabolites between cells, plasmodesmata are central in the function and development of plants as well as the integration of their responses to environmental signals. In plant cells, the cytoskeleton has a myriad of functions from orchestrating cytokinesis and directing cellulose deposition through to driving cytoplasmic streaming and vesicle and organelle motility. The identification of the cytoskeletal components actin (White et al, 1994, Blackman and Overall, 1998), myosin (e.g. Blackman and Overall, 1998, Reichelt et al, 1999), centrin (Blackman et al, 1998) and Arp3 (Van Gestel et al, 2003) in plasmodesmata suggests that the cytoskeleton also plays a role in the trafficking to and through plasmodesmata. Tropomyosin is an actin-binding protein thought to be involved in a range of functions associated with the actin cytoskeleton, including the regulation of myosin binding to actin filaments, but to date no tropomyosin-like proteins have been conclusively identified in plant genomes. Anti-tropomyosin antibodies localised to plasmodesmata in the green alga Chara corallina. These antibodies also localised to other structures including actin cables. Anti-tropomyosin antibodies labelled plasmodesmata of Arabidopsis thaliana and leek tissue. Western blot analysis identified a 75kDa and a 55kDa protein in Chara protein extracts, a single protein at 42.5 kDa in A. thaliana extracts and two proteins at 58.5 and 54 kDa in leek extracts. The 75 kDa protein from Chara was present in protein extracts from cell walls containing plasmodesmata (nodal complex walls) and was absent from the Theexternal cell wallsSecond of internodes that do notSymposium contain plasmodesmata, again suggesting that a tropomyosin-like protein is associated with plasmodesmata.
1. Blackmanon LM, Plant Harper JDI, Overall Neurobiology RL (1999) Centrin localisation in higher plant plasmodesmata. Eur J Cell Biol. 78, 297-304 2. Blackman LM and Overall RL (1998) Immunlocalisation of the cytoskeleton to plasmodesmata of Chara corallina. Plant J 14: 733-742
43 Abstracts for Oral Presentation Session 8
3. Reichelt S, Knight AE, Hodge TP, Baluska F, Samaj J, Volkmann D, Kendrick-Jones J (1999) Characterization of the unconventional myosin VIII in plant cells and its localization a the post-cytokinetic cell wall. Plant J 19: 555-567 4. Van Gestel K, Slegers H, von Witsch M, Samaj J, Baluska F, Verbelen J-P (2003) Immunological evidence for the presence of plant homologues of the actin-related protein Arp 3 in tobacco and maize: subcellular localisation to actin-enriched pit fields and emerging root hairs. Protoplasma 222: 45-52 5. White RG, Badelt K, Overall RL, Vesk M (1994) Actin associated with plasmodesmata. Protoplasma 180: 169-184
The Second Symposium
on Plant Neurobiology
44 Abstracts for Oral Presentation Session 8
Cloning and Characterization of a Ca2+-dependent Actin-binding Protein from Lily Pollen
Yun Xiang and Haiyun Ren
Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, and College of Life Science, Beijing Normal University, Beijing 100875, People’s Republic of China
As the only known Ca2+-dependent actin binding proteins, villin/gelsolin/fragmin superfamily proteins play important roles in pollen germination and the tube growth. However, bioinformatic analysis shows that separate genes for gelsolin/severin do not exist in Arabidopsis and rice (Oryza sativa L.), and it is generally accepted that these proteins might be encoded by mRNA splicing variants of villins. Here we succeeded in cloning an identical full-length cDNA of 989 bp from two different cDNA libraries of lily pollen by PCR, respectively. It encoded a predicted protein of 263 amino acids that shared 100% identity to N-terminus of P-135-ABP (a lily villin) except for the 6 amino acids in C-terminus. In addition, the entire 3’UTR of the cDNA was totally different from that of cDNA encoding P-135-ABP. The deduced LdABP29 contained G1, G2 and part of G3 domains. Biochemical analysis showed that the purified recombinant LdABP29 could accelerate actin nucleation, block barbed ends and sever actin filaments in a Ca2+-dependent manner in vitro. Over expression of LdABP29 in tobacco BY-2 cells resulted in fragmentation of actin filaments. These results suggest that there exists a separate gene that encodes the small molecular mass protein of villin/gelsolin/fragmin superfamily in plants and LdABP29 is a new member of the superfamily, which may participate in regulating the organization of actin cytoskeleton in living plant cells.
1. Huang S, Blanchoin L, Chaudhry F, Franklin-Tong VE, Staiger CJ (2004) A gelsolin-like protein from Papaver rhoeas pollen (PrABP80) stimulates calcium-regulated severing and depolymerization of actin filaments. J Biol TheChem 279:Second 23364–23375 Symposium 2. Fan X, Hou J, Chen X, Chaudhry F, Staiger CJ, Ren H (2004) Identification and characterization of a Ca2+-dependent actin filament-severing protein from lily pollen.on Plant PlantPhysiol 136: 3979–3989 Neurobiology 3. Yokota E, Takahara K, Shimmen T (1998) Actin-bundling protein isolated from pollen tubes of lily. Biochemical and immunocytochemical characterization. Plant Physiol 116: 1421–1429
45 Abstracts for Oral Presentation Session 9
Plant Prevacuolar Compartments: Secretory Trafficking and Endocytosis
Sheung Kwan Lam, Yu Chung Tse, Louse Lo, Junqi Wang, Hong-Ye Li, Yansong Miao and Liwen Jiang
Department of Biology and Molecular Biotechnology Program, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein trafficking in the secretory and endocytic pathways. We have previously demonstrated that vacuolar sorting receptor (VSR) proteins concentrated on PVCs [1] and VSR-marked PVCs in tobacco BY-2 cells were multivesicular bodies (MVBs) [2], which might also involve in receptor-mediated endocytosis because MVBs colocalized with internalized endosomal marker FM4-64 [2-4]. Using transgenic BY-2 cells expressing a GFP fusion involving endocytosis, we have also identified early endosomal compartment in BY-2 cells [5]. To further study PVC biogenesis and PVC-mediated protein traffic, we have purified PVCs from both tobacco BY-2 cells and Arabidopsis cells for proteomics analysis via both 2D gel MS/MS and 1D gel LC-MS/MS [6]. Current studies focus on characterization of newly identified PVC proteins for their subcellular localization and functional roles. Supported by grants from the Research Grants Council of Hong Kong to L Jiang.
1. Li YB, SW Rogers, YC Tse, SW Lo, SSM Sun, GY Jauh and Jiang L (2002) BP-80 and homologs are concentrated on post-Golgi, probable lytic prevacuolar compartments. Plant Cell Physiology 43: 726-742 2. Tse YC, BX Mo, S Hillmer, M Zhao, DG Robinson, SW Lo and Jiang L (2004) Identification of multivesicular bodies as prevacuolar compartments in tobacco BY-2 cells. The Plant Cell 16: 672-693 2. Lam SK, YC Tse, L Jiang, P Oliiusson, L Heinzerling, and Robinson DR (2005) Plant prevacuolar compartments and endocytosis. In: Plant Endocytosis (Edited by J. Samaj, F. Baluska and D. Menzel). Plant Cell Monographs 1: 37-61. The3. Lam SK, SHSecond Jang, GH An and Jiang LSymposium (2005) SCAMPs mediate endocytosis via prevacuolar compartments in transgenic tobacco BY-2 cells. Abstract presented at Plant Biology 2005, Seattle, WA, USA July 16-20, 2005 4. Lam onSK et al. (2006)Plant Identification Neurobiologyof early endosomal compartments in tobacco BY-2 cells (Submitted for publication) 5. Mo BX, YC Tse and Jiang L (2003) Organelle identification and proteomics in plant cells. Trends in Biotechnology 21: 331-332
46 Abstracts for Oral Presentation Session 9
An Important Pool of Sucrose Linked to Starch Biosynthesis is Taken up by Endocytosis in Heterotrophic Cells
Edurne Baroja-Fernandez1, Ed Etxeberria2, Francisco José Muñoz1, María Teresa Morán-Zorzano1, Nora Alonso-Casajús1, Pedro Gonzalez2 and Javier Pozueta-Romero1
1Agrobioteknologia Instituta, Nafarroako Unibertsitate Publikoa, Gobierno de Navarra and Consejo Superior de Investigaciones Científicas, Mutiloako etorbidea zenbaki gabe, 31192 Mutiloabeti, Nafarroa, Spain; 2University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA.
We have recently shown the occurrence of endocytic sucrose uptake in heterotrophic cells (Etxeberria et al. Plant Cell Physiol. 46, 474-481). Whether this mechanism is involved in the sucrose-starch conversion process was investigated by comparing the rates of starch accumulation in sycamore cells cultured in the presence or absence of the endocytic inhibitors wortmannin and LY294002. These analyses revealed a two-phase process involving an initial 120 min wortmannin- and LY294002- insensitive starch accumulation period, followed by a prolonged phase that was arrested by the endocytic inhibitors. Both wortmannin and LY294002 led to a strong reduction of the intracellular levels of both sucrose and the starch precursor molecule, the ADPglucose. No changes in maximum catalytic activities of enzymes closely linked to starch and sucrose metabolism occurred in cells cultured with endocytic inhibitors. In addition, starch accumulation was unaffected by endocytic inhibitors when cells were cultured with glucose. These results provide a first indication that an important pool of sucrose incorporated into the cell is taken up by endocytosis prior to its subsequent conversion into starch in heterotrophic cells. This conclusion was further substantiated by experiments showing that sucrose-starch conversion was strongly prevented by both wortmannin and LY294002 in both potato Thetuber discs and Second developing barley endosperms. Symposium
Etxeberria, E., Baroja-Fernandez, E., Muñoz, F.J. and Pozueta-Romero, J. (2005) Sucroseon inducible Plant endocytosis as a mechNeurobiologyanism for nutrient uptake in heterotrophic plant cells. Plant Cell Physiol. 46: 474-481
47 Abstracts for Oral Presentation Session 9
Multivesicular Compartments “in Action” in Response to Infection by the Barley Powdery Mildew Fungus
Qianli An1*, Ralph Hückelhoven2, Katrin Ehlers1, Karl-Heinz Kogel2, and Aart J. E. van Bel1
1Institute of General Botany, Justus-Liebig-University Giessen, Senckenbergstrasse 17, D-35390 Giessen, Germany 2Institute of Phytopathology and Applied Zoology, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany
The recent finding that Arabidopsis and barley syntaxins known to mediate membrane fusion are required for the papilla-associated penetration resistance to powdery mildew fungi highlights the function of vesicle trafficking in plant–pathogen interactions (Assaad et al., 2004; Collins et al., 2003). Plant resistance to powdery mildew fungi is performed by arrest of fungal penetration attempts through polarized secretion of defense materials and deposition of papillae, by restriction of fungal infection through resistance-gene-mediated hypersensitive cell death response, or a combination of both depending on the host-fungus combination. In barley leaves, light-microscopically visible vesicle-like bodies intensively
stained by H2O2-reactive dyes frequently accumulate around papillae, in which the penetration attempt of barley powdery mildew fungi is halted. By using transmission
electron microscopy in combination with cytochemical localization of H2O2, we
demonstrated that the conspicuous H2O2-containing vesicle-like bodies were actually small papillae instead of cytoplasmic vesicles and that large multivesicular compartments including multivesicular bodies and paramural bodies, some of which
contained H2O2, proliferated near papillae. Moreover, we observed intravacuolar multivesicular bodies with double limiting membranes, of which the outer one was seemingly derived from the tonoplast, and intravacuolar vesicle aggregates, which might result from a degradation of the limiting membranes of the intravacuolar multivesicular bodies (An et al., 2006). These vesicular structures also proliferated at Thethe periphery Secondof the intact cells neighbor Symposiuming the hypersensitive dying cells in the resistant MLA12-barley. All plasmodesmata between intact cells and hypersensitive cells were constricted or blocked by cell wall appositions frequently associated with paramuralon bodies. Plant Neurobiology Together, multivesicular bodies seemingly followed two distinct pathways: either they fused with the plasma membrane to release their internal vesicles into the paramural space or they were engulfed by the tonoplast for degradation in the vacuole. They appeared to secret building blocks for deposition of cell wall appositions to 48 Abstracts for Oral Presentation Session 9
prevent the fungal penetration and to contain the hypersensitive cell death. They may be also involved in internalization of deleterious materials, damaged membranes, elicitors, and elicitor receptors.
1. An Q, Hückelhoven R, Kogel K-H, van Bel AJE (2006) Multivesicular bodies participate in a cell wall associated defense response in barley leaves attacked by the pathogenic powdery mildew fungus. Cell Microbiol doi:10.1111/j.1462-5822.2006.00683.x 2. Assaad FF, Qiu J-L, Youngs H, Ehrhardt D, Zimmerli L, Kalde M, Wanner G, Peck SC, Edwards H, Ramonell K, Somerville CR, Thordal-Christensen H (2004) The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae. Mol Biol Cell 15: 5118–5129 3. Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu J-L, Hückelhoven R, Stein M, Freialdenhoven A, Somerville SC, Schulze-Lefert P (2003) SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425: 973–977
The Second Symposium
on Plant Neurobiology
49 Abstracts for Oral Presentation Session 9
Signal and Assimilate Phloem Translocation: Understand the Controlling Mechanisms of Nutrient Supply and Seed Development in the Economically Important Crops
Hong Li Wang
Department of Biology, University of Arkansas, Little Rock, Arkansas 72204, USA
It would greatly benefit our economics and society if we can develop crops that have higher biomass production which can be used for fuels and fibers; and high yield of grains with better nutritional quality for human and animal feeds. Biomass production of crop is mainly determined by plant’s ability to produce high levels of assimilates over a wide range of environmental conditions, while the crop yield is mainly depending on its capacity to efficiently transport and accumulate a high proportional assimilates into the harvesting organs such as seeds and grains. Phloem translocation of signals and assimilates plays significant roles on controlling of nutrient supply and seed development of economically important crops such as wheat. This work presents our long-term research on the elucidation of the cellular pathways of assimilate transport in the developing wheat grain, and our understanding on the physiological, cellular and molecular bases of phloem translocation in crop plants, especially their underlying controlling and regulatory mechanisms. Our work revealed that the transfer cells which are differentiated from the nucellar projection and the modified aleurone cells in the wheat grains enhance the preferential accumulation of assimilates into the endosperm tissues. Moreover, this mechanism can also be applied to improve nutrient assimilation (mineral nutrient acquisition and biomass production) in the source tissues. Our findings provide novel insights into the manipulation of the genes involved in the controlling points, for the breeding of ideal crops, the optimization of cropping systems and the improvement of agricultural management.
Keywords: Signals, assimilates, phloem translocation, controlling mechanisms, seed Thedevelopment; cropSecond yield, nutritional quality, Symposium transfer cells, wheat.
on Plant Neurobiology
50 Abstracts for Oral Presentation Session 10
Electrophysiological Studies on Mechano-perception and Wounding Response in Green Axon, Characean Cells
Teruo Shimmen
Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1297, Japan
Mechanical and wounding stresses are serious problem for plants. It is suggested that the plasma membrane first perceives such stresses and the electrical signal plays pivotal roles. In higher plants composed of complex tissues, it is hard to monitor signals from target cell. On the other hand, it is easy in Characeae because of its simple morphology. In addition, large cell size makes the electrical measurement easy and simple. Mechano-sensing: By dropping a glass rod on a internodal cell of Chara corallina, receptor potential was induced. The intensity of the stimuli can be simply controlled by either the weight of the glass rod or height from which the glass rod is dropped. Systematic analysis indicated that activation of chloride channel is involved in generation of the receptor potential. In addition, involvement of calcium channel was also indicated. Wounding response: When plants were suffered from wounding, the initial signal should be received by a cell neighboring to the killed cell. In higher plants, it is very hard to selectively record the electrical response of these cells. I prepared a specimen comprising two adjoining internodal cells. One internodal cell (victim cell) was killed by cutting and change of the membrane potential in another internodal cell (receptor cell) was analyzed. Upon cutting a victim cell, a receptor cell generated four kinds of depolarizing response; (1) rapid depolarization, (2) long-lasting depolarization, (3) action potentials and (4) small spikes. Both rapid and long-lasting depolarizations are generated at the distal end and action potential was at the flank region of the cell. At the peak of the long-lasting depolarization, an action potential was induced and transmitted along the internodal cell. Thus, the wounding Thesignal is perceived Second at the nodal end and Symposiumthe long-lasting depolarization is a kind of receptor potential. At the peak of the long-lasting depolarization, action potentials were induced and transmitted along the cell. The role of K+ released from the victim cell in inductionon ofPlant long-lasting depolarization Neurobiology was indicated. Characeae can be an ideal material for analysis of electrical signals in stress response.
51 Abstracts for Oral Presentation Session 10
Brassinosteroids and ABA Regulate Plasma Membrane Anion Channels in Addition to Proton Pumps in Arabidopsis Thaliana Cells
Jean-Pierre Rona, Mathias Brault, AM Pennarun, Zahia Amiar, Michèle Monestiez, Bernadette Biligui, Karine Madiona and Zongshen Zhang
Electrophysiologie des Membranes, EA 3514, Université Paris VII, 2 place Jussieu, 75005 Paris, France
The plant growth regulators 28-homobrassinolide (HBL) and abscisic acid (ABA) play key roles in the control of plant development and cell volume by regulating ion channel activities and water exchanges across the plasma membrane (PM). In Arabidopsis thaliana suspension cells, our results clearly show that both HBL and ABA had opposite effect on the modulation of the proton pump and anion channel activity. These modulations were associated with the control of the PM electrical gradient magnitude involved in phytohormones signaling pathways. Using experiments employing combined voltage clamping and continuous measurement of extracellular pH during PM phytohormone signalings on cells where physiological wall functions are maintained, we demonstrate that HBL induced both medium acidification (ΔpH ≈ 0.45 units in less than 10 min) and PM hyperpolarization (ΔEm ≈ -12 mV), whereas ABA simultaneously induced rapid alkalinization of the medium (ΔpH ≈ 0.06 units) and PM depolarization (ΔEm ≈ 6 mV). These data revealed that the PM H+-ATPase is activated by HBL (Zhang et al. 2005), but inhibited by ABA (Brault et al. 2004) in A. thaliana suspension cells. Upon ABA treatment, we observed an increase in the anion current (anion efflux) in suspension cells (ΔI ≈ 62%). This increase is abolished by a subsequent addition of the anion channel inhibitor 9-AC (ΔI ≈ 17%) or strongly reduced when ABA was added in the presence of 9-AC. In opposite manner, we observed HBL treatment decrease anion current in suspension cells (ΔI ≈ 70%) during the PM hyperpolarization. Therefore, Theanion channels Second may also be good candidate Symposiums, in addition to proton pumps for the controls of PM potential during the responses to phytohormones.
1. Zhangon Z, Ramirez Plant J, Reboutier D, Brault Neurobiology M, Trouverie J, Pennarun AM, Amiar Z, Biligui B, Galagovsky L, Rona JP. (2005) Brassinosteroids Regulate Plasma Membrane Anion Channels in Addition to Proton Pumps During Expansion of Arabidopsis thaliana Cells. Plant Cell Physiol. 46 (9): 1494-1504.
52 Abstracts for Oral Presentation Session 10
2. Brault M, Amiar Z, Pennarun, AM, Monestiez, M, Zhang, Z, Cornel, D, Dellis, O, Knight, H, Bouteau, F. and Rona, JP. (2004) Plasma membrane depolarization induced by abscisic acid in A. thaliana suspension cells involves reduction of proton pumping in addition to anion channel activation, which are both Ca2+ dependent. Plant Physiol. 135:231-243.
The Second Symposium
on Plant Neurobiology
53 Abstracts for Oral Presentation Session 10
On the Track of Ca2+ Permeable Channels
Karina Schulz, Bernd Mueller-Roeber, Barbara Koehler
University of Potsdam, Institute for Biochemistry and Biology, Molecular Biology, Karl-Liebknecht-Str. 25, H. 20, 14476 Golm, Germany
After the complete sequencing of the Arabidopsis genome the functional characterisation of unknown membrane proteins and to find out their physiological roles in the plant is one of the major challenges to date. We focus on the identification of putative plant ion channels involved in Ca2+-based signalling processes. Beyond dispute, Ca2+ is an important, ubiquitous second messenger during development and in responses to various stresses. Although candidate genes like cyclic-nucleotide activated channels and glutamate receptors are possible pathways for Ca2+ entry (Demidchick et al., 2002), the direct link to plant Ca2+ channels like they have been identified on the electrophysiological level (e.g. Hamilton et al., 2000; Köhler and Blatt, 2002; Dreyer et al., 2004) is still lacking. Based on sequence similarity to the conserved pore region of animal calcium channels and on the presence of at least six transmembrane spanning domains candidate genes from several multigene families were cloned. All of the candidate genes are of unknown function and do not belong to the cyclic-nucleotide activated channels or glutamate receptors. Candidate genes were investigated with respect to permeability for Ca2+ and monovalent cations (K+, Na+) in heterologous expression systems. Xenopus oocytes and yeast stably expressing reconstituted aequorin were used. Five of the candidate genes representing five multigene families with 2 to 13 members affected the intracellular Ca2+ homeostasis of yeast. These were investigated further. The knowledge of the subcellular distribution and the expression pattern within the plant is crucial for developing approaches to assign functions. Therefore, GFP-fusion proteins were transiently expressed in tobacco BY2 protoplasts to determine the intracellular localisation. Two of the candidate genes were located in the plasma membrane, two were located in Theendomembranes, Second and one was located in mSymposiumitochondria. Promoter-GUS studies of the candidate genes were done end partly extended to the whole gene family. Some of the genes were expressed throughout the plant. Others showed a very specific expression pattern. Availableon informationPlant from databa Neurobiologyses was compiled. A portrait comprising current knowledge of the selected gene families will be presented.
Demidchick V, Davenport RJ, Tester M (2002) Nonselective cation channels in plants. Annu Rev Plant Biol. 53:67-107 54 Abstracts for Oral Presentation Session 10
Dreyer I, Mueller-Roeber B, Köhler B (2004) Voltage gated ion channels. In: Annual Plant Reviews, Vol. 15, ed. M.R. Blatt, CRC Press Hamilton DWA, Hills A, Köhler B, Blatt MR (2000) Ca2+ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid. PNAS 97: 4967-72 Köhler B, Blatt MR (2002) Protein phosphorylation activates the guard cell Ca2+ channel and is a prerequisite for gating by abscisic acid. Plant J. 32: 185-94
The Second Symposium
on Plant Neurobiology
55 Abstracts for Oral Presentation Session 10
Mechano-perception: Thermodynamics of Mechanosensitive Ion Channels
V. S. Markin, M.I.Volkova-Gugeshashvili, A.G.Volkov.
Department of Chemistry, Oakwood College, 7000 Adventist Blvd. Huntsville, Alabama 35896 USA
Plants respond to external stimuli. This includes mechanical response of plants to mechanical stimuli that occurs via mechanosensory ion channels (MSC). These channels are activated by mechanical stress and then transduce this information into electrical signals. MS channels are involved in the growth, development and response to environmental stress in higher plants. Detailed analyses of the electrophysiology in higher plants are difficult because such plants are composed of complex tissues. The large cells of the charophytes facilitate electrophysiological measurements and allow studying MS ion channels at the level of single cells. This showed that their functioning is based on the same principles as much better studied MS channel in bacterial and animal cells, which are immersed into lipid matrix and are transducing mechanical stress in the membrane. Mechanosensitivity of ion channels are conventionally interpreted as being driven by a change of in-plane area Amsc of mechanosensitive comples. This, however, does not include any factors relating to membrane thickness, spontaneous curvature or changes in channel shape, length or stiffness. Since the open probability of a channel is sensitive to all these factors, we constructed a general thermodynamic formalism. Corresponding equations comprise the basis for the analysis of the behavior of mechanosensitive channels in lipids of different geometric and chemical properties ─ properties such as the hydrophobic mismatch at the boundary between the protein and lipid, and effects of changes in the bilayer intrinsic curvature caused by the adsorption
of amphipaths. This model predicts that the mid-point γ1/2 and the slope1/2 of the gating curve are not generally independent. Using this relationship, we predicted the line tension at the channel/lipid border of MscL as ~ 10 pN, much less than the line tension of aqueous pores in pure lipid membranes. The channel appears quite well matched to its lipid environment. Using gramicidin as a model system, we have explained its observed conversion from stretch-activated to stretch-inactivated gating Theas a function ofSecond bilayer thickness and composition. Symposium We have also identified two types of shape sensitive mechanotransduction: one-sided and two-sided shape activation. These effects are particularly relevant to the activity of amphipaths and to curvature sensitive channels. Electrophysiologyon Plant of mechanosensitive Neurobiology responses of Venus flytrap and Mimosa are analyzed using ultra fast data acquisition PXI system from National Instruments. Touching of mechanosensitive hairs of Venus flytrap induced action potentials between lobes and midrib with duration about 1 ms and speed of propagation about 10 m/s. 56 Abstracts for Oral Presentation Session 11
Properties and Physiological Roles of ROS-activated Cation Channels in Higher Plants
Vadim Demidchik
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO43SQ, United Kingdom
Reactive Oxygen Species (ROS) are involved in such important physiological phenomena in plants as stress responses, growth and development, regulation of stomatal aperture, gravitropism and others. One of mechanisms of ROS action on plant physiological processes is through their effect on plasma membrane cation channels – proteins catalyzing an exchange of cations between the cell and the environment (Demidchik et al., 2002, Annu Rev Plant Biol 53: 67). Here, I present data obtained in collaboration with several laboratories (Cambridge, Plymouth and John Innes Centre) on properties and physiological roles of Ca2+- and K+-permeable cation channels in Arabidopsis thaliana root cells. We showed that application of ROS (hydroxyl radicals) at the extracellular side of the plasma membrane in whole-cell patches caused an activation of inwardly rectifying Ca2+ conductance and outwardly rectifying K+ conductance (Demidchik et al., 2003, J Cell Sci 116: 81). ROS-induced conductances in elongation zone and root hairs were 2-3 times larger than in mature epidermis and 10-20 times larger than in pericycle. This suggested an involvement of ROS-activated cation channels of root epidermal cells to the perception of external stimuli, growth and development. Pharmacological analyses showed that ROS-activated Ca2+ conductance was mediated by a new group of Ca2+-permeable nonselective cation channels having different properties than nonselective cation channels involved in toxic Na+ influx (Demidchik & Tester, 2002; Plant Physiol 128: 379). ROS-activated K+ conductance was mediated by outwardly rectifying K+ channels because it was blocked by TEA+ and showed high selectivity to K+ over other the range of monovalent cations. Single-channel studies in Theoutside-out patches Second demonstrated an activation Symposium of hyperpolarisation-activated 14.5-pS channel (20 mM CaCl2 in the bathing solution) in response to H2O2 applied at the intracellular side of the plasma membrane. Properties of this ROS-activated single channel onresembled Plant properties of constiNeurobiologytutive hyperpolarisation-activated Ca2+ channels. Single-channel analyses of K+ outward conductances are in progress. Using ® MIFE technique we have found that H2O2 and hydroxyl radicals activate significant transient Ca2+ influx of and K+ efflux in intact roots. In elongation zone ROS-activated cation fluxes were larger than in mature epidermis. Experiments with 57 Abstracts for Oral Presentation Session 11
Ca2+-aequorin luminometry showed that extracellular ROS accumulation is induced by abiotic and biotic stresses and that this accumulation is accompanied by the activation Ca2+ influx through ROS-activated cation channels. Using K+ photometry we have found that oxidative stress-induced K+ release from plant tissues is mediated by ROS-activated K+ channels. A critical role of ROS-activated Ca2+ channels in plant elongation growth was demonstrated in experiments with Arabidopsis root hair deficient mutant (rhd2) (Foreman et al., 2003, Nature 422: 442). We have shown that impaired HADPH oxidase activity results in decreased production of extracellular ROS that leads to decreased Ca2+ influx through ROS-activated cation channels and stops Ca2+-dependent elongation of root cells (root hairs and cells of elongation zone). In conclusion, our data show that ROS-activated cation channels are critical systems for sensing environmental stresses and regulation of plant elongation growth.
The Second Symposium
on Plant Neurobiology
58 Abstracts for Oral Presentation Session 11
Myotubularins: Novel Members of Phosphoinositide-Based Signalling Pathways in Plants
Andrej Hlavacka1,2, Dieter Volkmann1, Diedrik Menzel1, Zoya Avramova3, František Baluška1
1 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany 2 LINV, Dip. Horticulture, Viale delle idee 30, 50019 Sesto F.no (FI), Italy 3 School of Biological Sciences, University of Nebraska, Lincoln, NE 68588-0118, USA
Myotubularins are dual-specificity phosphatases that use PI(3,5)P2 or PI(3)P as substrate and convert it into PI(5)P. They have been first identified in humans where the mutation of hMTM1 (human myotubularin 1) causes Charcot-Marie-Tooth syndrome that results into the muscular myopathy disease. By screening the Arabidopsis genome, we have identified 2 myotubularin homologues – AtF18K10 and AtT32M21. Based on bioinformatical studies, we have found, that both of these proteins exhibit similar structure and properties comparing to human ones. Despite the high similarities on the protein level and in the protein structure, these genes have evolved in the different way than the ones from animals and humans. They form a separate clade on the phylogenetical tree and they might also have a different roles.
Plant myotubularins posses a consensus active site Cx5R within the PTP (Protein Tyrosine Phosphatase) domain and a myotubularin-related domain. In the case of AtT32M21, there is also a PH-GRAM domain (domain found in glucosyltransferases, myotubularins and other putative membrane-associated proteins). We have also found a SET-interacting domain (SID) overlaping with PTP domain. This domain is interacting with proteins carrying well conserved SET (SuVar (3–9)-E(z)-trithorax) domain. Recently, we have reported (Alvarez-Venegas et al. 2006) that Arabidopsis trithorax homologue 1 (ATX1), belonging to the Trithorax gene family, contains SET domain. Moreover, we have also found that ATX1 binds specifically PI(5)P and is necessary for the proper plant development and stress response. Arabidopsis Themyotubularins Secondcould form a complex with Symposium ATX1 and provide a substrate for this protein. Therefore, through balancing the level of phosphoinositides, there might regulate the development of the plant body and stress response. on Plant Neurobiology Alvarez-Venegas R, Sadder M, Hlavacka A, Baluška F, Xia Y, Lu G, Firsov A, Sarath G, Moriyama H, Dubrovsky JG, Avramova Z (2006) The Arabidopsis homolog of trithorax, ATX1, binds phosphatidylinositol 5-phosphate, and the two regulate a common set of target genes. Proc Natl Acad Sci USA 103: 6049-6054 59 Abstracts for Oral Presentation Session 11
Transition Zone in the Root Apex: Oxygen Influx and Nitric Oxide Production During Temporary Changes in Gravity Conditions
S. Mancuso1, S. Mugnai1, E. Azzarello1, C.Pandolfi1, A. Hlavacka1,2, B.Voigt1,2, F. Baluska1,2, D. Volkmann2
1LINV (Laboratorio Internazionale Neurobiologia Vegetale), Department of Horticulture, University of Florence, Italy, 2Institut für Zelluläre und Molekulare Botanik, University of Bonn, Germany Email: [email protected]
Oxygen influx changed in the transition zone after varying root position from the vertical to the horizontal on ground, showing a gravity-regulated asymmetry. Specifically, 18 ± 2 sec after changing the root position oxygen influx increased only on the upper root side of the transition zone, remaining stable on the lower one.
Considering that the tilting procedure took around 15 s, the first O2 signal can be hypothesized to appear just few seconds after gravistimulation. This rapid change in the oxygen flux into root apices is by far the fastest ever reported plant response to gravity. In order to study this phenomenon in a real microgravity condition, an experiment has been set up on a parabolic flight. Oxygen and nitric oxide flushes have been monitored during normal, hyper- and microgravity conditions in roots of Zea mays seedlings. During parabolic flight a clear and distinct signal in oxygen and NO fluxes has been detected only in the apex zone, starting just 2.0 ± 0.5 s after the imposition of microgravity conditions. No significant changes have been noticed neither in normal nor in hypergravity conditions. The significance of these results on the nature of the graviperception will be discussed.
The Second Symposium
on Plant Neurobiology
60 Abstracts for Oral Presentation Session 12
Herbivore-Induced Volatiles in Plant Defense: Early and Late Events in Enemy-recognition and Response
Wilhelm Boland, Axel Mithöfer, Gen-Ichiro Arimura, Heiko Vogel, Jürgen Kroymann
Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, Germany Email: [email protected]
Herbivore feeding elicits defence responses in the infested plants, typically the emission of a blend of volatile organic compounds (VOCs) that mediates interactions with the parasites or enemies of the herbivore. To study the impact of individual factors which may contribute to the stimulation of volatile biosynthesis or control the composition of a blend, a mechanical caterpillar (MecWorm) has been engineered, which very closely resembles the herbivore-caused tissue damage in terms of a similar physical appearance and a long lasting wounding period on defined leaf areas. In many plants the mechanical treatment was sufficient to induce a blend of VOCs as known from real herbivore feeding.1 The defence patterns could be modified by addition of salivary secretions from the feeding insect to the wounded leaf, demonstrating that the salivary secretions also have a strong impact on the composition of the blend. This was further established by microarrays comprising the whole genome of A. thaliana. In total about 5000 genes were either up- or down regulated after simple mechanical damage. By Principal Component analysis the different treatments of the leaves of A. thaliana, such as mechanical damage, feeding by a specialized insect (Diamond Back Moth), and a generalist herbivore (Beet Army Worm), could be clearly distinguished by a typical set of differently affected genes. Interestingly, the salivary secretions of the feeding insects seem to silence locally the gene expression in the damaged leaf, compared to the effect of mechanical wounding, but in distant leaves a significant reprogramming occurs that is not observed after the MecWorm treatment.2 TheOther elements Second of damage recognition Symposium and internal signaling comprise membrane depolarization and influx of Ca2+-ions.3 Some of the effects can be induced by low molecular N-acyl glutamines, which are typical and widespread components within salivary onsections ofPlant Lepidopteran larvae. Neurobiology4 The N-acyl glutamines are amphiphilic compounds that cause depolarization and membrane destabilization resulting in a simultaneous up-regulation of the octadecanoid and the salicylate signaling pathways. Depending on the extent of the activation of the signal-transduction routes (jasmonic
61 Abstracts for Oral Presentation Session 12
acid, salicylic acid, ethylene, and Ca2+-influx) and depending on their syn- or antagonistic interaction, the biosynthesis of phytoalexins (volatiles) is either induced, suppressed or modulated.5 The complexity of interactions will be presented at the molecular level; consequences and the impact on plant-insect interactions will be discussed.6
1. Mithöfer, A.; Wanner G., Boland, W. (2005) Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. II. Mechanical Wounding Resembling Insect Feeding is Sufficient to Elicit Herbivory-related Volatile Emission. Plant Physiol., 13, 1-9. 2. Boland, W.; Mithöfer, A.; Vogel, H.; Kroymann, J., manuscript in preparation 3. Maffei, M.; Bossi, S.; Spiteller, D.; Mithöfer, A.; Boland, W. (2004)Effects of feeding Spodoptera littoralis on Lima beans leaves. I. Membrane potentials, intracellular calcium variations, oral secretions, regurgitants components, and signaling molecules. Plant Physiol., 134, 1-11. 4. Pohnert, G.; Jung, V.; Haukioja, E.; Lempa, K.; Boland. W. (1999) New Fatty Acid Amides from Caterpillar (Noctuidae, Geometridae) Regurgitants. Tetrahedron 55, 11275-11280. 5. Arimura, G.-G.; Mithöfer, A.; Maffei, M.; Uchtenhagen, H.; Bossi, S.; Starvaggi-Cucuzza, L.; Garms, S.; Schulze, B.; Leitner, M.; Boland, W.(2005) Synergistic and Antagonistic Cross-talks within Signaling Networks Mediating Herbivory-Induced Terpenoid Formation in Medicago truncatula. submitted. 6. Arimura, G.-I.; Kost, C.; Boland, W. (2005) Ecological networks and signaling in herbivore-induced plant response. Biochim. Biophys. Acta. 1734, 91-111.
The Second Symposium
on Plant Neurobiology
62 Abstracts for Oral Presentation Session 12
Role of Auxin Signaling in Communication among Maize Plants
E. Van Volkenburgh1,*, M. Fellner2, E.D. Ford1
1Biology Department and College of Forest Resources, University of Washington, USA 2Department of Cell Biology and Genetics, Palacky University in Olomouc, Czech Republic
Plants sense each other by communicating via changes in the ratio of red to far-red light (R:FR ratio) inside the canopy. In response to crowding, and thus warnings of competition from others, shade-avoiding plants invoke a syndrome of responses including stimulation of elongation growth, reduced branching, and a redistribution of leaves to the top of canopy (shade avoidance responses). It was proposed that extension growth induced by neighbor detection and shade is the result of R:FR-regulated auxin distribution (Morelli and Ruberti 2000). Plant breeders, when selecting corn (Zea mays L.) plants with high yield in dense plantings, created hybrids with reduced sensitivity to neighbors. We have tested the possibility that the physiological consequence of the selection involves changes in responsiveness to light and auxin (see also Fellner et al. 2003). Etiolated seedlings of a hybrid originally released in the 1930’s (307) elongated significantly more than seedlings of a modern hybrid released in the 1990’s (3394). The level of endogenous auxin and activity of polar auxin transport (PAT) were similar in both genotypes. The 1990’s hybrid shows resistance to auxin- and light-induced responses at the seedling, cell, and molecular levels. Intact 3394 plants exhibited less responsiveness to the inhibitory effect of R, FR, and W, auxin, antiauxin, and inhibitors of PAT. Excised mesocotyl tissue from 3394 seedlings also showed low responsiveness to NAA. Cells of 3394 were insensitive to auxin- and light-induced hyperpolarization of the plasma membrane. Expression of ABP4 was much less in 3394 than in 307, and in contrast Theto 307, it was Second not up-regulated by NAA, Symposium R and FR. Preliminary analysis of abp mutants suggests that ABPs may be involved in development of leaf angle in corn. Corn breeders inadvertently selected hybrids for reduced sensitivity to neighbors. Modern onplants have Plant reduced sensitivity Neurobiology to auxin, not to light. By analogy they are blind to each other, not because their eyes don’t see, but because their brains don’t process the image. Instead of having a signaling system responsive to changes in R:FR, these plants appear to be ‘hard-wired’ in a shade-avoidant posture with upright leaves. One consequence of the more vertical canopy architecture is that modern
63 Abstracts for Oral Presentation Session 12
hybrids shade themselves and each other less, allowing more light to power photosynthesis in the leaf subtending the developing ear, thus generating higher yield.
1. Fellner M, Horton LA, Cocke AE, Stephens NR, Ford ED, and Van Volkenburgh E (2003) Light interacts with auxin during leaf elongation and leaf angle development in young corn seedlings. Planta 216, 366-376. 2. Morelli G, Ruberti I (2000) Shade avoidance responses. Driving auxin along lateral routes. Plant Physiology 122, 621-626.
The Second Symposium
on Plant Neurobiology
64 Abstracts for Oral Presentation Session 12
Compatible Solutes as Regulatory Agents in Plant Adaptive Responses to Environment
Tracey Cuin, Zhonghua Chen and Sergey Shabala
School Agricultural Science, University of Tasmania, Private bag 54, Hobart 7001, Tasmania, Australia
When confronted with a hostile environment (such as salinity, drought or low temperatures), plants respond with a significant elevation in the level of compatible solutes in the cytosol, ameliorating the detrimental effects of stress on cell metabolism [1]. Mechanisms of such amelioration are not fully understood. It has been previously suggested that the functions of compatible solutes are not likely to be limited by conventional osmoprotection, but may also include a regulatory role in adjusting metabolic pathways to altered environmental conditions [2]. Physiological assessment of over 70 barley cultivars in our laboratory has suggested that the difference between salt-sensitive and salt-tolerant barley cultivars was conferred essentially by their ability to retain K+ and minimise the magnitude of NaCl-induced K+ efflux [3]. At the same time, salt-tolerant cultivars showed attenuated K+ efflux responses to a hydroxyl radical (OH•) -generating Cu2+/ascorbate (Cu/a) mixture. Therefore, it was hypothesised that one function of compatible solutes in stress-induced responses is in scavenging ROS and maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell. This hypothesis was investigated using the non-invasive MIFE ion flux measuring technique. In both barley and Arabidopsis plants, low (0.5 to 5 mM) concentrations of exogenously supplied proline or glycine betaine significantly reduced NaCl-induced K+ efflux from plant roots in a dose-response manner. The above mitigating effect was instantaneous, implying that large intracellular concentrations of compatible solutes are not required for an amelioratory role in salt tolerance. Exogenously supplied betaine also significantly enhanced NaCl-induced H+ efflux, but only in preincubated roots, implying some Thealternative mechanism Second of regulation. Sap KSymposium+ and Na+ analysis and membrane potential measurements are also consistent with the model that one function of compatible solutes is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, possibly through the enhanced activity of H+-ATPase, on Plant Neurobiology+ controlling voltage-dependent outward-rectifying K channels and creating the electrochemical gradient necessary for secondary ion transport processes. Root preincubation in low concentrations of compatible solutes also significantly reduced the extent of the OH•-induced potassium efflux. Importantly, such reduction was 65 Abstracts for Oral Presentation Session 12
found not only for osmolytes for which a role is free-radical scavenging has been demonstrated in vitro experiments, but also for compounds thus far not shown to act as free-radical scavengers. This indicates that compatible solutes must play some other (regulatory) roles in addition to free-radical scavenging, in mitigating the damaging effects of oxidative stress. Overall, these data provide the first direct evidence for regulation of ion fluxes across the plasma membrane by physiologically relevant low concentrations of compatible solutes. This may be important for understanding the mechanisms of stress tolerance in plants and development of salt-tolerant crop species.
1. Bray EA (1997) Trends Plant Sci 2: 48-54 2. Bonhert HJ, Sheveleva E (1998) Curr Opin Plant Biol 1: 267-274 3. Chen Z, Newman I, Zhou M, Mendham N, Zhang G, Shabala S (2005) Plant Cell Environ 28: 1230-1246
The Second Symposium
on Plant Neurobiology
66 Abstracts for Oral Presentation Session 12
Plants as Environmental Biosensors
Maya I. Volkova-Gugeshashvili1, Alexander G. Volkov1 and Vladislav S. Markin2
1Department of Chemistry, Oakwood College, 7000 Adventist Blvd., Huntsville, AL 35896, USA 2Department of Anesthesiology UT Southwestern Medical Center, Dallas, TX 75390-9068
Plants are continuously exposed to a wide variety of perturbations including variation of temperature and/or light, mechanical forces, gravity, air and soil pollution, drought, deficiency or surplus of nutrients, attacks by insects and pathogens, etc., and hence, it is essential for all plants to have survival sensory mechanisms against such perturbations. As a consequence, plants generate various types of intracellular and intercellular electrical signals mostly in the form of action potentials or variation potentials in response to these environmental changes (1-2). However, over a long period, only certain plants with rapid and highly noticeable responses to environmental stresses have received much attention from plant scientists. Of particular interest to our recent studies on ultra fast action potential in green plants, we discuss in this review the possibility of utilizing green plants as fast biosensors for molecular recognition of the direction of light, monitoring the environment, and detecting the insect attacks as well as the effects of pesticides and defoliants.
1. Volkov A G (Ed.) (2006) Plant Electrophysiology. Springer, Berlin, New York 2. Ksenzhek O S, Volkov A G (1998) Plant Energetics. Academic Press, San Diego
The Second Symposium
on Plant Neurobiology
67 Abstracts for Oral Presentation Session 12
Spatially Regulated Changes in Proton Pumping, Accumulation of Wall Phenolics, Wall Extensibility and Growth Along the Elongation Zone of Water Stressed Maize Roots
Peter M Neumann and 1Ling Fan
Plant physiology lab, Dept of water, environmental and agricultural engineering, Faculty of Civil and Environmental Engineering, Technion IIT, Haifa 32000, Israel 1Institute of Nuclear and Biological Technology, Xinjiang Academy of Agricultural Sciences, Urumqi 830000, China
Accelerating segmental growth rates from 0-3 mm behind the tips of intact maize (Zea mays L) seedling primary roots were well maintained under a 48 h water deficit and correlated with outward flux of protons (Fan and Neumann, 2004). However, segmental growth rates, proton flux and wall mechanical-extensibility decreased progressively 3 to 9 mm behind the tip and more so under water deficit. Since exogenous acidification did not reverse these decreases, additional growth-regulatory factors were involved. The root expression of 2 gene transcripts involved in lignin biosynthesis, cinnamoyl-CoA reductase 1 and 2, increased after only 1 h of water deficit and before the onset of decreases in wall extensibility. We therefore investigated the possibility that spatially-localized increases in deposition of wall-linked phenolic compounds such as lignins and ferulic esters might stiffen the expanding cell-walls and cause irreversible deceleration of root growth. Progressive increases in wall-linked phenolics were detected by comparing Fourier transform IR-spectra and UV-fluorescence images of isolated cell walls from control and water–stressed roots at 0 to 3, 3 to 6 and 6 to 9 mm behind the tip. The increases in UV fluorescence and lignin-staining induced under water deficit co-located to cell walls of vascular tissues in the stele. Longitudinal bisection of the elongation zone resulted in inward curvature, indicating that the inner stelar tissues were rate-limiting for growth (Fan et al., 2006). We suggest that spatially-localized wall stiffening and Thedeceleration ofSecond growth in the basal elongati Symposiumon zone involves regulated increases in the deposition of wall-bound phenolics. These changes may facilitate acclimation to water deficit by diverting resources to the tip meristem. They could be mediated by gradientson of growth Plant regulatory signals orNeurobiology of cell responsiveness to such signals.
Fan L, Neumann PM (2004) Plant Physiology 135:2291-2300 Fan L et al. (2006) Plant Physiology 140: 603-612
68 Abstracts for Oral Presentation Session 13
Plant Electrophysiology: Effects of Ion Channel Blockers on Signal Transduction in Green Plants
Alexander G. Volkov1,*, Vladislav S. Markin2, Maia I. Volkova-Gugeshashvili1
1Department of Chemistry, Oakwood College, Huntsville, AL 35896 2Department of Anesthesiology UT Southwestern Medical Center, Dallas, TX 75390-9068
Ion transport plays a fundamental role in many biophysical processes in plant cells, including the generation of cell turgor, energy, signal transduction, and metabolite distribution. Ion channels also play an important role in the signal transduction in higher plants. Action potentials in higher plants may be the information carriers in intercellular and intracellular communication in the presence of environmental challenges [1-3]. Action potentials take an active part in the expedient character of response reactions of plants. These impulses transfer a signal about the changes of conditions in a conducting bundle of a plant. Impulses travel in either direction, from the root system to the point of growth and or from the point of growth to the root system [2]. The response reactions of plant tissues and organs can be local or transmitted from cell to cell over long distances. The transfer of excitation has a complicated character accompanied by an internal change in cells and tissues. Inhibition of Ca2+ channels blocks propagation of action potentials induced by electrical stimulus or phototropism. We have found that tetraethylammonium chloride, a potassium channel inhibitor, blocks the propagation of action potentials induced by phototropism, thermal or mechanical stresses in soybean plants. (This work was supported by NASA Grant NAG8-1888.)
1. Volkov A G (Ed.) (2006) Plant Electrophysiology. Springer, Berlin, New York 2. Ksenzhek O S, Volkov A G (1998) Plant Energetics. Academic Press, San Diego 3. Volkov A G, Dunkley T, Labady A, Brown C (2005) Phototropism and electrified Theinterfaces Second in green plants. Electrochim Symposium Acta 50: 4241-4247
on Plant Neurobiology
69 Abstracts for Oral Presentation Session 13
Can Plants Discover Neighboring Plants by Volatile Signalling?
Velemir Ninkovic, Robert Glinwood, and Jan Pettersson
Swedish University of Agricultural Sciences, Department of Entomology, Box 7044, SE-750 07 Uppsala, Sweden
We have introduced the term ‘allelobiosis’ to describe a type of plant-plant interaction via chemicals (Pettersson et al. 2003; Ninkovic et al. 2006). The three key aspects of our definition of allelobiosis are (1) the interaction occurs between undamaged plants, (2) the interaction may be beneficial for the receiving plant and (3) the responses of the receiving plant affect organisms at other trophic levels. Aspect (1) distinguishes allelobiosis from plant-damage/stress signalling, in which chemicals are released by infected/infested plants, while aspect (3) distinguishes allelobiosis from allelopathy. We have studied a model system consisting of different cultivated and wild genotypes of barley (Hordeum vulgare spp.), aphids, and aphid natural enemies. Data on the temporal dynamics of plant responses volatile cues, in terms of aphid acceptance and leaf temperature, show major differences between allelobiosis-related volatiles and stress-related signals such as methyl salicylate and methyl jasmonate. Furthermore, allelobiosis interactions in Hordeum are genotype-dependent, and are manifested only in certain combinations of inducing and responding genotypes. Certain Hordeum genotypes that are already resistant to aphids show further significant reductions in aphid growth rate after exposure to volatiles from certain other genotypes. Changes in the pattern of biomass allocation in responding barley plants suggest that responses may be beneficial in the context of potential competition with neighbouring plants (Ninkovic 2003). The effects of allelobiosis are apparent over three trophic levels; certain barley cultivars become more attractive to the natural Theenemies of aphids Second after exposure to volat ilesSymposium from certain other barley cultivars. We believe that allelobiosis represents a mechanism by which certain barley genotypes can detect potential competition from neighbouring plants. The changes in plant statuson induced Plant by allelobiosis affect Neurobiology the interaction of the plant with both aphid herbivores and their natural enemies, and may therefore be ecologically important.
Ninkovic V., Glinwood, R. & Pettersson, J.(2006) Communication between undamaged plants by volatiles: the role of allelobiosis. In: Baluška, F., Mancusko 70 Abstracts for Oral Presentation Session 13
S, & Volkmann, D. (eds) Communication in plants neuronal aspects of plant life. Springer-Verlag Berlin Heidelberg, p. 411-434. Pettersson, J., Ninkovic, V. & Glinwood, R. (2003) Plant activation of barley by intercropped conspecifics and weeds: allelobiosis. BCPC Crop Science and Technology, 2, 1135-1144. Ninkovic, V. (2003) Volatile communication between barley plants affects biomass allocation. Journal of Experimental Botany, 54, 1931-1939.
The Second Symposium
on Plant Neurobiology
71 Abstracts for Oral Presentation Session 13
External Glutamate Modifies Root Growth in Arabidopsis
Pia Walch-Liu, Tony Remans and Brian Forde
Biological Sciences, Lancaster University, Bailrigg, Lancaster LA1 4YQ, United Kingdom
The sessile nature of plants demands a highly sophisticated sensory system monitoring environmental changes and an extreme flexible physiological and physical plasticity in order to respond to this changes efficiently. Plants are known to forage for localised supplies of nitrate by proliferating their lateral roots within nitrate-rich patches. There is increasing evidence that amino acids can be an important source of N for many plant species. Is it possible that roots also possess sensory mechanisms that enable them to more efficiently exploit heterogenous supplies of organic N? Studies with Arabidopsis have shown that exogenous L-glutamate (L-Glu), one of the most abundant forms of soluble organic N in the soil, is able to elicit complex changes in root architecture. L-glutamate, at µM concentrations, is perceived at the primary root tip in a stereospecific manner and inhibits mitotic activity in the root apical meristem. Surprisingly, mitotic activity in the developing lateral root is insensitive to L-Glu, but lateral roots acquire L-Glu sensitivity later in their development. The plant’s sensitivity to L-Glu is under the influence of a number of environmental factors (such as light intensity) and different ecotypes of Arabidopsis differ markedly in their L-Glu sensitivity. We have used populations of recombinant inbred lines (Ler x Col-0, C24 x Col-0), together with near isogenic lines (NILs), to identify major QTLs for L-Glu sensitivity to chromosomes 2, 3 and 5. A screen of a fast neutron bombardment population has yielded a number of mutants showing altered L-Glu sensitivity and the deletions responsible are being mapped. Through this combination of approaches we aim to elucidate the mechanism of L-Glu sensing in the Arabidopsis root tip.
The Second Symposium
on Plant Neurobiology
72 Abstracts for Oral Presentation Session 13
A 14-AA Peptide Derived from CLV3 is Sufficient to Regulate the Stem Cell Population in Arabidopsis
Martijn Fiers* and Chun-Ming Liu+
*Plant Research International, Wageningen, The Netherlands; +Center for Signal Transduction & Metabolomics, Institute of Botany, +Chinese Academy of Sciences, China
Stem cells positioned in the central zone of the plant shoot apical meristem (SAM) are the source of totipotent cells, which give rise continuously to new organs post-embryonically. These slow-dividing cells maintain simultaneously two antagonistic events, cell proliferation and cell differentiation in a similar manner to animal stem cells. The determination of the fate of the progeny cells is made by a population-based mechanism in which signals from neighboring cells play the most important role. Genetic experiments have shown that, as part of a feedback regulatory loop, the stem cell-promoting transcription factor WUSCHEL (WUS), which is expressed in the stem cell organizing center (OC), provides a positive signal to maintain an undifferentiated state; while CLAVATA3 (CLV3) interacts with the underlying CLV1/CLV2 receptor complex to act as a negative signal to restrict the number of stem cells. Previous work suggested that CLV3 encodes a mobile ligand that acts in a non-cell autonomous fashion for intercellular communication. CLV3 belongs to a family of small proteins, named CLV3/ESR (CLE), found in plants and parasitic nematodes. They share an N-terminal secretion signal (SS) and a conserved 14-amino acid (AA) CLE motif at or near their C-termini. Over-expression of several CLE genes, such as CLV3, CLE19 and CLE40 from Arabidopsis and SYV46 from nematode, causes a termination of the root meristem (Casamitjana-Martínez et al., 2003; Fiers et al., 2004). Recently we showed that CLE19p and CLE40p, peptides corresponding to the 14-amino acid CLV3/ESR (CLE) motif of CLV3 and CLE40, respectively, are able to mimic the over-expression phenotype of the corresponding genes, triggering Thethe consumption Second of root meristem in vitroSymposium (Fiers et al., 2005). Through deletion analyses, we demonstrate further that the CLE motif of CLV3, together with its secretion signal, is sufficient to complement clv3-2 defects. A synthetic peptide, CLV3p, oncorresponding Plant to this motif, is ableNeurobiology to restrict the size of the SAM in vitro in a CLV1-dependent manner. Peptides derived from CLE40, CLE5, CLE19 and CLE22 confer a various degree of complementation, as reported before in transgenic assay. Application of CLV3p restricts the expression of the stem-cell promoting
73 Abstracts for Oral Presentation Session 13
transcription factor WUSCHEL (WUS) in clv3-2. We thus propose that the CLE motif is the functional cue of CLV3, and the mode of action in different CLEs may be the same.
M. Fiers, E. Golemiec, J. Xu, L. van der Geest, R. Heidstra, W. Stiekema, and C.M. Liu (2005) The 14-Amino Acid CLV3, CLE19 and CLE40 Peptides Trigger Consumption of the Root Meristem in Arabidopsis through a CLAVATA2-Dependent Pathway. Plant Cell 17: 2542-2553. M. Fiers, G. Hause, K. Boutilier, E. Casamitjana-Marinez, D. Weijers, R. Offringa, L. van der Geest, M. van Lookeren Campagne, CM. Liu (2004) Mis-expression of the CLV3/ESR-like gene CLE19 in Arabidopsis leads to a consumption of root meristem. Gene, 327:37-49. E. Casamitjana-Martínez, H. F. Hofhuis, J. Xu, C.M. Liu, R. Heidstra and B. Scheres (2003) Root-Specific CLE19 Overexpression and the sol1/2: Suppressors Implicate a CLV-like Pathway in the Control of Arabidopsis Root Meristem. Current Biology, 13: 1435-1441.
The Second Symposium
on Plant Neurobiology
74 Abstracts for Poster Presentation
Sugar Signaling to Cytoskeleton
Agnieszka K. Banas, Weronika Krzeszowiec, Halina Gabrys
Department of Plant Physiology and Biochemistry, Faculty of Biotechnology, Jagiellonian
University, Kraków, Poland
Besides being a source of carbon skeletons sugars may also act as signal molecules. They are involved in the regulation of gene expression, enzyme activity and numerous cellular processes. Hexokinase is the only hexose sensor identified so far in plant cells. In the presence of glucose, it has been shown to induce microfilament depolymerization in vitro. Abundant evidence points to interactions between sugars/hexokinase and actin in vivo in animal cells. In contrast, very little is known about the influence of sugars on plant cytoskeleton. Glucose, sucrose and mannose have been shown to inhibit light-induced chloroplast movements in higher plants. These intracellular movements are based on actin cytoskeleton. The aim of this study was to investigate the effect of exogenously delivered sugars on actin cytoskeleton in leaf mesophyll cells of Arabidopsis thaliana and Nicotiana tabacum. Detached leaves were incubated on sugar-enriched agar plates for 2 days. Influence of glucose, sucrose and mannose on microfilaments was tested. Neither glucose nor sucrose affected the microfilament architecture in Arabidopsis. In contrast, mannose caused a complete destruction of actin cytoskeleton, reducing a branched network to fluorescent foci within 24 h. Interestingly, Nicotiana mesophyll cells turned out to be insensitive to mannose. This insensitivity was probably connected with the presence of phosphomannose isomerase in tobacco cells. Mannose is widely used as a selection marker for transformation of plants lacking enzymes responsible for the metabolism of mannose-6-phosphate. Exposure to this hexose is linked with DNA fragmentation and release of cytochrome c from mitochondria in Arabidospsis roots and in the maize suspension culture. Both responses are known as features of programmed cell death. TheHowever, no SecondDNA laddering was observed Symposium in Arabidopsis leaves exposed to mannose.
on Plant Neurobiology
75 Abstracts for Poster Presentation
Regulation of Starch Composition in the ArabidopsisLeaves by Pad1 and Pad2 Proteins
Chao Chen, Wei Cheng, Xiaojin Zhou and Yingdian Wang*
Laboratory of Plant Development Physiology and Molecular Biology, College of Life Sciences, Beijing Normal University, Beijing 100875, China *Corresponding author: Yingdian Wang (Tel: +86-10-58808195; Fax: +86-10-58809077; E-mail: [email protected])
Starch is the main component of the maize kernel, which is used for hundreds of food and nonfood products. Amylose extender (ae) of rice and maize are controlled by SBEIIb deficient. The ae mutants of rice have decreased levels of amylopectin chains (DP≦17), increased levels of intermediate and long amylopectin chains. To understand the molecular basis for the variations of the amylose contents, we examined transcripts for key starch branching enzyme (SBEIIb). SBEIIb functions as a critical master regulator in starch composition show low transcription in high amylose content maize plant, but high transcription in low amylose content maize plant. In the present study, using the yeast two-hybrid system, a protein named Zmpa4 that similar with Arabidopsis pad1 and pad2 was identified as a ZmSBEIIb-binding protein. Invitro specific interaction between SBEIIb and Zmpa4 was confirmed by E. coli and yeast expression products. When overexpressed in E. coli cells, the GST-Zmpa4 could co-precipitate with ZmSBEIIb from yeast proteins. This interaction requires the motif 1 and 2 of ZmSBEIIb and the C-terminus of Zmpa4. Plant lines carrying T-DNA insertions that disrupt the Arabidopsis pad1 or pad2 exhibited the altered starch composition in the leaves. The ZmSBEIIb interaction proteins identified in this screen are new targets for studies of starch metabolism in higher plants.
Key words: amylopectin, amylose, protein-protein interactions, starch branching Theenzyme, alph 4Second subunit of 20s proteasome. Symposium
on Plant Neurobiology
76 Abstracts for Poster Presentation
RACK1 Mediates Multiple Hormone Responsiveness and Developmental Processes in Arabidopsis
1,2* 2,7 4 Jin-Gui Chen , Hemayet Ullah , Brenda Temple , Jiansheng 2,6 1 5,8 5 Liang , Jianjun Guo , José M. Alonso , Joseph R. Ecker , and Alan 2,3 M. Jones
1 Department of Botany, University of British Columbia, Vancouver, British Columbia, V6T 1Z4 Canada 2 Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA 3 Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA 4 Structural BioInformatics Core Facility, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA 5 Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA 6 College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, P.R. China 7 Present address: Department of Biology, Howard University, Washington, DC 20059, USA 8 Present address: Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695, USA
E-mail: [email protected]
The scaffold protein RACK1 (Receptor for Activated C Kinase 1) serves as an integrative point for diverse signal transduction pathways. The Arabidopsis genome contains three RACK1 orthologs, however, little is known about their functions. We report here that one member of this gene family, RACK1A, previously identified as the Arabidopsis homolog of the tobacco arcA gene, mediates hormone responses and plays a regulatory role in multiple developmental processes. RACK1A expresses Theubiquitously inSecond Arabidopsis. Loss-of-functi Symposiumon mutations in RACK1A confer defects in multiple developmental processes including seed germination, leaf production, and flowering. rack1a mutants displayed reduced sensitivity to gibberellin and brassinosteroidon in Plantseed germination, Neurobiologyhypersensitivity to abscisic acid in seed germination and early seedling development, and hyposensitivity to auxin in adventitious and lateral root formation. These results provide the first genetic evidence that RACK1A may have a regulatory role in diverse signal transduction pathways.
77 Abstracts for Poster Presentation
Disruption of Actin Filaments by Latrunculin B Affects Cell Wall Construction in Picea meyeri Pollen Tube Via Vesicle Trafficking Disturbance
Tong Chen1,2, Nianjun Teng1,2, Xiaoqin Wu1,2, Yuhua Wang1,2, Wei Tang3, Jozef Šamaj4,5, František Baluška4,6 and Jinxing Lin1*
1 Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 2 Graduate School of Chinese Academy of Sciences, Beijing 100049, China 3 Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC27858-4353, USA 4Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany 5Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, SK-95007, Nitra, Slovak Republic 6Institute of Botany, Slovak Academy of Sciences, Dubravska 14, SK-84223, Bratislava, Slovak Republic
*E-mail: [email protected]
The involvement of actin filaments (AFs) in vesicle trafficking, cell wall construction and tip growth were investigated during pollen tube development of Picea meyeri. Pollen germination and tube elongation were inhibited in a dose-dependent manner by the latrunculin B treatment. The fine actin filaments (AFs) were broken down into disorganized fragments showing a tendency of aggregation. FM4-64 labelling revealed that the dynamic balance of vesicle trafficking was perturbed due to F-actin disruption and the fountain-like cytoplasmic pattern changed into disorganized Brownian movement. The configuration and/or distribution of cell wall components, such as pectins, callose, cellulose as well as arabinogalactan proteins also dramatically changed after the LATB application. FTIR analysis further established significant changes in the chemical composition of the wall material. Our Theresults indicated Second that depolymerization ofSymposium actin filaments affects the distribution and configuration of cell wall components in Picea meyeri pollen tube via vesicle trafficking disturbance. on Plant Neurobiology
78 Abstracts for Poster Presentation
Characterization of Differentially Expressed Pollen Tube Proteins: Towards Understanding of the Regulation of Ca2+-Calmodulin in Pollen Tube Development in Conifer
Tong Chen1, Xiaoqin Wu1, František Baluška2,3, Jozef Šamaj2,4, Jinxing Lin1*
1 Key Laboratory of Photosynthesis and Molecular Environmental Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 2Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany 3Institute of Botany, Slovak Academy of Sciences, Dubravska 14, SK-84223, Bratislava, Slovak Republic 4Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, SK-95007, Nitra, Slovak Republic
*E-mail: [email protected]
We investigated the roles of Ca2+-CaM signaling pathway in the development of pollen tube of Picea meyeri Rehd. et Wils., using calmodulin antagonist trifluoperazine (TFP) under quantitatively controlled conditions. Proteomic approach was employed to analyze protein expression profile changes during pollen germination and subsequent tube growth after inhibition of calmodulin functions. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed over 800 protein spots. A total of 76 of these were reproducibly differentially displayed at different hours with varying doses of TFP, and 57 differentially expressed proteins were identified by tandem mass spectrometry. These proteins were grouped into distinct functional categories including carbohydrate and energy metabolism, signaling, cell expansion, defense and stress response, etc. Moreover, perturbation of Ca2+-calmodulin signaling dissipated the tip-focused 2+ [Ca ]c gradient and dramatically increased calcium concentration in the cytoplasm. TheMorphology Secondof mitochondria, Golgi stacks Symposium along with a differential expression of proteins involved in their functions was also affected. In the meantime, the patterns of endocytosis/exocytosis and cell wall construction were obviously changed after inhibitoron application. Plant These findings Neurobiologyprovide new insights into the sophisticate mechanism of calmodulin functions in pollen tube development and its interaction with energy-producing pathways, signaling and cell expansion machinery.
79 Abstracts for Poster Presentation
Cloning and Expression Analysis of the Hexokinase Family in the Developing Spikelets of Rice (Oryza sativa L.)
Wei Cheng, Xiaojin Zhou, Jie Li, Chao Chen, Yingdian Wang*
Laboratory of Plant Development Physiology and Molecular Biology, College of Life Sciences, Beijing Normal University, 19 XinJieKouWai Avenue, Beijing 100875, China *Corresponding author: Yingdian Wang (Tel: +86-10-58808195; Fax: +86-10-58809077; E-mail: [email protected])
The carbon metabolism and sugar signal transduction is critical in the plant growth and development processes. Plant hexokinase not only catalyzes the production of hexose 6-phosphates but also acts as a sugar sensor plays an important role in sugar sensing and signaling (Jang et al., 1997; Coruzzi and Bush, 2001; Moore et al., 2003). To identify the hexokinase gene family in rice, we searched the databases of both the whole genome and full-length cDNAs of rice carried out with the known OsHXK I and OsHXK II sequences, and isolated eight new rice hexokinase cDNAs designated OsHXK1-OsHXK8 (AY884164-AY884171). The rice hexokinase gene family is to be comprised by those ten genes. All OsHXKs have a highly conserved genomic structure consisting of nine exons, except for OsHXK1 with single exons. Those genes mapped to one of three chromosomes: OsHXK2, OsHXK4, OsHXK5 and OsHXK8 to chromosome 1; OsHXK3, OsHXK6, OsHXK7 and OsHXK I to chromosome 5; OsHXK1 OsHXK II to chromosome 7. The deduced proteins of obtained HXK genes were predicted to contain adenosine phosphate binding sites and substrate recognition sequences, which were highly conserved compared with known HXKs, suggesting the ten OsHXKs are likely to encode functional HXKs. The predicted molecular mass of those OsHXKs are much closer in size, except OsHXK4 and OsHXK6. Isoelectirc point values were calculated to be ranged from 4.81 to 6.77. Multi-sequence alignment analysis of hexokinase proteins in rice and other species suggested that all Theplant hexokinases Second are to be grouped into fourSymposium classes. The transcript levels of the HXK family were confirmed in several organs and developing spikelets in rice. Different expression patterns of OsHXKs were observed at the differenton stages Plant of plant development. Neurobiology The expression results of OsHXKs in different tissues suggest that those ten HXKs may possess diverse physiological functions in different tissues, whereas the function of specific HXKs in metabolism and sugar sensing during rice caryopsis development still need to be further clarified.
80 Abstracts for Poster Presentation
Jang, J.-C., Leon, P., Zhou L., and Sheen, J. (1997) Hexokinase as a sugar sensor in higher plants, Plant Cell 9 ,5 -19. Coruzzi, G.M., and Bush, D.R. (2001) Nitrogen and carbon nutrient and metabolite signaling in plants. Plant Physiol. 125, 61-64. Moore, B., Zhou, L., Rolland, F., Hall, Q., Cheng, W.H., Liu,Y.X., Hwang, I., Jones, T. and Sheen, J. (2003). Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling. Science 300, 332–336.
The Second Symposium
on Plant Neurobiology
81 Abstracts for Poster Presentation
Fluid Phase Endocytotic Uptake of Artificial Nano-Spheres and Fluorescent Quantum Dots by Sycamore Cultured Cells: Evidence for the Distribution of Solutes to Different Intracellular Compartments
Ed Etxeberria1,* Pedro Gonzalez1, Edurne Baroja-Fernandez2 and Javier Pozueta-Romero2
1 University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA, Department of Horticultural Sciences, 2 Agrobioteknologia Instituta, Consejo Superior de Investigaciones Cientificas and Nafarroako Unibertsitate Publikoa, Mutiloako etorbidea zembaki gabe, 31192 Mutiloabeti, Nafarroa, Spain;
E-mail: [email protected]; [email protected] Fluid phase endocytic uptake of external solutes in plant cells was further substantiated using artificial polystyrene nano-spheres (40 nm) and CdSe/ZnS quantum dots (20 nm). Both types of artificial nano-particles were taken up by sycamore cultured cells. However, whereas polystyrene nano-spheres were delivered to the central vacuole, CdSe/ZnS nano-dots were sequestered into cytoplasmic vesicular structures. Using dextran-Texas Red (mw, 3,000; d-TR) as additional marker, confocal micrographs confirmed the distinct topographic distribution of CdSe/ZnS quantum dots within the cell. Initially, d-TR and CdSe/ZnS quantum dots co-localized within cytoplasmic vesicles. After 18 h incubation, d-TR was distinctly localized in the vacuole whereas CdSe/ZnS quantum dots remained sequestered in cytoplasmic membranous compartments. The data provide a first evidence for the rapid distribution of solutes taken up by endocytosis to distinctive intracellular compartments.
The Second Symposium
on Plant Neurobiology
82 Abstracts for Poster Presentation
Short-term Systemic Reactions of Photosynthetic Apparatus of Tobacco Plants to Local Burning in Relation to Propagating of Electrical Signal and Accumulation of Signaling Molecules in the Distant Leaves
Vladimíra Hlaváčková1,*, Pavel Krchňák1, Martin Kubala1, Jan Nauš1, Ondřej Novák2, Miroslav Strnad2
1 Laboratory of Biophysics, Department of Experimental Physics, Palacký University, tř. Svobody 26, 771 46 Olomouc, Czech Republic 2 Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71 Olomouc, Czech Republic
*E-mail: [email protected]
Local burning events have been shown to induce short-term changes in photosynthetic parameters of distal leaves in various plants that appear to be mediated by electrical signals. Longer-term changes (within hours) in hormone and heat-shock proteins levels have also been reported. However, the mechanisms whereby electrical and/or chemical signals induce the short-term photosynthetic (or heat-shock protein) responses are largely unknown. We studied short-term (up to 1 h) systemic responses of tobacco (Nicotiana tabacum cv. Samsun) plants to local burning of an upper leaf by measuring the following variables in a distant leaf: extracellular electrical potentials (EEPs); gas exchange parameters; fast chlorophyll fluorescence induction; endogenous concentrations of three putative chemical signaling compounds – abscisic (ABA), jasmonic (JA), salicylic (SA) acids and content of heat-shock proteins (Hsp70 family). A decrease in EEPs in the distant leaves started to decline within 10-20 s of the beginning of the treatment, fell sharply for ca. 1-3 min, and then tended to recover within the following hour. The measured gasometric parameters (stomatal
conductance and the rates of transpiration and CO2 assimilation) started to decrease 5 - 7 min after local burning, suggesting that the electrical signals may induce stomatal closure. Simultaneously, systemic increases in the endogenous ABA concentration Thewere followed Second by huge systemic rises in endogeSymposiumnous JA levels started after ca. 15 min, providing the first evidence of short-term systemic accumulation of these plant hormones in responses to local burning. Furthermore, JA appears to have an inhibitory effect onon CO2 assimilation. Plant Also an increase Neurobiology in Hsp70 content was detectable both locally and systemically within 1 hour after local burning. The correlations between the kinetics of the systemic EEP, stomatal, photosynthetic, ABA, JA and Hsp70 responses suggest that (i) electrical signals (probably induced by a propagating
83 Abstracts for Poster Presentation
hydraulic signal) may trigger chemical defence-related signaling pathways in tobacco plants; (ii) both electrical and chemical signals are interactively involved in the induction of short-term systemic stomatal closure and subsequent reductions in the
rate of transpiration and CO2 assimilation; (iii) systemic accumulation of heat-shock proteins may be induced by chemical and/or electrical signals propagating after local burning events.
The project was supported by grant from the Ministry of Education of the Czech Republic, No. MSM 6198959215.
The Second Symposium
on Plant Neurobiology
84 Abstracts for Poster Presentation
Opposite Patterns of ABA and IAA in Relation to the Cambium Periodicity in Eucommia ulmoides Oliv
Xinqiang He，Kalima-N’Koma Mwange, Hongwei Hou, Wanfeng Li, Keming Cui
College of Life Sciences, Peking University, Beijing 100871, China
E-mail: [email protected]
The seasonal change of abscisic acid (ABA) and Indole-3-acetic acid (IAA) and their relationship with the cambial activity in Eucommia ulmoides tree were investigated by ABA and IAA immunolocalization, quantification, and systematic monitoring of vascular cell layers production. ABA and IAA clearly displayed opposite annual distribution patterns. In the active period, both immunolocalization and HPLC detected an abrupt decrease of ABA, reaching its lowest level in summer. During dormancy, ABA started increasing in the first quiescence (Q1) (autumn), peaked in the rest (winter), and gradually decreased from the onset of the second quiescence (Q2) (end winter). IAA showed a reverse pattern to that of ABA: it sharply increased in the active period, but noticeably decreased from the commencement of the first quiescence. The concomitant IAA-ABA distribution and seasonal changes in vascular tissues greatly correlated with xylem and phloem cell production, and late wood differentiation and maturation. Meanwhile, A cDNA clone of auxin binding protein 1 (ABP1), the putative auxin receptor, was isolated. Spatio-temporal expression patterns showed that ABP1 transcript abundance in the cambium tissues was high, low and remarkably scarce respectively in active, quiescent and resting tree. Results strongly support the role of ABP1 in mediating the IAA signals, which could boost cambium reactivation during cambium quiescence, and the results could also explain why IAA could not reactivate a resting cambium. This ABP1 expression pattern correlated positively with that of IAA, but negatively with that of ABA. It also correlated closely with auxin sensitivity during cambial activity periodicity. TheKeywords: ABA, Second ABP1, cambium periodicity, Symposium dormancy, Eucommia ulmoides, IAA
This study was supported by the National Natural Science Foundation of China (30471367,on 30530620). Plant Neurobiology
85 Abstracts for Poster Presentation
Analysis of Metabolite Content in Arabidopsis GABA Shunt Mutants
Anke Hüser, Ulf-Ingo Flügge, Frank Ludewig
Botanical Instiute, University of Cologne, Gyrhofstr, 15, D-50931 Cologne, Germany
Email: [email protected]
GABA (gamma-amino butyric acid) is a four carbon amino acid, found in all organisms. In plants, mutants of both catabolic genes (gaba-t and ssadh) display phenotypic deviations to WT. ssadh knock-out plants are severely affected in growth, probably due to the accumulation of a toxic compound. Two candidate substances (GHB and SSA), that can be converted into each other, were examined for their capacity to affect plant growth. WT and mutant plants were grown on half-strength MS-media containing different amounts of either SSA or GHB to test which of the compounds are causing the ssadh phenotype. Plant growth was observed and the metabolite content was determined using GC/MS techniques. WT or gaba-t mutant plants were affected in growth by increased concentrations of SSA or GHB added to the media, but to a lesser extent than gaba-t ssadh double knock-out plants. By determining the GHB content in plant extracts using GC/MS, higher amounts of GHB were found in plants grown on GHB containing media. These results indicate that SSA might be the causative substance for the observed ssadh phenotype. To verify these results, knock-out mutants in the ghbdh gene were isolated and analyzed for their response to GHB or SSA in comparison to WT plants.
The Second Symposium
on Plant Neurobiology
86 Abstracts for Poster Presentation
Aluminium sensitivity of Arabidopsis roots: unique status of the transition zone in plasma membrane properties, endosomal behaviour, nitric oxide production and internalization of aluminium
Peter Illéš1, Markus Schlicht2, Ján Pavlovkin1, Irene Lichtscheidl3, František Baluška1,2*, Miroslav Ovečka1
1 Institute of Botany, Slovak Academy of Sciences, Bratislava 2 Institute of Cellular and Molecular Botany, University of Bonn, Bonn 3Institution of Cell Imaging and Ultrastructure Research, University of Vienna, Vienna
*E-mail: [email protected]
One of the most relevant problems of recent research on aluminium phytotoxicity is to define the primary site of aluminium action on a cellular and subcellular level. In addition, data on the fate of internalized aluminium during plant recovery are completely missing. We studied the extent of aluminium internalization during the recovery from aluminium stress in living roots of Arabidopsis thaliana by non-invasive in vivo microscopy in real time. We document evidently distinct sensitivity of the cells passing through different developmental zones. Aluminium exposure caused rapid depolarization of the plasma membrane. It was much more extensive in cells of the distal portion of the transition zone than in the proximal portion of the transition zone. Also full recovery of the membrane potential after removal of external aluminium was slower in cells of the distal portion of the transition zone compared to cells of its proximal part. Apoplastic aluminium internalized extensively during the recovery phase into endosomal/vacuolar compartments in the most aluminium sensitive cells of the distal portion of the transition zone. Importantly, internalization of aluminium is spatially restricted to the pectin-recycling zone. Aluminium interfered with endosomal behaviour and inhibited the formation of BFA-induced compartments in these cells. Moreover, cells of the distal portion of the transition zone emitted large amounts of nitric oxide (NO) and this was blocked by aluminium treatment. All these data suggest that the most sensitive status of the distal Theportion of the Second transition zone towards aluminium Symposium reflects the specific mode of aluminium sensing within the root apex.
Thison work wasPlant supported in part Neurobiology by the Grant Agency VEGA (grants No. 2/5085/25 and 2/5086/25) and by a Marie Curie European Reintegration Grants No. MERG-CT-2005-031168 within the 6th European Community Framework Programme.
87 Abstracts for Poster Presentation
Comparison Analysis of the ADP-ribosylation Factors Gene Family in Rice
Jie Li, Wei Cheng, Chao Chen, Xiaojin Zhou, Yingdian Wang*
Laboratory of Plant Development Physiology and Molecular Biology, College of Life Sciences, Beijing Normal University, 19 XinJieKouWai Avenue, Beijing 100875, China *Corresponding author: Yingdian Wang
(Tel: +86-10-58808195; Fax: +86-10-58809077; E-mail: [email protected])
ADP-ribosylation factors (Arf), a subfamily of the Ras superfamily of GTP-binding proteins, was originally described as a cytosolic cofactor activity required for cholera toxin to ADP-ribosylate the α-subunit of the Gs heterotrimeric G-protein (Munro, 2005). They play important roles in intracellular trafficking and regulate a wide variety of intracellular signaling in eukaryotic cells including plant cells (Memon, 2004). In rice, the development of endosperm may be closely related with the process of vesicle trafficking. Vesicle trafficking delivers proteins to intracellular and extracellular compartments, cellulose synthase to the plasma membrane, and non-cellulosic polysaccharides to the cell wall (Gebbie et al., 2005). Therefore, the vesicle trafficking is essential in the process of cellulization and proliferation of endosperm cells. Few studies about ARF gene family were reported in rice now. To clarify the molecular mechanisms of endosperm development process, the members of ARF gene family were predicted according to the rice genomic database. To search ARF genes associated with the development of rice endosperm, the various analysis were made within those predicted ARF genes. In this study, a genome-wide analysis of rice ARF genes was carried out, and eight new rice ARF genes were identified according the known the sequences of OsARF1 and OsARF2. The ten putative OsARFs could fall into two classes based on the comparison of the gene structures and the sequences of deduced amino acid with Theother known plantSecond and animal ARFs. Six ofSymposium the ten OsARFs are highly analogous with the class1 ARF subfamily members in animals and Arabidopsis, while the other ARF genes are only identified in plants. The analysis of the six class1 ARF gene sequences reveal thaton they are Plant consist of six exons Neurobiology and five introns. Moreover their amino acid sequences are also highly conserved with similarity over 97%. The other four ARFs are less conserved than the class1 ARF subfamily members, but contain the similarity more than 80% in their amino acid sequences. RT-PCR analysis was performed in order to investigate the mRNA expression pattern of each member of the ARF gene 88 Abstracts for Poster Presentation
family in rice. The results show that one of the OsARF genes, OsARF3, which belongs to the class1 ARF subfamily specifically expressed in young leaf, root and seed. It was demonstrated that OsARF3 could complement yeast arf1 arf2 mutants and its GFP-fusion is localized to the Golgi apparatus in plant cells like its animal counterpart. The physiological role of OsARF3 in rice will be further analyzed.
Gebbie, Leigh K.,Burn, Joanne E., Hocart, Charles H.and Williamson, Richard E. (2005) Gene encoding ADP-ribosylation factors in Arabidopsis thaliana L.Heyn.;genome analysis and antisense suppression. Journal of Experimental Botany 56, 1079-1091. Memon, A. R. (2004) The role of ADP-ribosylation factor and SAR1 in vesicular trafficking in plants. Biochimica et Biophysica Acta 1664, 9-30. Munro, S. (2005) The Arf-like GTPase Arl1 and its role in membrane traffic. Biochemical Society Transactions 33, 601-605.
The Second Symposium
on Plant Neurobiology
89 Abstracts for Poster Presentation
Intracellular Localization of Integrin-like Protein and Its Roles in Osmotic Stress-induced ABA Biosynthesis in Zea Mays
Bing Lv1, Feng Chen1, Zhonghua Gong1, Hong Xie1, Jianhua Zhang2, Jiansheng Liang1*
1College of Bioscience and Biotechnology, Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Yangzhou University, Yangzhou, People’s Republic of China, 225009 2Biology Department, Hong Kong Baptist College, Kowloon Tang, Hong Kong
*E-mail: [email protected]
Plants have evolved many mechanisms to cope with adverse environmental stresses. Abscisic acid (ABA) accumulates significantly in plant cells in response to drought conditions, which has been considered as a major mechanism for plants to enhance drought tolerance. In this study, we explore the possible mechanisms how plant cells perceive the osmotic stress, and as a consequence, induce cells to biosynthesize ABA. Our results showed that a great difference existed between protoplasts and cells in responses to osmotic stress in induction of ABA biosynthesis, implying that cell wall and/or cell wall-plasma membrane interaction play important roles in perceiving osmotic stress. Western Blotting and immuno-fluorescence localization experiment, using polyantibody against rabbit integrin β1, showed that there existed a protein in roots of Zea Mays, which was similar to integrin protein of animals and it mainly localized in insoluble fraction of plant cells. Treatment with GRGDS, a synthetic pentapeptide containing RGD domain, which interacted specifically with integrin protein and thus blocked the cell wall-plasma membrane interaction, significantly inhibited osmotic stress induced ABA biosynthesis in cells, Thebut not in protoplasts.Second Accordingly, weSymposium concluded that cell wall and/or cell wall-plasma membrane interaction mediated by integrin-like protein played important roles in osmotic stress- induced ABA biosynthesis in Zea Mays.
on Plant Neurobiology
90 Abstracts for Poster Presentation
Cloning, Expression and Characterization of a Plastidial N-glycosylated ADP-glucose Hydrolase
Yohei Nanjo1, Hiromasa Oka1, Noriko Ikarashi1, Kentaro Kaneko1, Aya Kitajima1, Toshiaki Mitsui1, Francisco José Muñoz2, Milagros Rodríguez-López2, Edurne Baroja-Fernández2, Javier Pozueta-Romero2
1 Laboratories of Plant and Microbial Genome Control and Department of Applied Biological Chemistry, Niigata University, 2-8050 Ikarashi, Niigata 950-2181, Japan; 2 Agrobioteknologiako Instituta, Nafarroako Unibertsitate Publikoa, Gobierno de Navarra and Consejo Superior de Investigaciones Científicas, Mutiloako etorbidea zenbaki gabe, 31192 Mutiloabeti, Nafarroa, Spain
*E-mail: [email protected] ; [email protected]
A nucleotide pyrophosphatase/phosphodiesterase (NPP) activity has been shown to occur in the plastidial compartment of both mono- and di-cotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) (Rodríguez-López et al. 2000 Proc. Natl. Acad. Sci. USA 97, 8705-8710). To further our knowledge about the protein entity responsible for this activity, two NPPs from rice and barley seedlings have been purified and characterized. Both enzymes are glycosylated, since they bind to Concanavalin A, stain with periodic acid-Schiff reagent and can be digested by Endo-H. A complete rice NPP encoding cDNA, designated as OsNPP1, was isolated, characterized and overexpressed in transgenic plants displaying high ADPG hydrolytic activity. Computer searches of data banks revealed that OsNPP1 belongs to a functionally divergent group of plant nucleotide hydrolases. OsNPP1 contains numerous N-glycosylation sites and a cleavable hydrophobic signal sequence that does not match with the N-terminal part of the mature protein. Both immunocytochemical analyses and confocal-fluorescence microscopy of rice cells expressing OsNPP1 fused with the green fluorescent protein (GFP) revealed that OsNPP1-GFP occurs in the plastidial compartment. The Second Symposium Rodríguez-López, M., Baroja-Fernández, E., Zandueta-Criado, A., Pozueta-Romero, J. (2000) Adenosine diphosphate glucose pyrophosphatase: a plastidial phosphodiesteraseon Plant that prevents starchNeurobiology biosynthesis. Proc. Natl. Acad. Sci. USA 97, 8705-8710
91 Abstracts for Poster Presentation
Effects of Auxin Inhibitors and Glutamate on Electrotropism of Maize Roots
Camilla Pandolfi, Elisa Azzarello, Sergio Mugnai, Stefano Mancuso*
LINV, Dip. Ortoflorofrutticoltura, University of Florence, viale delle Idee 30, 50019 Sesto Fiorentino, Italy
* Email: [email protected]
Like all living tissues, the cells of plant roots produce an electric field due to the activities of ion transporters. This ion movement creates a flow of current through the tissue and the formation of electrical potential differences across the membrane. As a consequence of this ion transport activities, plant roots generate long-lasting electric fields in the apoplast and rhizosphere (Weisenseel et al., 1992). These electric fields can polarize cells and tissues and can affect growth of the root. For instance, electric fields may generate a lateral asymmetry of ions and hormones in the elongating zone. Application of electric fields can modify the direction of growth of certain plant cells or organs. This phenomenon, known as electrotropism, has been reported in fungi (McGillavray and Gow NAR 1986) and algae (Brower and Giddings 1980) as well as in roots (Fondren and Moore 1987; Schrank,1959), and shoots (Schrank,1959) of higher plants. The preferred direction of growth relative to the applied electric field varies with the type of cell or organ tested and, in some cases, is species dependent. In our experiments we examined the effect of electric field on the primary root of maize (Zea mays L.). At first we used different intensity of voltage, observing a root curvature towards cathode. Electric field greater than 1 V/cm caused important reduction in root elongation. Then we added other substance to the medium using an electric field of 0,2 V/cm not to interfere with root growth . Electrotropism was partially inhibited when we used different concentration of TheTIBA and NPA. Second Symposium Another interesting phenomenon was observed adding different concentration of glutamate to the medium. Glutamate is one of the 20 standard aminoacids but it is also the most abundant excitatory neurotransmitter in the nervous system. Numerous on Plant Neurobiology glutamate receptor-like (GLR) genes have been identified in plant genomes, and plant GLRs are predicted, on the basis of sequence homology, to retain ligand-binding and ion channel activity (Davenport 2002). Electrotropic effect on root growth was inverted in presence of glutamate: roots turn towards anode and show a corkscrew 92 Abstracts for Poster Presentation
kind of growth. BMAA, a glutamate antagonist, inhibited this glutamate effect on the root apex electrotropism.
Romola Davenpor 2002, Glutamate Receptors in Plants Annals of Botany 90: 549-557. Schrank AR (1959) Electronasty and electrotropism. In E Bunning, ed, Physiology of Movements, Encyclopedia of Plant Physiology, New Series, Vol 17. Springer, New York, pp.148-163. Fondren WM, Moore R (1987) Collection of gravitropic effectors from mucilage of electrotropically-stimulated roots of Zea mays L. Ann Bot 59: 657-659 McGillavray AM, Gow NAR (1986) Applied electrical fields polarize the growth of mycelial fungi. J Gen Microbiol 132: 2515-2525 Brower DL, Giddings TH (1980) The effects of applied electric fields on Micrasterias. II. The distributions of cytoplasmic and plasma membrane components. J Cell Sci 42: 279-290 Weisenseel MH, Becker HF, Ehlgotz JG (1992) Growth, gravitropism and endogenous ion currents of cress roots (Lepidium sativum L.). Measurements using a nove1 three-dimensional recording probe. Plant Physiol 100: 16-25
The Second Symposium
on Plant Neurobiology
93 Abstracts for Poster Presentation
Effect of Secondary Metabolites Associated with Anaerobic Soil Conditions on Ion Fluxes and Electrophysiology in Barley Roots
Jiayin Pang1, Tracey Cuin1, Lana Shabala1, Meixue Zhou1, Neville Mendham1, Jiansheng Liang2, Sergey Shabala1
1School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, Tasmania, 7005, Australia 2College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, 225009, China
*Email: [email protected]
Waterlogging stress is traditionally associated with O2 depletion. In addition, a significant accumulation of toxic substances from the microbial reduction processes has been widely reported in waterlogged soil. In this study, the effects of several secondary metabolites (phenolic acids, monocarboxylic acids and Mn2+) on nutrient (K+, H+ and Ca2+) acquisition of barley roots were investigated using the non-invasive MIFE® system. All three lower monocarboxylic acids (formic, acetic and propionic acids) and three phenolic acids (benzoic, 2-hydroxybenzoic, 4-hydroxybenzoic acids) caused immediate net influx of H+ and the reduction of K+ uptake, while Mn2+ treatment caused K+ to quickly return to the initial level following the net efflux in the first few minutes and gradual increase of H+ influx. Phenolic acids slightly increased the influx of Ca2+ immediate after treatment, but not in other chemicals. Plant roots showed different responses of ion fluxes and membrane potential to these chemicals in the long term (24 h). 24 h treatment with all chemicals significantly reduced the K+ uptake, and the adverse effects of phenolic acids were smaller than with monocarboxylic acids and Mn2+. Treatment with monocarboxylic acids for 24 h reversed H+ from net efflux to net influx, while all three phenolic acids did not cause significant effects compared with the control. Phenolic acids caused significant net TheCa2+ efflux from Second roots pre-treated for 24 h.Symposium The possible model explaining effects of secondary metabolites on membrane transport activity is suggested.
on Plant Neurobiology
94 Abstracts for Poster Presentation
IBA Promotes Lateral Root Formation Via IBA-induced NO Formation and ß-oxidation-like IBA–to–IAA Conversion in Peroxisomes of Pericycle and Endodermis Cells
Markus Schlicht1, Diedrik Menzel1, František Baluška1
1 Institute of Cellular and Molecular Botany, University of Bonn, Bonn
Indole butyric acid (IBA) is considered to be an inactive form of auxin, which had to be converted to indole acetic acid (IAA) to show biological activities relevant for the plant growth and development. A process similar to the ß-oxidation of fatty acids within peroxisomes is implicated in this conversion. Currently valid model suggest that ß-oxidation-like process converts IBA to IAA to cause any biological effects. However, this model can not convincingly explain why IBA induces stronger effects, than IAA, on the formation of lateral roots. Furthermore the lrt1-mutant of rice is resistent against the inhibitory effect of auxins (IAA, 2,4-D and IBA) on the root elongation and only IBA, but not IAA, can induce lateral roots (Chhun et al. 2003). The same phenomenon is true also for the lrt1-mutant of maize (this work). Moreover, levels of free IBA are nearly equal to the levels of free IAA in young seedlings (Ludwig-Müller and Cohen 2002). Astonishingly, a polar transport of IBA in roots takes place which is independent of PIN1 and AUX1 proteins and not inhibited by the IAA efflux inhibitors like NPA (Rashotte et al. 2003). Mutants with a disturbed polar IBA transport, but normal polar IAA transport, are showing phenotypes which are typical for the polar IAA transport related mutants. For example, these seedlings show disturbed gravisensing (rib1, Poupart et al. 2005) or fewer lateral roots (arm2, Chhun et al. 2005). Altogether, IBA emerges as biologically active auxin. Our present results, based on the IAA specific antibody (Schlicht et al. 2006), are showing a first direct proof for a possible IBA-conversion within peroxisomes of endodermis and pericycle. In addition, we show that this tissue-specific ß-oxidation-like conversion is needed for the IBA activity stimulating the lateral root Theinduction via Secondnitric oxide (NO) produc tion.Symposium The IBA-induced NO production, in combination with the tissue specific IBA-to-IAA conversion, explain why the weak auxin IBA strongly promotes the lateral root formation despite showing only weak activitieson for other typiPlantcal actions of IAA. Neurobiology
Chhun T, Taketa S, Ichii M, Tsurumi S (2005) Involvement of ARM2 in the uptake of indole-3-butyric acid in rice (Oryza sativa L.) roots. Plant Cell Physiol 46, 1161-1164
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Ludwig-Muller J, Cohen JD (2002) Identification and quantification of three active auxins in different tissues of Tropaeolum majus. Physiol Plant 115, 320-329 Poupart J, Rashotte AM, Muday GK, Waddell CS (2005) The rib1 mutant of Arabidopsis has alterations in indole-3-butyric acid transport, hypocotyl elongation, and root architecture. Plant Physiol 139, 1460-1471 Rashotte AM, Poupart J, Waddell CS, Muday GK (2003) Transport of the two natural auxins, indole-3-butyric acid and indole-3-acetic acid, in Arabidopsis. Plant Physiol 133, 761-772 Schlicht M, Strnad M, Scanlon MJ, Mancuso S, Hochholdinger F, Palme K, Volkmann D, Menzel D, Baluška F (2006) Auxin immunolocalization implicates vesicular neurotransmitter-like mode of polar auxin transport in root apices. Plant Signal Behav 1, In press
The Second Symposium
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96 Abstracts for Poster Presentation
Interaction of Photoreceptors: Signal Memory in Mougeotia
Tadeusz Walczak, Halina Gabryś*
Department of Plant Physiology and Biochemistry, Faculty of Biotechnology, Jagiellonian University, Kraków, Poland
Although most plants use blue light for activating chloroplast movements, some ferns, mosses and green algae use two spectral regions, red and blue. Among these species the filamentous alga Mougeotia scalaris rotates its ribbon-shaped chloroplast towards a face position in weak blue light and in red light irrespective of its fluence rate, and towards a profile position in strong blue light alone or combined with red. According to the classical hypothesis developed by Haupt in the sixties, the red-controlled rotation is mediated by a gradient of Pfr form of phytochrome bound in spirally ordered arrays at the plasma membrane. Evidence for the involvement of an independent blue-absorbing photoreceptor (phototropin?) in the control of weak-light activated response has been provided more than two decades ago [1]. Interaction between the two photoreceptors in the generation of the strong light (face-to-profile) response was investigated by using high energy blue and red light pulses. This technique provided a time-resolved excitation of both photoreceptor systems. The results demonstrated an indirect nature of the interaction between the photoreceptors [2,3]. First, a stable product was formed in a blue light-mediated reaction, having a lifetime of ca. 2 min at room temperature. Subsequently, this blue light signal carrier interacted with phytochrome in its physiologically active, far-red absorbing Pfr form. As a result of this interaction a long-lived product was formed capable of inducing the face-to-profile rotation. The induction required elimination of the Pfr gradient. The information about the latent face-to-profile response could be stored in the cell for at least 40 min, and displayed after application of far-red light eliminating the phytochrome gradient. Even if the classical phytochrome will be substituted by a recently discovered neochrome as a novel partner of the reaction, its interaction with a phototropin seems to be a prerequisite of the response of Mougeotia chloroplasts to strong light.
The1. Gabrys H., SecondWalczak T., Haupt W.(1984) Symposium Blue light induced chloroplast orientation in Mougeotia. I. Evidence for a separate sensor pigment besides phytochrome. Planta 160:21-24 2. Gabryson H.,Walczak Plant T.,Haupt W.(1985) Neurobiology Interaction between phytochrome and the blue light photoreceptor system in Mougeotia. Photochem. Photobiol. 42:731-734 3. Gabrys H.,Walczak T.,Scheuerlein R.(1988) Time characteristics of the interaction between phytochrome and the blue light photoreceptor system in Mougeotia. J.Photochem. Photobiol. 2:467-474 97 Abstracts for Poster Presentation
The Subcellular Localization and Blue Light-induced Dynamic Relocalization of Phototropin 1 in Root and Shoot Cells of Arabidopsis thaliana
Yinglang Wan1, William R Eisinger3, Weronika Krzeszowiec4, Halina Gabrys4, František Baluška1, David W. Ehrhardt2, Winslow R Briggs2
1Institute of Cellular and Molecular Botany,University of Bonn, Kieschallee 1, D-53115 Bonn, Germany 2Carnegie Institution Department of Plant Biology, 260 Panama Street, CA94305 Stanford, USA 3 Santa Clara University, 500 El Camino Real, CA 95053 Santa Clara, USA 4The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, Krakow, Poland
The blue light receptor phototropin 1 (phot1) is a flavin-binding light-activated protein kinase. It serves as the major photoreceptor for phototropism in both roots and shoots. Although the activation of the C-terminal kinase domain has been characterized, the signal transduction pathway between perception of blue light by phot1 and phototropic responses remains unresolved. In continuation of our previous study with phot1-GFP-transformed Arabidopsis plants lacking phot1 (Sakamoto et al., 2002), here we have investigated patterns of subcellular localizations of phot1-GFP in different cell types in shoots and roots. Driven from the Phot1 promoter, the PHOT1:GFP fusion protein is expressed in almost all plant cells. Expression is especially strong in cells of shoot and root tips. We observed different patterns of phot1 expression and localization in cells of different tissues. For example, in cortical cells of the shoot elongation zone, PHOT1:GFP can be detected mainly at the plasma membrane (PM). GFP signal was especially strong at the apical pole of the cell formed irregular longitudinal stripes along the regions of lateral walls in closest contact with the neighboring cells. In root cortical cells, PHOT1:GFP was mainly observed at the cell poles, where it formed a semicircle pattern. The localization to lateral walls observed in the hypocotyl was not detected in root cortical cells. Following blue-light irradiation, PHOT1:GFP relocalized within approximately xx minutes, forming sharp punctae at the PM and also a marked increase in signal in the cytosol. The extent of relocalization is related to the intensity of the blue light and the Theexposure time. Second Experiments with brefeldin Symposium A demonstrated that the increased signal in the cytosol does not arrive from the ER, and suggested that blue-light activates endocytosis and recycling of membrane-bound PHOT1:GFP. In support of this notion, both coldon treatment Plant and depolymerization Neurobiology of all F-actin with latrunculin B inhibit PHOT1:GFP internalization. Moreover, internalized PHOT1:GFP co-localized with FM4-64 labeled compartments within the cytoplasm, suggesting that PHOT1:GFP may become associated with endosomes in this process. We propose that the BL-induced PHOT1 recycling between the plasma membrane and endiosomes may be involved in the signal-transduction pathway for the phototropism of plant organs. 98 Abstracts for Poster Presentation
Dynamic Imaging of Secretory Vesicles in Living Pollen Tubes of Picea meyeri using Evanescent Wave Microscopy
Xiaohua Wang1,2, Yan Teng 3, Qinli Wang1,2, Xiaojuan Li1,2, Maozhong Zheng1,2, Jozef Šamaj4,5, František Baluška4,6 and Jinxing Lin1*
1 Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 2 Graduate School of Chinese Academy of Sciences, Beijing 100049, China 3 Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC27858-4353, USA 4Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany 5Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, SK-95007, Nitra, Slovak Republic 6Institute of Botany, Slovak Academy of Sciences, Dubravska 14, SK-84223, Bratislava, Slovak Republic
*E-mail: [email protected]
Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through a ~300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional (2-D) trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the 2-D diffusion coefficient D (2). The velocity and Thedirection of vesicleSecond motions, frame-to-frame Symposium displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time that two types of motion are present, and that vesicles in living pollen tubes exhibit complicatedon behaviors Plant and oscillations Neurobiologythat differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.
99 Abstracts for Poster Presentation
Reorganization of Cytoskeleton in Rice Cells during the Early Stages of Disease Development Caused by Magnaporthe grisea
Min-He Yang1, Zhong Zheng2
1College of Life Science, Fujian Normal University, Fuzhou, Fujian 350007, China; 2Department of Plant Protection, Zhejiang University, Hangzhou, Zhejiang 430070 China
The procedures of immunostaining of microtubules and microfilaments were examined in inner epidermal cells of rice leaf sheath. The results demonstrated that microtubules were successfully detected in detached single layer of rice leaf sheath after fixed in a mixed solution of 4% paraformaldehyde and 1% glutaraldehyde combined with enzyme digestion of rice cell wall for 1-2 min. Netted distribution of microtubules was observed in inner epidermal cells of rice leaf sheath. Microfilaments were distributed in parallel with the longitudinal axis of rice cell. The results also suggested that actin filaments were sensitive to paraformaldehyde and glutaraldehyde fixation. And pretreatment of rice tissue with 1mmol/L MBS (3-maeimidobenzoic acid N-hydroxysuccinimide ester) for 30 min was critical for the detection of microfilaments in cells of rice leaf sheath. M. grisea penetration resulted in rapid and striking reorganization of microtubules and microfilaments in cells of rice leaf sheath. The distribution patterns of cytoskeleton in the incompatible interaction of rice and M. grisea were different from those in compatible interaction. In cells undergoing resistant response, microtubules and microfilaments of rice cells radiated to fungal penetration site during the process of fungal penetration. Then they were gradually fragmented and eliminated from host cells after hypersensitive cell death occurred. However, in cells undergoing susceptible response, the cytoskeleton was severely damaged and fragmented quickly at the initial stage of fungal penetration. As the disease development, fine bundles of cytoskeleton were seldom observed in host cells although the cytoskeleton of M. grisea was normally detected. The Second Symposium
on Plant Neurobiology
100 Abstracts for Poster Presentation
Glutamic Acid Decarboxylase is Involved in the Regulation of Tobacco Pollen Tube Growth
Guanghui Yu, Mengxiang Sun*
Key Laboratory of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China
E-mail: [email protected] ; [email protected]
Calcium is a key regulator of pollen tube growth, but little is known concerning the downstream components of the signaling pathways involved. Glutamic acid decarboxylase (GAD) ， as a down-regulatory molecule of Calmodulin, was investigated in present work. To analyze its potential roles in the pollen tubes growth, both Ca2+ chelator ethyleneglycol-bis (β-aminoethyl ether)-N,N΄-tetraacetic acid (EGTA) and Calmodulin antagonist trifluoperazine (TFP) were employed in the experiments. The results showed that GAD was constituently expressed in the pollen grains and pollen tubes. Moreover, it was tip localized and asymmetrically distributed in the pollen tubes. The inhibitory effect of the EGTA and TFP on the growth of pollen tubes could be partially ameliorated by the addition of 1mmol/L exogenous γ-Aminobutyric acid (GABA). This was further confirmed by the use of 3-mercaptopropinic acid (3-MPA), which is a relatively specific inhibitor of GAD. The experiments demonstrated that the exogenous GABA could also decrease the inhibitory effect of 3-MPA (at lower concentration) on pollen tube growths (statistical significance p<0.01). Additionally, coming along with the morphological alteration of pollen-tube tip after 3-MPA treatment at higher concentration, actin filaments disorganized, the speed of pollen tubes growth and cytoplasmic streaming were much decreased, orientation of vesicles trafficking was also misleaded. Our results suggest that GAD is a down modulatory molecular of Ca2+/CaM and plays an essential role in the regulation of pollen tube growth possibly via controlling the traffic of secretory vesicles. 1. Baum G., Lev-Yadun S, Fridmann Y et al. Calmodulin binding to glutamate Thedecarboxylase Second is required for regulati Symposiumon of glutamate and GABA metabolism and normal development in plants. EMBO J. 1996, 15:2988–2996 2. Fu Y, Wu G, and Yang Z.B. Rop GTPase-dependent dynamics of tip-localized F-actinon controls Plant tip growth in pollenNeurobiology tubes. Journal. Cell Biol. 2001, 152, 1019–1032 3. Jin H, Wu H, Osterhaus G et al. Demonstration of functional coupling between γ-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Proc. Natl. Acad. Sci. USA. 2003.100: 4293-4298 101 Abstracts for Poster Presentation
Roles of the Ubiquitin/Proteasome Pathway in Pollen Tube Growth with Emphasis on MG132-Induced Alterations in Ultrastructure, Cytoskeleton and Cell Wall Components
Xianyong Sheng 1, 2, Zhenghai Hu 2, Hongfei Lü1, Xiaohua Wang1, František Baluška3, 4 Jozef Šamaj3, 5, and Jinxing Lin1
1 Institute of Botany, The Chinese Academy of Sciences, Key Laboratory of Photosynthesis and Molecular Environment Physiology, Beijing 100093, China; 2 College of life Science, Northwest University, Xi’an, 710069, China; 3 Institute of Cellular and Molecular Botany, Rheinische Friedrich- Wilhelms- University Bonn, Department of Plant Cell Biology, Kirschallee 1, D-53115 Bonn, Germany; 4 Institute of Botany, Slovak Academy of Sciences, Dubravska 14, SK-84223, Bratislava, Slovak Republic; 5 Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, SK-95007, Nitra, Slovak Republic The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner; while hardly similar effects were detected when Cys-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins was also observed using immunoblotting. TEM revealed that MG132 induces ER-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of ubiquitinated proteins in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with FITC-phalloidin and β-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules, and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with Theanti-pectin antibodies Second and calcofluor indicated Symposiumthat MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by FTIR analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatmenton promotesPlant the accumulation Neurobiology of ubiquitinated proteins in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton, and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.
102 Abstracts for Poster Presentation
Proteomic Analysis of Differentially Expressed Protein by Mannitol-induced Osmotic Stresses in Rice
Xin Zang*, Setsuko Komatsu** and Tuansheng Shi*
*Bioengineering Department, Zhengzhou University, Zhengzhou 450052, China **National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan
Plants are continuously exposed to biotic and abiotic stresses that endanger their survival. Among abiotic stresses, osmotic stress is one of the most severe, caused by drought, high salinity and cold stresses in nature. In this study, to investigate the response of rice to osmotic stress induced by mannitol, proteins from basal part of rice leaf sheath were screened by a proteomics approach. Two-week-old rice seedings were treated with 400 mM mannitol for 48 h. After separation of proteins from basal part of leaf sheath by two-dimensional polyacrylamide gel electrophoresis, 327 proteins were identified. Among them, 15 proteins significantly responded to osmotic stress by up- or down regulation, as 12 proteins were increased while 3 proteins were decreased. The expression profile of unique proteins was further confirmed by dose- and time-dependent parameters. The changed protein spots were analyzed by protein sequencer and mass spectrometry. Homology searches were carried out using the FastA or Mascot search tools. N-terminal sequences were successfully obtained for eight of these proteins. They were 26S proteasome regulatory subunit, calreticulin precursor, heat shock protein, dnaK-type molecular chaperone, uroporphyrinogen decarboxylase, two proteins encoded by Oryza sativa genomic DNA and a function unknown protein. All the rest protein spots were N-terminally blocked. One of them was identified as glutathione S-transferase by internal sequencing. Among the other seven proteins, three were not identified while four were identified as endosperm lumina bnding protein, lipid transfer protein, glyoxalase I and alpha subunit of 20S proteasome. TheMoreover, Second comparative proteomics apprSymposiumoach was carried out among different stress treatments, different tissues and different cultivars. These findings have important implications for understanding the biochemical and molecular mechanisms of plant adaptation and response to osmotic stress. on Plant Neurobiology
103 Abstracts for Poster Presentation
Interactions Between Shoot Derived Auxin Transport, Root Auxin Metabolism and Phenolic Accumulation in the Regulation of Iron-deficiency Stress Responses in Red Clover
Shao Jian Zheng1, Chong Wei Jin2
1Key State Laboratory of Plant Physiology and Biochemistry, College of Life Science; 2College of Environmental and Resource Science, Zhejiang University, Hangzhou 310058, China
In the present research, an Fe-efficient plant species, red clover (Trifolium pratense L.), was used to investigate the role of shoot-derived signals and phenolic metabolism in regulating Fe-deficiency responses. Both the application of the inhibitor of polar auxin (IAA) transport, TIBA (2, 3, 5-triiodobenoic acid) to the stem as well as decapitation of the shoot apex, significantly decreased root ferric reductase activity induced by Fe deficiency. The inclusion of exogenous IAA in the nutrient solution bathing the roots only slightly enhanced the ferric reductase activity in the Fe-sufficient plant, but remarkably enhanced this activity in Fe-deficient plants. In addition, both stem TIBA application and shoot decapitation completely inhibited the stimulated root proton extrusion and subapical root hair development. Exogenous IAA application to roots of Fe-sufficient plants also induced subapical-root hair development, further supporting the role of shoot-derived IAA in these responses. Another of the Fe deficiency stress responses, root phenolic secretion, was not affected by stem TIBA treatment, suggesting that shoot-derived IAA is not involved in regulating this Fe-deficiency stress response. While Fe deficiency enhanced root phenolic levels, it inhibited root IAA-oxidase activity and increased the endogenous root IAA levels significantly. A link between these two processes is suggested by the demonstration that, phenolics extracted from roots of Fe deficient plants inhibited IAA-oxidase activity in vitro, and this inhibition was greater than with phenolic extracted from roots of Fe sufficient plants. Based on these observations, we propose a model where under Fe deficiency stress in dicots, an increase in root phenolic concentrations plays a role in regulating root IAA levels through an inhibition of root TheIAA oxidase activity.Second This response, along Symposium with a stimulation of shoot to root IAA transport in response to Fe deficiency, leads to an increase in root IAA levels, which in turn help induce increased root ferric reductase activity, proton extrusion and subapical-root hair development. This is the first report to demonstrate a significant role for theon commonly Plant recognized phenomena Neurobiology of phenolic accumulation in dicot roots under Fe deficiency in helping regulate a suite of Fe deficiency responses through interactions with root IAA oxidase activity. These findings are helping to advance our understanding of the signaling pathways associated with the response of Strategy I plants to Fe deficiency. 104 Abstracts for Poster Presentation
Cytoskeleton Antagonistic effects on Mitochondria Motilities Revealed by Dynamic Imaging of Mitochondria in Living Pollen Tubes
Maozhong Zheng1, Yan Teng 2, Xiaohua Wang1, Qinli Wang1 and Jinxing Lin1
1 Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 2 Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Mitochondria has been revealed to serve as fundamental elements in intracellular signaling, such as in calcium signal and participants in cell death programs, except for being envisioned to be cellular power plants. Previous investigations showed that actively mitochondrial movements were indispensable to fulfill strategic mitochondrial localization at particular subcellular sites. Up to date, mitochondrial dynamics have been studied extensively using yeast or animals, and are known to vary considerably depending on the cell type and organism studied. For plant cells, few studies have been conducted and therefore little is known about dynamics of plant mitochondria. None has been reported on pollen tubes with actively cytoplsamic streaming. In order to determine how cytoskeleton and the relevant molecular motors affect mitochondrial motility and to study the mechanisms underlying the mitochondrial distribution, transporting and positioning in Picea wilsonii pollen tubes, the dynamic nature of mitochondria motion was characterized after the pollen tubes were stained with mito-tracker Red dye and treated with three cytoskeleton inhibitors and relevant molecular motor inhibitors by means of laser scanning confocal microscope and evanescent wave microscope.. The results showed that the actin filament disrupting drug latrunculin B and the myosin ATPase inhibitor 2, 3-butanedione 2-monoxime severely decreased the mitochondrial motility (decreased 82.2% and 61.0% respectively). Interestingly, Themicrotubules Seconddisrupting agent oryzalin Symposiumand dynein inhibitor vanadate slightly increased the motility, while kinesin inhibitor AMP-PNP showed no affect and taxol slightly inhibited mitochondrial motility. Further observations through evanescent microscopeon provided Plant the two-dimensional Neurobiology (2-D) trajectories, 2-Ddiffusion coefficient, velocity and direction of mitochondria motions. It was found that mitochondria can move toward the tip, away from the tip or across the tube in different trajectories. Four classes of mitochondrial movement are detected: rapid movements at average
105 Abstracts for Poster Presentation
velocity of 7.1μm/s, slower movement at average velocities of 1.6μm/s, wiggling mitochondria at average velocities 0.74μm/s and no movement at all. Drug treatment complicatedly altered mitochondrial dynamics, including velocity and distribution. This research demonstrated that microtubules and microfilaments are both responsible for mitochondrial motility and distribution in Picea wilsonii pollen tubes. Actin filaments served as track for mitochondrial transport, which was primarily powered by myosin ATPase while the most of mitochondria perhaps can use microtubule polymerization dynamics for the positionging. In the other hand, microtubule also may be affected mitochondria motility by mediating, direct and indirect, microfilament organization.
The Second Symposium on Plant Neurobiology
106 3rd International Symposium on Plant Neurobiology
(14-18 May 2007, Štrbské pleso, SLOVAKIA)
Book of Abstracts
PROGRAMME
2 Monday May 14
08:30 – 09:00 Opening session Fedor Čiampor: Wellcome address for the Presidium of Slovak Academy of Sciences
Morning session (Chairman Elizabeth Van Volkenburgh) 09:00 – 09:40 Ladislav Kováč: Information in biology: A time for rethinking the fundamentals 09:40 – 10:10 Virginia Shepherd: From semi-conductors to the rhythms of sensitive plants: The research of J.C. Bose 10:10 – 10:40 Mark Staves: Responses to environmental stimuli by internodal cells of Chara corallina
10:40 – 11:00 Coffee break
11:00 – 11:40 František Baluška: Plant neurobiology: paradigm shift in plant sciences 11:40 – 12:10 Fatima Cvrčková: Plant intelligence: why, why not, or where? 12:10 – 12:40 Paco Calvo Garzón: Are eukaryotes truly intelligent?
12:40 – 14:00 Lunch
Afternoon session (Chairman Dieter Volkmann) 14:00 – 14:40 Frank Telewski: A unified hypothesis of mechanoperception in plants 14:40 – 15:10 Stefano Mancuso: Spatio-temporal dynamics of the electrical network activity in the root apex. A multi-electrode array (MEA) study 15:10 – 15:40 Peter Barlow: The minimum set of cells required to enervate the ‘root brains’ of plants
15:40 – 16:00 Coffee break
16:00 – 16:40 Paul Galland: Mechanisms of magnetoreception in plants and fungi 16:40 – 17:10 Daniel Robert: Insect hearing and nanoscale mechanoreception
17:10 – 17:30 General Discussion
20:00 Welcome party
3 Tuesday May 15
Morning session (Chairman Viktor Žárský) 09:00 – 09:40 Akihiko Nakano: Roles of endocytosis regulation in plant physiology and development 09:40 – 10:10 Lukáš Synek: EXO70A1, a putative exocyst subunit, is important for polar growth and plant development 10:10 - 10:30 Jan Martinec: Inositol trisphosphate receptor in plants – is it real?
10:30 – 11:00 Coffee break
11:00 –11:40 Bruce Veit: Stem cell signaling networks in plants 11:40 – 12:10 Patrick Masson: A novel class of microtubule-binding proteins control root growth behavior and anisotropic cell expansion in Arabidopsis 12:10 – 12:40 Przemyslaw Wojtaszek: Domain-specific cell wall-plasma membrane interface
12:40 – 14:00 Lunch
Afternoon session (Chairman Bruce Veit) 14:00-14:40 Julian Schroeder: Guard cell ion channel signaling 14:40 – 15:10 Nan Yao: Endogenous programmed cell death triggers in plants 15:10 – 15:40 Toshiaki Mitsui: Plastid targeting of glycoproteins in rice cells
15:40 – 16:00 Coffee break
16:00 – 16:40 François Chaumont: Plant aquaporin regulation and cell signaling
16:40 – 17:10 Thomas Paul Jahn: Controlled and facilitated diffusion of H2O2 as a potential mechanism involved in signaling and ROS scavenging 17:10 – 17:40 Sakiko Okumoto: The role of glutamate in plants and its potential function as a signaling molecule 17:40 – 18:10 Frank Ludewig: Plant GABA metabolism - approaches to identify genes in vivo 18:10 – 18:40 Ian B. Cole: Indoleamines and flavonoids in neuroprotective plant physiology
19:30 – 22:00 Poster session with beer and wine
4 Wednesday May 16
Morning session (Chairman Julian Schroeder) 09:00 – 09:40 Jutta Ludwig-Müller: Indole-3-butyric acid as a signal in early events of arbuscular mycorrhizal associations 09:40 – 10:10 Günther Scherer: A role for phospholipase A in auxin gene regulation and auxin responses. The receptor may not be TIR1 10:10 – 10:30 Michal Grunt: Evolutionary history of the domain architecture of plant formins
10:30 – 11:00 Coffee break
11:00 – 11:40 Teun Munnik: Phospholipid-based signaling - 'seeing is believing' 11:40 – 12:10 Susan Murch: The role of human neurotransmitters 12:40 – 12:40 General Discussion
12:40 – 14:00 Lunch
Afternoon session (Chairman Teun Munnik) 14:00 – 14:40 Axel Mithöfer: Jasmonates as inducers of Ca2+ signals in the nucleus and the cytosol of plant cells 14:40 – 15:10 Viktor Žárský: Plasma membrane NADPH oxidases (NOXs) in plants – beyond ROS signaling 15:10 – 15:40 Jianping Hu: The role for PEX11 and dynamin-related proteins in Arabidopsis peroxisome proliferation
15:40 – 16:00 Coffee break
16:00 – 16:40 Jinxing Lin: Myosin and actin function in directing mitochondria movement in living pollen tubes of Picea wilsonii 16:40 – 17:10 Sonia Philosoph-Hadas: Actomyosin-mediated gravisensing and early transduction events in gravistimulated snapdragon spikes 17:10-17:40 Sergio Mugnai: Temporary changes in gravity conditions affect oxygen influx at root level
17:40 – 18:30 General discussion (especially on the name Plant Neurobiology)
19:30 – 22:00 Poster session with beer and wine
5 Thursday May 17
Morning session (Chairman Mary-Jane Beilby) 09:00 – 09:40 Minoru Ueda: Chemical factors inducing leaf-movement in Fabaceae and carnivorous plants 09:40 – 10:10 Arnaldo Schapire: Vesicular trafficking as a mechanism of abiotic stress tolerance in plants 10:10 – 10:40 Amit Levy: A plasmodesmata associated β-1,3-glucanase in Arabidopsis regulates plasmodesmata function
10:40 – 11:00 Coffee break
11:00 – 11:40 Ralph Hueckelhoven: Cellular polarization for membrane dynamics in interaction of barley with pathogenic Blumeria graminis 11:40 – 12:10 Hans Thordahl-Christensen: Syntaxin SYP121 is involved in a number of pathogen defence mechanisms 12:10 – 12:40 Yangdou Wei: Mining iron for host defense and pathogen virulence
12:40 – 14:00 Lunch
Afternoon session (Chairman Irene Lichtscheidl) 14:00 – 14:40 Mary-Jane Beilby: Action potentials in Charophytes 14:40 – 15:10 Alexander Volkov: Electrophysiology of Venus flytrap (Dionaea muscipula Ellis) 15:10 – 15:40 Mary A. Bisson: Effects of acetylcholine on the blue-light response of dark- grown Arabidopsis seedlings
15:40 – 16:00 Coffee break
16:00 – 16:30 Edgar Wagner: Photoperiodic adaptation by systemic control of growth and rates and planes of cell division via systemic electrophysiological communication from the cellular to the organismic level 16:30 – 17:00 Elizabeth Van Volkenburgh: Mesophyll cells are the driving force for light- and acid-induced leaf blade expansion of Pisum sativum var. Argenteum 17:00 – 17:30 Ed Etxeberria: The linear phase of sucrose uptake concentration curve in sink organs is largely mediated by fluid phase endocytosis 17:30 – 18:00 Miroslav Kaminek: Cytokinin oxidase/dehydrogenase activity in oat xylem sap
18:00 – 18:30 General discussion
20:00 Farewell party
6 Friday May 18
Morning session (Chairman Wilhelm Boland) 09:00 – 09:40 Mark Mescher: Host-location by parasitic plants 09:40 – 10:10 Renata Bogatek: Allelochemicals as a signaling molecules in the negative plant-plant interaction 10:10 – 10:40 De Oliveira R.F.: Chemical communication between roots and shoots in tomatoes 10:40 – 11:10 Ralf Oelmueller: Molecular analysis of the interaction between Arabidopsis thaliana and the growth-promoting fungus Piriformospora indica
11:10 – 11:30 Coffee break
11:30 – 12:10 Ton Timmers: Common cellular mechanisms of endosymbiotic root infection 12:10 – 12:40 Charlotte Poschenrieder: Neurotoxicity of aluminium: parallelism between plants and animals (including men) 12:40 – 13:10 Heiko Maischak: Ion channel-forming compounds in caterpillar regurgitate: A way to manipulate the plant plasma membrane potential during herbivory? 13:10 – 13:40 Lukas Schreiber: Cutinized and suberized plant/environment interfaces: structure, biosynthesis and function
13:40 - 14:00 Closing session
14:00 Lunch
7
GENERAL TOPICS
8 Information in biology: a time for rethinking the fundamentals
Ladislav Kováč
Centre for cognitive biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia
Email: [email protected]
All biological species live in their own species-specific world (Umwelt), delimitated by their sensors. They acquire knowledge in their species-specific manner and construct their own species-specific reality. The human species is no exception. Humans live in a world of medium dimensions (macroworld). The worlds of small dimensions (microworld), of large dimensions (megaworld), and of great complexity (multiworld) are inaccessible to them, lying outside Kant’s barriers [1]. Humans are the exceptional species on Earth due to artefacts: artefacts empower humanity to gain knowledge on the world that exists behind the boundary erected by human biological sensors. To describe this unfamiliar world, humans use the concepts of their life world (Lebenswelt), and these concepts function as metaphors [2]. Science is replete with metaphors no less than is art. Biology of the second half of the 20th century has been dominated by the metaphor of information. It has been customary to consider cognition at the exclusive property of humans, with the human mind as an organ of conscious perception, thinking, and memory, busy with “information processing”. Cognition has often been analyzed in terms of formal systems, and, accordingly, it has been thought that, in principle, cognition might be embodied in any kind of “hardware”, including the human-made computers. Upon new discoveries in biology (restricted number of genes, not much different in flies, plants, and humans; organization of genes, proteins and metabolites as scale-free networks; histone code; the heredity of frames; multiple controlling roles of small RNAs) the paradigm of information in biology may need a revision.
(1) The notion of “information” is vague not only in common life, but in biology itself. It should be used in an unambiguous restricted manner introduced by Claude Shannon. He defined information entropy, or uncertainty, in terms of well defined question Q (in which all possible answers ∑Ai are implicated) and an apriori knowledge Ka (assigning probabilities ∑pi to the answers) as S(Q|Ka) = - const ∑ pi ln pi. Information I in a message is difference between two entropies, one associated with apriori knowledge before a message Ka and another associated with aposteriori knowledge Kp after a message: I = S(Q|Ka) – S(Q|Kp) [3]. It is obvious that information is a variable the magnitude of which depends on the nature of well defined question. As the question depends on the receiver, information is a subjective quantity. A different question is being posed by a communication engineer, a human patient addressing a physician, a sperm cell heading toward an ovum, a ribosome translating messenger RNA, a molecular sensor specific for a particular ligand. Information is measured in bits, but it is neither a thing nor a sequence of digits or other units. It can be neither “processed” nor “stored”. What can be stored, embodied in sequences of DNA, in texts, in structures, is knowledge, not information. Information need not be compared with “substances” like matter or energy, but rather with “processes” like heat and work. Information is a specific, subject- dependent process of transforming data into knowledge.
(2) Life on Earth, natural (n-) life, is not a formal system, but a chemical system, ruled and constrained by the laws of chemistry. Chemical interactions differ fundamentally from other kinds of interactions, such as mechanical combinations of Lego parts. Chemistry is a science of emergence. Electromagnetic interactions between atoms produce profusion of molecules, with properties qualitatively distinct from those of their constituents. Molecules combine and/or self- assemble into supramolecular structures. Specific conditions of the environment admit specific sets of chemical construction processes, driven thermodynamically or kinetically. But the environment has also another function: it selects, or, to use a more telling metaphor, culls the products of the processes, according to their stability in the environment, their capacity to
9 “survive” under the particular conditions. This capacity is largely dependent on a degree of isomorphism, functional rather than structural, between the products of the constructions processes and the environment. This isomorphism represents knowledge. Selection for stability introduces into chemical dynamics the second “time arrow”, in addition to the first one imposed by the second law of thermodynamics: evolution. Chemical evolution on Earth has produced chemical systems with particularly great stability, which we call, somewhat arbitrarily, living systems. To maintain stability, organisms are unceasingly performing ontic work, assisted by epistemic work. A specific manner of maintaining stability is the reproduction of a system as a whole, or of its “construction plans”, or “construction algorithms”, present in the form of genes, frames, memes, or other kind of “book-keepers” [4]. They all are storing knowledge, not information. Biological evolution is a progressing process of knowledge acquisition (cognition) and, correspondingly, of growth of complexity. The acquired knowledge is embodied in constructions of organisms. The structural complexity of those constructions which carry embodied knowledge corresponds to their epistemic complexity [1].
(3) There are two kinds of knowledge and of cognition and, correspondingly, two kinds of well posed questions. There is knowledge that reduces uncertainty; the corresponding questions are inquisitory questions, and the process of knowledge acquisition consists in assimilating data by the process of information. Another kind of knowledge serves to reduce ignorance; its corresponding variables are exquisitory questions and exformation (which enables novel inquisitory questions). Because of steady accumulation of knowledge, biological evolution is advancing as a Bayesian ratchet. There is much less “information processing” than it is assumed by the “life-as-information” or “life-as-computation” metaphor [5]. Constructions at all levels, from protein molecules, through cells, tissues, individual organisms, up to social institutions and culture, represent embodied knowledge. Triggering of pre-determined responses (usually as one- bit information) seems to be a more appropriate description of life functioning than information processing.
(4) The information metaphor in biology rendered a valuable service in unravelling processes of protein synthesis and topogenesis and in deciphering nucleic acid and protein sequences. In the postgenomic era, the concept of information may become retardant or misleading. Organisms may be viewed as multihierarchical chemical systems, consisting of loosely bound modules. In their evolution, distinct selections operate at each level of hierarchy. Biological individuality is hierarchically nested, from molecular sensors up to individual organisms, communities, species and terrestrial life as a whole (Gaia), an individual at each level of hierarchy being a distinct cognitive subject engaged in ontic and epistemic work. At the deepest and most elementary level, the loosely bound modules constitute sets of molecular engines. Engine, work, embodied knowledge, and triggering may become four metaphors of a new conceptual armoury.
REFERENCES
[1] Kováč L (2000) Fundamental principles of cognitive biology. Evol Cogn 6: 51-69 [2] Lakoff G, Johnson M (2003) Metaphors we live by. University of Chicago Press, Chicago, Ill. [3] Tribus M, McIrvine EC (1971) Energy and information. Sci Amer 225: 179-186 [4] Wimsatt WC (1980) Reductionistic research strategies and their biases in the units of selection controversy. In: Nickles, T. (Ed.) Scientific discovery, Vol. 2, Historical and scientific case studies, pp. 213-259. Reidel, Dordrecht, Holland [5] Kováč L (2006) Life, chemistry and cognition. EMBO Rep 7: 562-566
10 From semi-conductors to the rythmns of sensitive plants: the research of J.C. Bose
V.A. Shepherd
Department of Biophysics, School of Physics, The University of NSW, NSW 2052, Sydney, AUSTRALIA
Email: [email protected]
Jagadish Chandra Bose (1858-1937) was one of India’s first modern scientists, and one of the world’s first biophysicists. His work with semi-conductors, radio, and microwave technology, published between 1985 and 1900 in journals including the Proceedings of the Royal Society, the “Philosophical Magazine” and “The Electrician”, was well-respected then, and remains so today. In 1900, after winning the admiration of physicists such as Rayleigh and J.J Thompson, Bose crossed the border into plant biophysics. He became a controversial figure in the West. Inventing unique instruments for simultaneously measuring bioelectric potentials and for quantifying plant movements, Bose studied plants that made rapid movements, such as the touch-sensitive Mimosa pudica and the Indian Telegraph plant Desmodium, as well as “ordinary” plants that did not make obvious rapid movements (e.g. Nauclea, the mango and the carrot). Against the tide of the times, Bose concluded that plants and animals have essentially the same fundamental physiological mechanisms. All plants have a well-developed nervous system. All plants co- ordinate their movements and responses to the environment through electrical signalling. All plants are sensitive explorers of their world, responding to it through a fundamental, pulsatile, motif involving coupled oscillations in electric potential, turgor pressure, contractility, and growth. Bose’s overall conclusion that plants have an electromechanical pulse, a nervous system, a form of intelligence, and are capable of remembering and learning, was not well received in its time. A century later, some of these concepts have entered the mainstream literature.
REFERENCES
Shepherd VA (2005) Cell Mol Biol 51: 607-619
11 Insect hearing and nanoscale mechanoreception
Daniel Robert
University of Bristol, School Biological Sciences, Woodland Road, Brsitol BS8 1UB, UK
Email: [email protected]
In animals, sensory systems can operate at the limits of what is considered physically possible (1). The mechanosensitive neurons of auditory systems are sensitive to extremely low levels of incident stimulus energy. In effect, thresholds of detection can be at energy levels close to thermal noise (kBT), or some 4•10-21 Joules (4zJ). In insects, hearing has been shown to be exquisitely sensitive, relying on mechanically well balanced receivers (2) capturing sound energy associated with a collection of neurones sensitive to mechanical stimuli (3, 4). In mosquitoes and Drosophila, the hearing organs are the antennae, with a mechanosensory organ at their base – Johnston’s organ (5). For these animals, as for any other insect and crustacean, hearing relies on the mechanotransduction performed by ciliated neurones embedded in multicellular assemblies called scolopidia. Previous research has shown that active mechanisms are at work in the hearing organs of vertebrates (review in 6), enhancing sensitivity to faint sounds and sharpening frequency selectivity. In Drosophila and the mosquito Toxorhynchites brevipalpes, hearing has been shown to be an active mechanism and several nonlinear response characteristics have been identified, such as nonlinear dynamic compression and autonomous vibrations (7, 8). Functionally, these active sensory mechanisms contribute to the nanoscale sensitivity and the response dynamics of the auditory organ (9). In insects, the basis of such active mechanisms resides in the mechanical motility of the mechanoreceptive neurons; the first neurons demonstrated to be mechanically motile (8). The molecular machinery subtending motility in insect mechanosensitive neurons is not entirely known, but it is deemed to rely on the function of the axonemal structure of the ciliated scolopidial neurons. Using mutant analysis in Drosophila, it was shown that the action of the motor molecule dynein on microtubules pairs was required, and that the transducer channels nompc contributed to the nonlinear, active response (10). Interestingly, the oscillation energy of the entire receiver –eg the antenna- could be shown to fluctuate above thermal noise under the concerted action of the receptor neurons (7-10). This suggests that cellular metabolism alone can modulate or adjust the sensitivity of response coherence of the receptor cell. It also shows that the process can take place at low energy levels. A telltale sign of such process is the presence of autonomous vibrations (7). Remarkably, autonomous vibrations have been observed in yeasts (11), yet their function is still unknown. In the opinion of the author it is likely that nanoscale mechanical vibrations will be discovered in yet other organisms. Whether they are related to the reception of mechanical energy remains to be tested. Enticingly, the increasingly refined knowledge on plant mechanoreception (see contributions by F. Telewski and F. Baluska) offers novel possibilities for comparative research. Because of their respective experimental amenability at the genetic level, tantalizing comparisons could be drawn between mechanoreception in Drosophila and that of Arabidopsis. The outcome of future research will be an integrated and general understanding of mechanisms of cellular motility and information processing, including the processes of energy dissipation and transduction at the molecular, cellular and systemic levels.
REFERENCES
1. Bialek W (1987) Physical limits to sensation and perception. Ann Rev Biophys Biophys Chem 16: 455-478 2. Göpfert MC, Robert D (2001) Turning the key on audition. Nature 411: 908. 3. Robert D, Göpfert MC (2002) Novel schemes for hearing and acoustic orientation in insects. Curr Opin Neurobiol 12: 715-720 4. Göpfert MC, Robert D (2000) Nanometre-range acoustic sensitivity in male and female mosquitoes. Proc R Soc London B 267: 453-458
12
5. Clements AN (1999) The Biology of Mosquitoes, Vol. 2 Sensory Reception and Behaviour New York: CABI Publishing. 6. Hudspeth AJ (1997) Mechanical amplification of stimuli by hair cells. Curr Opin Neurobiol 7: 480-486 7. Göpfert MC, Robert D (2001) Active auditory mechanics in mosquitoes. Proc R Soc London B 268: 333-339 8. Göpfert MC, Robert D (2003) Motion generation by Drosophila mechanosensory neurons. Proc Natl Acad Sci USA 100: 5514-551 9. Jackson JC, Robert D (2006) Nonlinear auditory mechanism enhances female sounds for male mosquitoes. Proc Natl Acad Sci USA 103: 16734-16739 10. Göpfert MC, Humphris ADL, Albert JT, Robert D, Hendrich O (2005) Power gain exhibited by motile mechanosensory neurons in Drosophila ears. Proc Natl Acad Sci USA 102: 325-330 11. Pelling A et al. (2004) Local nanomechanical motion of the cell wall of Saccharomyces cerevisae. Science 305: 1147-1150
13 Plant neurobiology: a paradigm shift in plant sciences
František Baluška1, Dieter Volkmann1, Peter W. Barlow2, Stefano Mancuso3
1 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY 2 School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK 3 LINV, University of Florence, Viale delle idee 30, 50019 Sesto f.no(FI), ITALY
Email: [email protected]
Sensory plant biology and plant electrophysiology were two lively disciplines up until the 1970s (Bünning 1959, Haupt and Feinleib 1979) but then, for somewhat obscure reasons, they showed no further development. In the last few years, however, there have been numerous advances in plant sciences which necessitate not just a revival of plant sensory biology but also the introduction of plant neurobiology (Baluška et al. 2006). First of all, and contrary to all ‘mechanistic’ predictions based on the high turgor pressure of plant cells, endocytosis has been found to be an essential process of plant cells which impinges upon almost all aspects of plant life (Šamaj et al. 2005, 2006). Moreover, recent advances in the plant molecular biology have identified, besides classical neurotransmitters, also several proteins typical of animal neuronal systems, such as acetylcholine esterases, glutamate receptors, GABA receptors and endocannabinoid signalling components, as well as indicating signalling roles for ATP, NO and ROS (Baluška et al. 2006). Importantly, plant action potentials have turned out to control processes such as actin-based cytoplasmic streaming, plant organ movements, wound responses, respiration and photosynthesis, as well as flowering (Wagner et al. 2006, Fromm and Lautner 2007). Last, but not least, there have been significant advances in ecological studies on plant-plant and plant-insect communications, in behavioral studies on memory and learning phenomena in plants (Trewavas 2005a,b), as well as the revelation that complex plant behaviour implicates neuronal signal perception, processing, and the integration of ambient signals. Plants perform neuronal-like computation not just for rapid and effective adaptation to an ever-changing physical environment but also for the sharing of information with other plants of the same species. Plants societies increase their immunity to damage after receiving warnings from attacked neighbours (Engelberth et al. 2004, Ton et al. 2007). Strategies involve, among others, the release of volatiles which then attract the enemies of the attacking herbivores (D’Alessandro et al. 2006). Moreover, there are examples of ‘war-like’ phenomena whereby invading plants kill other plants via the release of toxic allelochemicals from their root apices (Bais et al. 2006). That this hostility can be caused by root apices of other plants is a new discovery. However, roots are also well known for their ability to avoid dangerous places by actively growing away from hostile soil patches. Also in war-like mode, the root apices of parasitic plants actively recognize the roots of their prey, grow towards them and then, in order to gain control over them, send out root-hair-like processes that later develop into parasitic haustoria (Tomilov et al. 2005). Thus, by using a vast diversity of volatiles, plants are able to attract or repel diverse insects and animals, and thereby are able to shape their biotic niche. The number of volatile compounds released and received by plants for biotic communication is immense, requiring complex signal-release machinery, as well as an unprecedent ‘neuronal’ decoding apparatus for correct interpretation of received signals. These aspects of plant activity have not yet been much studied. The plant neurobiological perspective reveals several surprises when the classical plant hormones like auxin, abscisic acid, ethylene, and salicylic acid are considered from this angle. Auxin and abscisic acid elicit immediate electric responses if applied to plant cells from outside (Pickard 1984, Felle et al. 1991, Roelfsema et al. 2004, Pei and Kuchitsu 2005), suggesting that their regulated release within plant tissues may be a part of neurotransmitter-like cell-to-cell communication (for auxin see Schlicht et al. 2006). Abscisic acid signaling pathway is conserved between plants and animals and this signalling molecule both stimulates and is endogenously produced in human granulocytes in a way suggesting that it acts as endogenous proinflammatory cytokine (Bruzzone et al. 2007). Importantly, biologically active abscisic acid was isolated from brains of vertebrates (Le Page-Degivry et al. 1986) indicating possible roles of abscisic acid in
14 the central nervous system. Salicylic acid activates similar subset of MAPKs as voltage pulses (Link et al. 2002). Ethylene, a classical plant hormonone, is an anaesthetic (Campagna et al. 2003), a fact that plant physiologists have ignored. Interestingly, anaesthetics used on animals including man, induce anaesthetising effects on roots similar to those of ethylene (Powell et al. 1973). Ethylene is released in mechanically stressed plant tissues, and structurally diverse anaesthetics activate mechanosensitive channels (Martinac et al. 1990, Patel and Homore 2001, Patel et al. 2001). As ethylene is released immediately after wounding, it might act to relieve ‘pain’ in plants. Similarly, ethanol is known to relieve pain (Benedikt et al. 2007), and plants, especially roots, synthesize ethanol under stress conditions such as hypoxia and anoxia. There are numerous other plant-derived substances which manipulate the pain receptors in animals, such as capsaicin, menthol, camphor. Interestingly, the monoterpene volatiles, menthol and camphor induce oxidative stress and inhibit root growth in maize (Zunino and Zygadlo 2004), indicating that they, too, act as plant signalling molecules. Finally, plants express inhibitors that are specific to the neuronal nitric oxide synthases (Lowe et al. 2007, Osawa et al. 2007). Another example of neuronal behavior of plants is the report that prevention of nyctinastic movements of leguminous leaves causes their death while leaves allowed to ‘sleep’ stayed healthy (Ueda and Nakamura 2006). This resembles the situation in animals (Cirelli et al. 2005). Although melatonin was discovered in plants more than ten years ago (Kolár and Machácková 2005, Arnao and Hernandez-Ruiz 2006, Pandi-Perumal et al. 2006), there no scores for melatonin in the highest ranking plant journals, despite the fact that it is biochemically closely related to auxin. Melatonin mimics auxin in the induction of lateral root primordia from pericycle cells (Arnao and Hernandez-Ruiz 2007). The Arabidopsis genome encodes ten NADPH oxidases (RbohA-J) of which six are expressed only in root apices (A, B, C, E, G, I) and two (D, F) are expressed in whole seedlings including the root apices (Sagi and Fluhr 2006). Expression of eight of these molecules in root apices makes this one of the most complex signal-mediated ROS-generating organs. It is currently unknown for what developmental and signalling purposes so many different NADPH oxidases in roots are needed. A similar perplexing complexity, unique also for root apices, concerns polar auxin transport. Five types of PIN molecule (PIN1,2,3,4,7) are expressed in root apices (Blilou et al. 2005), whereas only one PIN (PIN1) is sufficient for the morphologically more complex shoot apices (Reinhardt et al. 2003, Reinhardt 2006)! What, then, is so special about root apices? This is a tough question, but answers seem to be emerging in the multitude of recent data, not easily interpretable by the classical plant physiological approach, but comprehensible from the approach of plant neurobiology (Baluška et al. 2005, Brenner et al. 2006). One of them involves the idea that the transition zone of root apices acts as some kind of ‘command centre’ (Baluška et al. 2004). Despite a relatively simple body organization, plants need sophisticated sets of coordinative processes. Besides their root-shoot coordination, there is also need for coordination amongst radial tissues, especially within and between the cortex and stele. Action potentials run preferentially in an axial direction and they link root and shoot apices. Despite the modular and apparently decentralized organization of the plant body, there are several critical situations requiring ‘centralized’ decisions, such as, for instance, the onset of flowering as well as the onset and breakage of dormancy. Although these decisions are based on information retrieved via numerous distant organs, they imply some central ‘processor’ which would reliably control the whole plant body. Importantly, any wrong decision would have detrimental consequences for the whole plant. Moreover, internal circadian pacemakers of animals are located in their brains. The transition zone of root apices is the only zone in the plant body showing ‘brain-like’ oscillatory patterns of cellular activities responding also to leaf wounding (Mancuso and Marras 2006). Moreover, cells of this zone are the only ones to express up to five different PIN efflux carriers (Verbelen et al. 2006, Bandyopadhyay et al. 2006). Across the F-actin and myosin VIII-enriched plant synapses (Baluška et al. 2005), PINs drive complex transcellular patterns of polar auxin transport. As this auxin transport is driven via vesicular secretion (Schlicht et al. 2006), auxin elicits electrical responses in adjacent cells (Felle et al. 1991), and it synchronizes cell activities within a cell file (Nick 2006, Maisch and Nick 2007), auxin fulfils the minimum criterion for being a neurotransmiter-like signalling molecule in plants. Human perception of the outside world relies on a so-called ‘neural code’ which links sensory signals and neuronal responses. Similarly, in plants, numerous parameters of the physical environment, especially, light, temperature, and gravity, are continuously monitored. Polar auxin transport translates perceived and processed sensory information into adaptive physiological and 15 motoric responses. New concepts are needed, and new questions must be asked, for advancing our rudimentary understanding of the communicative nature of sensory plants.
REFERENCES
Arnao MB, Hernandez-Ruiz J (2006) Plant Signal Behav 1: 88-94 Arnao MB, Hernandez-Ruiz J (2006) J Pineal Res 42: 147-152 Baluška F, Šamaj J, Menzel D (2003) Trends Cell Biol 13: 282-285 Baluška F, Mancuso S, Volkmann D, Barlow PW (2004) Biologia 59: 9-17 Baluška F, Volkmann D, Menzel D (2005) Trends Plant Sci 10: 106-111 Baluška F, Mancuso S, Volkmann D (2006) Communication in Plants: Neuronal Aspects of Plant Life. Springer-Verlag Bandyopadhyay A et al. (2006) Biochenm Soc Trans 35: 137-141 Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco J (2006) Annu Rev Plant Biol 57: 233-266 Benedikt J, Teisinger J, Vyklicky L, Vlachova L (2007) J Neurochem 100: 211-224 Blilou I et al. (2005) Nature 433: 39-44 Brenner E et al. (2006) Trends Plant Sci 11: 413-419 Bruzzone S et al. (2007) Proc Natl Acad Sci USA 104: 5759-5764 Bünning E (1959) In: Handbuch der Pflanzenphysiologie. Springer Verlag Campagna JA, Miller KW, Forman SA (2003) N Engl J Med 348: 2110-2124 Cirelli C et al. (2005) Nature 434: 1087-1092 D’Alessandro M, Held M, Triponez Y, Turlings TC (2006) J Chem Ecol 32: 2733-2748 Engelberth J, Alborn HT, Schmelz EA, Tumlinson JH (2004) Proc Natl Acad Sci USA 101: 1781-1785 Felle H, Peters W, Palme K (1991) Biochim Biophys Acta 1064: 199-204 Fromm J, Lautner S (2007) Plant Cell Environm 30: 249-257 Haupt W, Feinleib ME (1979) In: Encyclopedia of Plant Physiology, Springer Verlag Maisch J, Nick P (2007) Plant Physiol, In press Mancuso S, Marras AM (2006) Plant Cell Physiol 47: 401-409 Nick P (2006) Plant Biol 8: 360-370 Le Page-Degivry et al. (1986) Proc Natl Acad Sci USA 83: 1155-1158 Link VL, Hofmann MG, Sinha AK, Egness R, Strnad M, Roitsch T (2002) Plant Physiol 128: 271-281 Lowe ER, Everett AC, Lau M, Dunbar AY, Michner D, Osawa Y(2007) Phytomedicine 1: 1-7 Kolár J, Machácková I (2005) J Pineal Res 39: 333-341 Osawa Y, Lau M, Lowe ER (2007) Plant Signal Behav, In press Pandi-Perumal SR et al. (2006) FEBS J 273: 2813-2838 Pei Z-M, Kuchitsu K (2005) J Plant Growth Regul 24: 296-307 Pickard BG (1984) Plant Cell Environm 7: 171-178 Powell JN, Grant CJ, Robinson SM, Radford SG (1973) Brit J Anaesth 45: 682-690 Reinhardt D (2006) Curr Opin Plant Biol 8: 487-493 Reinhardt D et al. (2003) Nature 426: 255-260 Roelfsema MRG, Levchenko V, Hedrich R (2004) Plant J 37: 578-588 Sagi M, Fluhr R (2006) Plant Physiol 141: 336-340 Šamaj J, Read ND, Volkmann D, Menzel D, Baluška F (2005) Trends Cell Biol 15: 425-433 Šamaj J, Baluška F, Menzel D (2006) Endocytosis in Plants. Springer-Verlag Tomilov AA, Tomilova NB, Abdallah I, Yoder J (2005) Plant Physiol 138: 1469-1480 Ton J et al. (2007) Plant J 49: 16-26 Schlicht M et al. (2006) Plant Signal Behav 1: 122-133 Trewavas A (2005a) Trends Plant Sci 10: 413-419 Trewavas A (2005b) Naturwissenschaften 92: 401-413 Ueda M, Nakamura Y (2006) Nat Prod Rep 23: 548-557 Verbelen J-P, De Cnodder T, Le J, Vissenberg K, Baluška F (2006) Plant Signal Behav 1: 296-304 Wagner E, Lehner L, Normann J, Veit J, Albrechtova J (2006) In: Communication in Plants: Neuronal Aspects of Plant Life, F Baluška, S Mancuso, D Volkmann (eds), Springer-Verlag, 369-389 Zunino MP, Zygadlo JA (2004) Planta 219: 303-309
16 Plant intelligence: why, why not, or where?
Fatima Cvrckova1, Viktor Zarsky1,2
1 Department of Plant Physiology, Faculty of Sciences, Charles University, Vinicna 5, CZ 128 43 Praha 2, Czech Republic 2 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojova 135, CZ 160 00 Praha 6, Czech Republic
Email: [email protected]
The authors are supported by the MSM 0021620858 project.
The concept of plant intelligence, as proposed by Anthony Trewavas, has raised considerable discussion that has contributed also to the birth of plant neurobiology (see Trewavas 2002, 2003, 2004, 2005a,b, Firn 2004, Brenner et al. 2006). However, plant intelligence remains loosely defined, and, as a result of attempts to persuade its opponents, it became either practically synonymous to Darwinian fitness (“adaptively variable behaviour” or “ability of an individual to perform in its environment”), or reduced to a mere decorative metaphor. A more strict view can be taken, with emphasis on individual memory and learning. Even this has to be done cautiously, the main problem being the definition of memory itself. To qualify as memories, traces of past events have to be not only stored, but also actively accessed (or at least accessible). We propose a variety of Occam´s razor approach for eliminating false candidates of possible plant intelligence phenomena in this stricter sense: a particular behavior of the plant may be considered “intelligent” only if it cannot be approximated by an algorithmic model that does not require recourse to stored information about past states of the individual or its environment. Re-evaluation of the phenomena previously presented as examples of plant intelligence shows that only some of them pass our test, while others do not.
REFERENCES
Brenner E, Stahlberg R, Mancuso S, Vivanco J, Baluska F, Van Volkenburgh E (2006) Trends Plant Sci 11: 413-419 Firn R (2004) Ann Bot 93: 345-351 Trewavas A (2002) Nature 415: 841 Trewavas A (2003) Ann Bot 92: 1-20 Trewavas A (2004) Ann Bot 93: 353-357 Trewavas A (2005a) Naturwissenschaften 92: 401-413 Trewavas A (2005b) Trends Plant Sci 10: 413-419
17 Are eukaryotes truly intelligent?
Paco Calvo Garzón
Department of Philosophy, University of Murcia, Murcia 30100, Spain
Email: [email protected]
Although Plant Neurobiology emphasizes the interdisciplinary effort whose ultimate target is the study of the complex patterns of behaviour of plants qua information-processing systems, it is not clear what we mean by “information-processing system”. Most researchers adopt a computational perspective, according to which information-processing boils down to the manipulation of symbols /subsymbols according to algebraic or statistical rules. We may nevertheless adopt an embodied, embedded perspective (Thelen and Smith, 1994), and interpret information-processing systems in non-computational and/or non-representational terms (Calvo Garzón, in press). According to this view, cognition is to be understood in the continuous interplay of brain, body and environment. In my talk, I propose to study the integration of contemporary scientific knowledge in Cognitive Neuroscience (Gazzaniga et al., 2002) and Plant Neurobiology (Baluška et al., 2006) under this lens in order to assess whether eukaryotes can be interpreted as genuinely intelligent. Trewavas has defended the integration of scientific knowledge of plants and animals in a number of works (2005; and references therein), arguing that plants do indeed count as intelligent organisms in much the same way as animals do. However, although sympathetic to Trewavas (2005) position, I shall turn his framework upside down. In particular, I shall consider time-estimation in relation to the distinction between online plant behaviour (flower heliotropism) and offline plant behaviour (leaf heliotropism) - specifically, plants’ nocturnal reorientation in the absence of solar-tracking (Schwartz and Koller, 1986). This case neatly illustrates why plants and animals’ (anticipatory) competencies can indeed be interpreted as two sides of the same coin: Both animals and plants can solve complex problems and react adaptively to environmental contingencies. In my view, nevertheless, once an embodied-embedded picture is granted, all eukaryotic organisms, although subject to an information-processing analysis with individual cells as computational building blocks, should be interpreted in non-computational terms. This view will allow us to reassess Trewavas’ insights under a new light. An embracing picture of eukaryotic anticipatory capacities will allow us to place amoebae, plants, and animals (human and non-human) along a continuum. Once we look at the shared cellular and molecular mechanisms of these life forms, we have a reason to unify the knowledge obtained along the spectrum; in non-computational and non- representational terms, however. The ultimate aim is to discuss the sort of novel predictions that such a model may generate in order to test intelligence.
REFERENCES
Baluška F, Mancuso S, Volkmann D (2006) Communication in Plants: Neuronal Aspects of Plant Life: Springer-Verlag Calvo Garzón P (2007) Towards a general theory of antirepresentationalism. British J Philos Sci, In press Gazzaniga M, Ivry R, Mangun G (2002) Cognitive Neuroscience: The Biology of the Mind: Norton. Schwartz A, Koller D (1986) “Diurnal Phototropism in Solar Tracking Leaves of Lavatera cretica”, Plant Physiol 80: 778-781 Thelen E, Smith L (1994) A Dynamic Systems Approach to the Development of Cognition and Action: MIT Press. Trewavas A (2005) Green plants as intelligent organisms. Trends Plant Sci 10: 413-419
18 A unified hypothesis of mechanoperception in plants
Frank W. Telewski
W.J. Beal Botanical Garden, Department of Plant Biology, Michigan State University, East Lansing, MI 48824, U.S.A.
Email: [email protected]
The ability to sense and respond to physical environmental stimuli is of key importance to all living things. Among the common environmental stimuli detected by living organisms are light, temperature, and a variety of chemical signals. A number of environmental stimuli appear to be closely related and can be considered as physical-mechanical stimuli, requiring the perception of a differential mechanical force or pressure gradient by the living cell. These include the perception of gravity, self-loading and internal growth strains, mechanical loading, touch, sound, and the state of hydration within a cell (turgor pressure). Recent advances have lead to the proposal of a plant-specific mechanosensory network within plant cells that is similar to that previously described in animal systems (Jaffe et al. 2002, Baluška et al. 2003). This sensory network is the basis for a unifying hypothesis which may account of the perception of numerous mechanical signals including gravitropic, thigmomorphic, thigmotropic, self-loading, growth strains, turgor pressure (drought and flooding stress), xylem pressure potential, and sound (Telewski 2006). The current state of knowledge of a mechanosensory network in plants is reviewed and considerations given to two different mechanoreceptor models: a plasmodesmata- based cytoskeleton-cell membrane-cell wall (CMCW) network (Baluška et al. 2003) vs. stretch- activated ion channels (Ding and Pickard 1993, Pickard and Fujiki 2005). Post-mechanosensory physiological responses to mechanical stresses are also reviewed along with recommendations for directing future research in the area of mechanoperception and response.
REFERENCES
Baluška F Šamaj J, Wojtaszek P, Volkmann D, Menzel D (2003) Plant Physiol 133: 483-491 Ding, JP Pickard BG (1993) Plant J 3: 83-110 Jaffe MJ, Leopold AC, Staples RA (2002) Am J Bot 89: 375-382 Pickard BG, Fujiki M, (2005) Funct Plant Biol 32: 863-879 Telewski FW (2006) Am J Bot. 93: 1306-1316
19 Spatio-temporal dynamics of the electrical network activity in the root apex. A multi- electrode array (MEA) study.
Masi E1, Ciszak M2, Montina A2,3, Malachovska V1, Mugnai S1, Azzarello E1, Pandolfi C1, Renna L1, Stefano G1, Voigt B1, Hlavacka A1, Arecchi FT2,3, Mancuso S1
1 LINV - Laboratorio Internazionale di Neurobiologia Vegetale - University of Florence, Viale delle idee 30, 50019 Sesto F.no (FI), ITALY 2 Department of Physics, University of Florence, Via G. Sansone 1, Sesto F.no (FI), ITALY 3 INOA - Istituto Nazionale di Ottica Applicata, Largo E. Fermi 6, 50125 Firenze, ITALY
Email: [email protected]
Root cells have been a popular research tool for decades because they allow easy access to individual cells for electrophysiological recording and stimulation, pharmacologic manipulations and high resolution microscopic analysis. However, it is technically difficult to record from and stimulate more than three cells using standard intracellular microelectrodes, and those cells usually die within minutes or, barely, hours. Thus any distributed/synchronized electrical activity is missed without a multi-unit approach. Multi-electrode arrays (MEAs) provide a tool to record from and stimulate many cells (up to hundreds) of the same root apex, concurrently and non-invasively. Since the array substrate is made of transparent glass, cell morphology can be easily monitored by the use of an inverted microscope or using fluorescent labels and a confocal. Here, for the first time in plant science, we use a 60-channels MEA to study in thick root apex slices the spatio-temporal characteristics of the electrical network activity. We observed an intense spontaneous electrical activity as well as stimulation-elicited bursts of spikes locally propagating. Our data indicate that synchronous activity of the cells emerges spontaneously throughout the time evolution. The strict similarity of the electrical behaviour recorded with the behaviour showed by neural cell culture may reflect an intrinsic capacity of the root apex to generate functional networks.
20 The minimum set of cells required to enervate the ‘Root Brains’ of plants
Peter Barlow1, Jacqueline Lück2
1 School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK 2 Atelier de Structuralisme Végétal, 1226 Chemin du Val d’Arenc, 83330 Le Beausset, FRANCE
Email: [email protected]
When a root system – which includes the collective ‘heads’ of a plant – burrows down into the soil, all its ‘brains’, which are continually being created as the root system extends and ramifies, have to be supplied (‘enervated’) with nervous tissue [1] that connects with the posterior portion of the plant. So, two important questions are: what are the minimal tissue and neuro-physiological systems that enervate a ‘root brain’, and how are these systems continuously developed? A minimal ‘nervous’ tissue can be discerned in so-called ‘hair roots’ – modified roots which are characteristic of the plant families Ericaceae and Epacridaceae [e.g., 2, 3]. Published papers on hair-root anatomy [e.g., 3] allow analysis of the structure of the plerome (vascular cylinder), a tissue constituting the Channel and Net of the nervous system which supplies the root brain at the root apex. To support this anatomical analysis, a stereotypic cell lineage of the plerome was gleaned from the literature on Arabidopsis [4]. Here, the plerome is derived from four apically located stem cells. Each stem cell gives rise, via radial divisions, to one quadrant of plerome whose cell numbers are subsequently amplified by transverse divisions. One pair of self-similar plerome quadrants contains both xylem and phloem cells; the other pair of quadrants contains xylem only; present in all four quadrants are parenchyma and pericycle cells, some of which are probably essential for root nervous function. On the basis of the above analysis of Arabidopsis, we can now see that the anatomy of the hair roots helps to define the minimal plerome tissue composition which is required by any root. Thus the hair-root plerome indicates that hair roots can be of two types: the first contains just one of the xylem-only quadrants; roots of the second type bear one of the xylem-plus- phloem-containing quadrants (which include one sieve tube plus a strand of companion cells, and one file of tracheids). We presume that the single xylem-containing quadrant could not support much root growth, and so roots of this first type are expected to have a limited life. By contrast, one xylem-plus-phloem-containing quadrant can evidently support viable root extension and solute uptake, as well as providing an integrated ‘nervous’ function for the plant. Although aspects of our analysis pertaining to hair roots are somewhat conjectural owing to lack of direct evidence from this material, especially from the crucial embryogenic stages of development, we suggest that a root which contains only one of the four plerome quadrants has had three of its plerome stem cells deleted sometime after the establishment of the complete root organ either in the embryo or in a lateral root primordium. The remaining single plerome stem cell can support development of a root if the tissue derived from it includes phloem. However, such a root has a much reduced diameter – i.e. it is a hair root. This putative stem-cell death scenario reminds us of that which attends animal neural tissue development [5]. The continued differentiation and penetration of plerome into the growing hair-root apex, even after much of the usual plerome tissue complement has been deleted, is due to the phenomenon of homeogenetic induction [6, 7]. This process allows the retention of a diminished plerome, but one that possesses a neuronally active xylem-plus-phloem quadrant.
REFERENCES
1 Baluška F, Volkmann D, Hlavacka A, Mancuso S, Barlow PW (2006) In: Communication in plants. Neuronal aspects of plant life (F Baluška, S Mancuso, D Volkmann, eds), pp 19-35. Springer- Verlag, Berlin 2 Burgeff H (1961) Mikrobiologie des Hochmoores mit besonderer Berücksichtigung der Erikazeen-Pilz-Symbiose. G Fischer-Verlag, Stuttgart 3 Allaway WG, Ashford AE (1996) Ann Bot 77: 383-388
21 4 Mähönen AP, Bonke M, Kauppinen L, Riikonen M, Benfey PN, Helariutta Y (2000) Genes Devel 14: 2938-2943 5 Bessis A, Béchade C, Bernard D, Roumier A (2007) Glia 55: 233-238 6 Lang A (1974) Brookhaven Symp Biol 25: 129-143 7 Barlow PW (1984) In: Positional controls in plant development (PW Barlow, DJ Carr, eds), pp 281-318. Cambridge University Press, Cambridge
22 Mechanisms of magnetoreception in plants and fungi
Paul Galland
Fachbereich Biologie, Philipps-Universität Marburg, Karl-von-Frisch Str. 8, D-35032 Marburg, GERMANY
Email: [email protected]
The ability to respond to magnetic fields is ubiquitous among the five kindoms of organisms. Apart from the mechanisms that are at work in bacterial magnetotaxis (ferrimagnetism) none of the numerous magnetobiological effects is as yet completely understood in terms of the underlying physical principles. Plants react in many ways to the geomagnetic field and to strong continuous as well as alternating magnetic fields (Galland and Pazur 2005). Because of a lack of model organisms and model reactions the magnetobiology of plants, fungi and microorganisms has remained largely on a phenomenological level. The problem is compounded by the fact that magnetic effects are observed for a huge range of magnetic flux densities that cover more than 10 orders of magnitude. To come to grips with such a huge dynamic range which is similar to that of human vision and numerous photoresponses of plants one would expect the study of dose-response relationships to be of paramount importance. It comes thus as a surprise that such studies are practically nonexistent. They would be particularly needed in view of the fact that responses are elicited by weak magnetic fields, such as the geomagnetic field, whose energy content is several orders of magnitude below the thermal energy content (kT- paradox). As a result most of the studies are characterized by a lack of mechanistic insight even though physics provides several theories that serve as guideposts for biological experimentation and that offer solutions for the kT-paradox. Beside ferrimagnetism, which is well proven for bacterial magnetotaxis and some cases of animal navigation, three further mechanisms for magnetoreception receive currently major attention: (i) the “radical-pair mechanism” consisting in the modulation of singlet-triplet interconversion rates of a radical pair by weak magnetic fields, and (ii) the “ion cyclotron resonance” mechanism, and (iii) the “coherence” mechanism. Recent studies with Arabidopsis (Ahmad et al. 2007) and Phycomyces show that blue-light reception and magnetoreception are intimately connected, an observation that is best explained in the context of the radical-pair mechanism.
REFERENCES
Ahmad M, Galland P, Ritz T, Wiltschko R, Wiltschko W (2007) Planta, In press Galland P, Pazur A (2005) J Plant Res 118: 371-389
23 Responses to environmental stimuli by internodal cells of Chara corallina
Mark P. Staves
Biology Department, Grand Valley State University, 1 Campus Dr., Allendale, MI 49401, USA
Email: [email protected]
The giant (2-10 cm long, 0.5-1.0 mm wide) internodal cells of Chara corallina provide a simple, easily manipulated tool for investigating responses to environmental stimuli in single cells. We have identified three responses to environmental stimuli: polarity of cytoplasmic streaming induced in response to gravity or hydrostatic pressure; action potential generation in response to mechanical or electrical stimulation and; tropistic growth in response to light and gravity. As can be predicted by their large size, Chara internodal cells exhibit a rapid (ca. 100 μm s-1) rotational cytoplasmic streaming. Streaming proceeds at equal rates basipetally and acropetally in horizontal cells. In contrast, gravity induces a polarity of cytoplasmic streaming in vertically- oriented cells such that the downwardly-directed stream moves ca. 10% faster than the upwardly-directed stream – regardless of the morphological identity of the cell ends. This gravity-induced polarity of cytoplasmic streaming can be mimicked by the application of a unilateral hydrostatic pressure to either end of a horizontal cell. Hydrostatic pressure applied to the bottom of a vertically-oriented cell can eliminate or even reverse the gravity-induced response. The induction of a polarity of cytoplasmic streaming by both gravitational pressure and hydrostatic pressure is Ca2+-dependent and requires both ends of the cell to be intact. Cytoplasmic streaming in Chara internodal cells ceases in response to an action potential. The ability of electrical and mechanical stimulations to generate action potentials in Chara is Ca2+-dependent and the response to each stimulus may be inhibited in a similar way by Ca2+ antagonists. Since ligated cells exhibit cessation of cytoplasmic streaming in response to mechanical and electrical stimulation, intact cell ends are not required for this response. Chara internodal cells exhibit tropistic growth in response to both light and gravity signals. In the absence of light, Chara internodal cells are negatively gravitropic. A light stimulus, opposite to the vector of gravity, will induce phototropic growth which will inhibit (at a flux of ca. 1 μmol m-2 s-1) or reverse (at a higher flux) the gravity-induced response. Intact cell ends are required for gravitropism but not photropism of internodal cells. Because of their large size and responsiveness to environmental stimuli, the internodal cells of Chara are particularly suited as a model system to elucidate signal transduction pathways in plants. Some progress in revealing insights into interactions between signal transduction pathways in these cells will be discussed.
24
MOLECULES, SIGNALLING & CELL BIOLOGY
25 Roles of endocytosis regulation in plant physiology and development
Akihiko Nakano1,2, Takashi Ueda1
1 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo and 2 Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, Wako, Japan.
Email: [email protected]
Crucial roles of endocytosis in various plant functions are emerging recently, but its molecular mechanism and physiological significance still remain largely unknown. Using a model plant, Arabidopsis thaliana, we have been studying the molecular mechanism of endocytosis with a special focus on Rab5 GTPases. Three Rab5 members, Ara7, Rha1 and Ara6, are encoded in the Arabidopsis genome, which are all involved in endocytosis. Ara7 and Rha1 are orthologs of mammalian Rab5, and Ara6 is a plant-unique type of Rab5 member. Through genetic analysis, we have found that these two subgroups function antagonistically in various developmental stages, although they are all activated by the practically sole GEF, AtVps9a. Moss and spikemoss also have the Ara6-type Rab5, thus this subgroup is well conserved among land plants. These data indicate that land plants have evolved a quite unique mechanism for the regulation of endocytosis, which is essential for the plant life.
26 EXO70A1, a putative exocyst subunit, is important for polar growth and plant development
Lukáš Synek1, Nicole Schlager3, Marek Eliáš2, Edita Drdová1, Marie-Theres Hauser3, Viktor Žárský1,2
1 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, CZECH REPUBLIC 2 Department of Plant Physiology, Faculty of Sciences, Charles University, Vini?ná 5, 128 44 Prague 2, CZECH REPUBLIC 3 Institute of Applied Genetics and Cell Biology, BOKU - University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, AUSTRIA
Email: [email protected]
The exocyst is a hetero-oligomeric protein complex involved in exocytosis and has been extensively studied in yeast and animal cells. Current analyses of mutations in genes encoding plant homologs of three subunits (A. thaliana SEC8, EXO70A1, and maize sec3) support the notion that an exocyst complex is also present in plant cells. Our bioinformatic analysis revealed that the Arabidopsis genome contains 23 EXO70 genes. Based on expression analysis, we identified EXO70A1 as the main EXO70 gene in Arabidopsis. We characterized two independent T-DNA insertional mutants in EXO70A1 gene. Heterozygous EXO70A1/exo70A1 plants appear normal and segregate in the 1:2:1 ratio. However, exo70A1 homozygotes exhibit multiple phenotypic defects. Polar growth of root hairs and stigmatic papillae is disturbed. Organs are generally smaller, plants show loss of apical dominance and an indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that EXO70A1, the putative exocyst subunit, is involved in cell and organ morphogenesis.
27 Inositol trisphosphate receptor in plants – is it real?
Ondřej Krinke1, Jan Martinec2
1 Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Technická 3, 166 28 Prague 6, Czech Republic 2 Institute of Experimental Botany, The Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic
Email: [email protected]
Our research is supported by Ministry of Education, Youth and Sports, grants No. LC 06034.
The receptor for D-myo-inositol 1,4,5-trisphosphate (InsP3-R) has been well documented in animal cells. It constitutes an important component of the intracellular calcium signalling system. Today the corresponding genes in many species have been sequenced and the antibodies against some of the InsP3-Rs are available.To the contrary very little is known about its plant counterpart. Only few published works have dealt directly with this topic. We have summarized the available relevant data and figured out some properties of the putative plant receptor(s) including the in vivo evidence, its electrophysiology, parameters of the InsP3-induced calcium release and InsP3 binding, its immunological cross-reactivity and its subcellular localization. Phosphatidylinositol-specific phospholipase C is undoubtedly parts of plant signalling pathways. Nevertheless, it is not sure that the InsP3-R is present in plant cells unless any corresponding gene is identified.
28 Identification of novel abscisic acid signal transduction components and ion channel regulation mechanisms in guard cells
Julian I. Schroeder, Tae-Houn Kim, Izumi Mori, Maik Böhmer, Yingzhen Yang, Yoshiyuki Murata1, Erwin Grill2, Yong-Fei Wang
Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0116 1 Dept. of Agriculture Okayama University, Okayama 700, Japan 2 Botany Institute, T.U. Munich, Germany
Email: [email protected]
Guard cells have been developed as a model system for dissecting ion channel functions and regulation mechanisms. Previous studies have shown that two classes of calcium-induced stomatal closing can be separated: rapid Ca2+ reactive and long term Ca2+ programmed stomatal closing (G. Allen et al., 2001, Nature 411). However, genetic evidence has been lacking for Ca2+ sensor mutants that disrupt Ca2+- and abscisic acid-regulated stomatal movements. In addition, a Ca2+-independent pathway functions in the abscisic acid (ABA) response. We have recently identified two calcium-dependent protein kinases (CDPKs) that function in abscisic acid (ABA) and Ca2+ regulation of guard cell ion channels and stomatal closing (I. Mori et al., 2006 PLoS Biol.). Furthermore, several independent signal transduction analyses suggest a new model for how plant cells can achieve specificity in calcium signaling through “priming” and “de- priming” of Ca2+ sensitive mechanisms (J. Young et al., 2006 PNAS). Further evidence that correlates with this “Ca2+ sensor priming” hypothesis will be presented. An important target of ABA and cytosolic Ca2+ signaling is the activation of S-type anion channels in guard cells. Progress at identifying new genes that are essential for mediating this response will be presented. Evidence for a parallel pathway that functions in the ABA signaling network will also be presented. Genetic, genomic and signal transduction analyses in several laboratories indicate that genetic redundancies and robustness exist within the abscisic acid signal transduction network. To address this complexity we have pursued gain-of-function genetic screens (e.g. J. Kuhn et al., 2006 Pl. Physiol.) and genomic approaches (e.g. N. Leonhardt et al., 2004 Pl Cell; Mori et al., 2006 PLoS Biol.). More recently we have developed a chemical genetics approach that allows high-throughput screening for molecules and mutants that affect ABA signal transduction. Progress at isolating a small molecule that blocks ABA responses and isolation and characterization of mutants in ABA signaling that are insensitive to this compound will be presented.
29 A novel class of microtubule-binding proteins control root growth behavior and anisotropic cell expansion in Arabidopsis
Patrick H. Masson1, Robyn M. Perrin1,2, Yan Wang1,3, Christen Y.L. Yuen1,4, Jessica Will1
1 Laboratory of Genetics, University of Wisconsin-Madison, 425G Henry Mall, Madison WI 52706, USA Current addresses: 2 Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706, USA 3 110 Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA 4 Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI96822, USA
Email: [email protected]
Research funded by NASA, HATCH funds and the UW-Madison Graduate School
In plants, anisotropic cell expansion is a tightly regulated process that contributes to morphogenesis. In addition to modulating overall growth rates, this process orchestrates directional growth responses to environmental cues, allowing plant organs to grow toward environments that are better suited for their primary functions. A screening for Arabidopsis thaliana mutants displaying defective seedling-root growth behavior on hard surfaces allowed us to identify WVD2, a gene that regulates both anisotropic cell expansion and the spiral growth of most organs. WVD2 over-expressing plants also display enhanced thigmomorphogenesis and sensitivity to salt and sucrose treatments, suggesting a role in the transduction of these signals. WVD2 encodes a microtubule (MT)-binding protein that promotes the bundling of MTs in vitro, and affects the organization of cortical MTs in expanding cells of the root (Yuen et al, 2003; Perrin et al, 2006). This protein shares a 95 amino-acid motif with 7 other Arabidopsis proteins, and initial phenotypic analyses of mutants that either over-express or are defective in one or several of these genes suggest distinct, though overlapping, roles for the WDL proteins in the regulation of MT-dependent morphological processes. We hypothesize that WVD2 and related WDL proteins contribute to the control of organs growth behavior in response to environmental and endogenous cues by regulating the organization and/or dynamic properties of cortical MTs in expanding cells, thereby modulating the patterns of anisotropic cell expansion.
REFERENCES
Perrin RM, Wang Y, Yuen CYL, Will J, Masson PH (2006) Plant J, In press Yuen CYL, Pearlman R, Silo-Suh L, Hilson, P, Carroll KL, Masson PH (2003) Plant Physiol 131: 493- 506
30 Domain-specific cell wall-plasma membrane interface
Przemysław Wojtaszek1,2, Anna Kasprowicz1, Magdalena Łuczak2, Marta Derba1, Ewelina Rodakowska1, Agnieszka Łapa2, Paweł Zawadzki1, Daniel Kierzkowski1
1 Department of Molecular and Cellular Biology, Adam Mickiewicz University, Międzychodzka 5, 60-371 Poznań, POLAND 2 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, POLAND
Email: [email protected]
The research presented has been supported by the Ministry of Science and Higher Education grants 3 P04C 011 22, PBZ-KBN-110/P04/2004, 2 P04C 055 29, N302 028 32/2243 to P.W.
At the end of XX century, Phil Lintilhac, discussing various concepts of cellularity, proposed that the idea of the basic unit of life could be reduced to “event surface” – the boundary separating internum from externum (Lintilhac 1999). Autopetic theory added one additional requirement to this concept: the boundary has to be constructed and maintained by the living system itself (Varela et al. 1974). Here, we will use these metaphors as a useful tool for description of some of the functionalities of the cell wall-plasma membrane-cytoskeleton (WMC) continuum with special attention paid to the plant wall component. Cell walls are the outermost functional zone of plant cells. Although they surround the individual cells, at the same time they form a part of supracellular structure – the apoplast. In suspension-cultured cells, cell walls are also embedded in the culture medium which can be thought of as a kind of superapoplast. Cell walls have been usually considered as a structural component of the cell and of the plant. Here we would like to draw attention to other wall functions, namely the signalling one and that of physical anchor for other cellular components. To illustrate this, some recent data indicating the possibility of extracellular generation of signals affecting the cell fate will be presented. Moreover, potential other routes for auxin transport will also be discussed. On the other hand, we will also present data showing that the WMC continuum can be treated as a complex sensory medium detecting and transmitting information from the walls to the cytoskeleton for signalling and regulation.
REFERENCES
Lintilhac P (1999) BioScience 49: 59-68 Varela FG, Maturana HR, Uribe R (1974) BioSystems 5: 187-196
31 Stem cell signalling networks in plants
Bruce Veit1, Natasha Forester1, Suzanne Lambie1,2, Vernon Trainor1,2, Karen Wilson1
1 Forage Biotechnology, AgResearch, Private Bag 11008, Palmerston North, New Zealand 2 Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand
Email: [email protected]
Although many aspects of multicellularity differ between plants and animals, both feature groups of cells that enable indeterminate patterns of cell division and growth. In an effort to understand how the behaviour of these cells is regulated, we review different types of meristematic tissues in higher plants, with a particular focus on the shoot and root apical meristems (SAM and RAM) (1). We consider whether concepts developed to explain stem cell behaviour in animals may also have relvance to plants, particularly with regard to how such groups of cells are established and maintained. Molecular genetic data is reviewed that suggests that while the establishment and organogenic related functions of the SAM and RAM differ, conserved mechanisms operate that help maintain initial cells in a pluripotent state. Finally we consider how a unique plant specific family of RNA binding proteins, termed MEI2-like, may function as part of larger signalling networks to regulate differentiaton related processes (2,3,4).
REFERENCES
1) Veit BE (2006) Stem Cell Signalling Networks in Plants. Plant Mol Biol 60: 793-810 2) Kawakatsu T, Itoh J, Miyoshi K, Kurata N, Alvarez N, Veit B, Nagato Y (2006) PLASTOCHRON2 regulates leaf initiation and maturation in rice. Plant Cell 18: 612-625 3) Anderson G, Veit B, Hanson M (2005) The Arabidopsis AtRaptor genes are essential for post- embryonic plant growth. BMC Biology 3:12 4) Alvarez N, Gilman C, Jeffares D, Trainor V, Hanson M, Veit B (2004) Diversification of genes encoding mei2-like RNA binding proteins in plants. Plant Mol Biol 54: 653-670
32 Endogenous programmed cell death triggers in plants
Nan Yao1, Jean T. Greenberg2
1 State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China 2 Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA
Email: [email protected]
It has been clear that the precursors and breakdown products of both chlorophyll and heme, such as porphyrins and related molecules, are extremely phototoxic; thus, their synthesis and degradation are highly compartmentalized and regulated. Accumulation of porphyrin compounds is known to cause cell death in both plants and animals. Moreover, ceramides and their related sphingolipid derivatives are bioactive lipids that play important roles as second messengers and as dampening signals for apoptosis in animals (Hannun and Obeid 2002). In an effort to search plant programmed cell death (PCD) triggers, we used Arabidopsis two mutants termed accelerated cell death 2 (acd2) and acd5. ACD2 and ACD5 encode red chlorophyll catabolite (RCC) reductase (Mach et al., 2001) and ceramide kinase (Liang et al., 2003), respectively. We found that protoporphyrin IX (PPIX, a precursor to heme and chlorophyll) and C2 ceramide trigger an apoptotic-like response in Arabidopsis protoplasts (Liang et al., 2003; Yao et al., 2004). PPIX was enhanced in ACD2-deficient plants and reduced in ACD2-overexpressing plants, indicating that PPIX triggers apoptotic cell death dependent on ACD2. Furthermore, PPIX induced altered ACD2 localization and levels (Yao et al., 2006). We also found that C2 ceramide induced PCD via its effect on mitochondrial permeability transition. The data suggest that a mitochondrial membrane potential loss was commonly induced early during plant PCD and was important for PCD execution, as evidenced by the concomitant reduction of the change in mitochondrial membrane potential and PCD by cyclosporin A. Our data suggest that RCC (and related porphyrin compounds such as PPIX) and ceramides are endogenous cell death triggers.
REFERENCES
Hannun YA, Obeid LM (2002) J Biol Chem 277: 25847–25850 Liang H, Yao N, Song JT, Luo S, Lu H, Greenberg JT (2003) Genes Dev 17: 2636–2641 Mach JM, Castillo AR, Hoogstraten R, Greenberg JT (2001) Proc Natl Acad Sci USA 98: 771– 776 Yao N, Eisfelder B, Marvin J, Greenberg JT (2004) Plant J 40: 596–610 Yao N, Greenberg JT (2006) Plant Cell 18: 397–411
33 Plastid targeting of glycoproteins in rice cells
Toshiaki Mitsui1, Aya Kitajima1, Kentaro Kaneko1, Tuyoshi Asakura1, Francisco José Muñoz2, Edurne Baroja-Fernández2, Javier Pozueta-Romero2, Akihiko Nakano3,4
1 Niigata University Department of Applied Biological Chemistry, Niigata 950-2181, JAPAN 2 IdeA, Consejo Superior de Investigaciones Científicas, 31192 Mutiloabeti, Nafarroa, SPAIN 3 RIKEN Discovery Research Institute, Saitama 351-0198, JAPAN 4 University of Tokyo Graduate School of Science, Tokyo 113-0033, JAPAN
Email: [email protected]
Nuclear-encoded plastidial proteins are usually synthesized in the cytosol and posttranslationally imported into the organelle. However, recent our investigations revealed that some starch metabolism-related enzymes are transported into the plastid through unusual pathway. A rice novel ADP-glucose hydrolytic nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) was shown to be glycosylated, since it contains numerous N-glycosylation sites, binds to Concanavalin A, stains with periodic acid-Schiff reagent and can be digested by Endo-H. Both immunocytochemical analyses and confocal-fluorescence microscopy of rice cells expressing NPP1-GFP revealed that NPP1 occurs in the plastidial compartment. Brefeldin A treatment to NPP1-GFP expressing cells prevented NPP1-GFP accumulation in the chloroplasts (Nanjo et al. 2006). Rice α-amylase I-1 (AmyI-1) is a well-known secretory enzyme bearing typical N-linked oligosaccharide chain. We found that AmyI-1 also occurs in the plastids in living rice cells (Asatsuma et al. 2005). In the targeting of AmyI-1, the effects of the dominant mutants of AtSAR1 and AtARF1 GTPases, which are engaged in the protein traffic from the ER to the Golgi apparatus, were tested in onion epidermal cells. These AtARF1 and AtSAR1 mutants severely arrested the targeting of AmyI-1 into plastids. These experiments provide strong evidence that the plastidial N-glycosylated glycoproteins are transported from the Golgi to the plastid through the secretory pathway in rice cells.
REFERENCES
Asatsuma et al. (2005) Plant Cell Physiol 46: 858-869 Nanjo et al. (2006) Plant Cell 18: 2582-2592
34 Plant aquaporin regulation and cell signaling
François Chaumont
Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 5-15, B-1348 Louvain-la-Neuve, Belgium
Email: [email protected]
Plant growth and development are dependent on the tight regulation of water uptake and transport across cellular membranes and tissues. This water movement can be controlled by the regulation of water channels or aquaporins. These channels are widespread in biological membranes and plant aquaporins are believed to act as “cellular plumbers” allowing plants to rapidly alter the membrane permeability in response to environmental cues (Hachez et al., 2006). In addition, plant aquaporins can also facilitate the transport of other important molecules such as CO2, ammonia, H2O2, boron and silicon. Recent data indicate that plant aquaporins are regulated by many different mechanisms modifying their subcellular localization and gating (Chaumont et al., 2005; Tornroth-Horsefield et al., 2006). The factors affecting aquaporin trafficking and gating behavior possibly involve phosphorylation, heteromerization, pH, calcium, pressure, solute gradient, temperature. For instance, plasma membrane aquaporins (PIP) are phosphorylated by calcium-dependent protein kinases resulting in the pore opening and an increase of the water channel activity. Interestingly, high osmotic pressure in the apoplast induces a decrease in the phosphorylation status of PIPs, probably preventing water exit from the cells. We also recently showed that non-functional and functional PIPs form heteromers in oocytes and plant cells leading to a relocalization of inactive PIPs from the secretory pathways to the plasma membrane. Recent progress in the elucidation of plant aquaporin regulation and cell signaling will be discussed.
REFERENCES
Chaumont F, Moshelion M, Daniels MJ (2005) Biol Cell 97: 749-764 Hachez C, Zelazny E, Chaumont F (2006) Biochim Biophys Acta 1758: 1142-1156 Tornroth-Horsefield S, Wang Y, Hedfalk K, Johanson U, Karlsson M, Tajkhorshid E, Neutze R, Kjellbom P (2006) Nature 439: 688-694
35 Controlled and facilitated diffusion of H2O2 as a potential mechanism involved in signaling and ROS scavenging
Gerd Patrick Bienert, Thomas Paul Jahn
Plant and Soil Science Laboratory, Department of Agricultural Sciences, Faculty of Life Sciences, Copenhagen University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
Email: [email protected]
The production of reactive oxygen species (ROS) is the natural consequence of aerobic metabolism. ROS are inter-convertible molecules with various degrees of reactivity and the potential to damage cellular components such as lipids, nucleic acids and proteins. Hydrogen peroxide (H2O2) is a rather stable ROS and has recently been shown to be involved in various signaling pathways. To minimize potential damage by ROS and to control a signaling role of H2O2 and other ROS, the concentration of ROS has to be tightly regulated. Such control may involve mechanisms of production and scavenging as well as transport across membranes. Mechanisms for production and scavenging have been studied in great detail. However, the aspect of transport of H2O2 is of particular interest, because a mechanism of transport is part of the definition of signaling molecules. Until recently it was assumed that H2O2 crosses the membrane by simple diffusion. Contrary to the concept of simple diffusion it was suggested that water channels facilitate the diffusion of H2O2. Henzler and Steudle (2000) showed that mercury, an aquaporin blocker, repressed H2O2 accumulation in internodial cells of the algae Chara corallina. The authors therefore suggested that some aquaporins in Chara served as peroxoporins. We have used the heterologous expression system yeast to test the hypothesis that specific aquaporins may flux H2O2 across membranes. Yeast is a very useful tool for this type of study, since many mutants are available that differ in the ability to metabolize and detoxify ROS. This allowed us to control the scavenging capacity of the yeast cells while asking the question, if the heterologous expression of aquaporins increased the sensitivity to externally supplied H2O2. In a comprehensive screen testing 24 aquaporin isoforms from plants and mammals we found that expression of plant aquaporins of the TIP1 group and human AQP8 increased the sensitivity of yeast cells towards H2O2 in the medium. Aquaporin-mediated H2O2 transport was further investigated in a fluorescence assay with intact yeast cells using an intracellular ROS-sensitive fluorescent dye. Our data provide molecular genetic evidence that human AQP8 and plant aquaporin AtTIP1 have the potential to facilitate the diffusion of H2O2 across membranes. The challenge is now to demonstrate a physiological role of hydrogen peroxide transport through aquaporins. We will present current strategies.
REFERENCES
Henzler T, Steudle E (2000) J Exp Bot 51: 2053-2066
36 The role of glutamate in plants and its potential function as a signaling molecule
Sakiko Okumoto1, Loren Looger1,2, Kristina Micheva3, Richard Reimer4, Stephen Smith3, Wolf Frommer1
1 Carnegie Institution of Washington, Panama St. Stanford, CA 94305, USA 2 HHMI, 4000 Jones Bridge Road, Chevy Chase, MD 20815 USA 3 Molecular and Cellular Physiology, Stanford University, Beckman B141, Stanford, CA 94305, USA 4 Departments of Neurology and Neurological Sciences and Molecular and Cellular Physiology, Stanford University, P211 MSLS, Stanford, CA 94305, USA
Email: [email protected]
Glutamate is one of 20 proteogenic amino acids, and serves as an acceptor molecule in primary ammonium assimilation in plants. It is also one of five major translocated amino acids found in phloem and xylem. Metabolism and synthesis of glutamate by enzymes such as glutamine synthetase (GLN1) and asparagine synthetase (ASN1) are regulated by light, carbon and nitrogen, suggesting that the regulation of glutamate level plays a significant role in the control of C/N balance in plants. In addition to its role as a nutrient and nitrogen transportation form in plants, glutamate might play a role in intracellular signaling. Externally applied glutamate induces membrane depolarization and cytosolic calcium spikes. Arabidopsis genome encodes for 20 putative glutamate receptor homologs, and the disruption of one of these glutamate receptors, GLR3.3, partially abolishes membrane depolarization and cytosolic calcium spikes (Qi et al. 2006). AtGLR1.1 deficient plants (antiAtGLR1.1) exhibit conditional germination phenotype that is sensitive to C:N ratio in the growth media (Kang and Turano 2003). The role of glutamate as a signaling molecule, however, is unclear. For most plant glutamate receptors the ligand specificity has not been identified. In addition, it is not known whether plants have mechanism that control local glutamate concentration. In order to understand the role of glutamate and glutamate receptors in plants, it is important to know the glutamate level in specific cell types or in subcellular compartments. A method to measure the concentration of glutamate in all cell types would provide valuable information. We have developed a protein- based, Fluorescence Resonance Energy Transfer (FRET) nanosensors for glutamate (Okumoto et al. 2005). Glutamate sensors, when expressed in mammalian cells, were able to detect real-time glutamate concentration change. Results from Arabidopsis plants expressing glutamate sensors anchored to the outside of the plasma membrane suggest that these sensors can detect glutamate concentration changes in the apoplast. We will introduce a new technique for the analysis of in vivo glutamate fluxes in plants and discuss our latest results.
REFERENCES
Qi Z, Stephens NR, Spalding EP (2006) Plant Physiol 142: 963-971 Kang J, Turano FJ (2003) Proc Natl Acad Sci USA 100: 6872-6877 Okumoto S, Looger LL, Micheva KD, Reimer RJ, Smith SJ, Frommer WB (2005) Proc Natl Acad Sci USA 102: 8740-8745
37 Plant GABA metabolism - approaches to identify genes involved
Anke Hüser, Ulf-Ingo Flügge, Frank Ludewig
University of Cologne, Botanical Institute, Gyrhofstr. 15, D-50931 Cologne, GERMANY
Email: [email protected]
GABA metabolism is compartmentalized. Anabolism takes place in the cytosol and catabolism occurs in mitochondria. The GABA catabolic succinic semialdehyde dehydrogenase (ssadh) mutant is strongly impaired in growth, most likely due to the accumulation of a toxic compound. The ssadh phenotype can be rescued by simultaneously knocking out the GABA-transaminase (gaba-t) gene, the gene upstream in GABA catabolism. This phenotype suppression can be explained by preventing a toxic intermediate to accumulate in double knock-out plants. Based on this finding and searching for unknown genes that are involved in GABA metabolism, an ssadh suppressor screen has been performed, where ssadh mutants have been mutagenized using EMS. Under short day conditions, unlike normal growth conditions, ssadh/gaba-t double mutants display a phenotype that resembles the one of ssadh mutants. To explain this phenotype, a second suppressor screen has been performed, mutagenizing ssadh/gaba-t double knock-out plants with EMS. Suppressor mutants of either screen have been collected and are currently analyzed. Ultimately, rescueing EMS mutations will be mapped and cloned to assign functions to the respective genes in GABA metabolism or regulation of the pathway. To further gather more information on the GABA pathway in plants, a recombinant inbred line (RIL) analysis was performed. RILs were grown in the presence of potentially toxic intermediates of the pathway to determine quantitative trait loci (QTLs) for resistance/sensitivity against/for the respective substances. Overlapping findings with those of suppressor screens are deliberately taken into account.
38 Indoleamines and flavonoids in neuroprotective plant physiology
Ian B. Cole, Susan J. Murch
Chemistry, I.K. Barber School of Arts & Sciences, University of British Columbia Okanagan, Kelowna, BC, Canada
Email: [email protected]
The plant genus Scutellaria is a rich source of neurologically active phytochemicals and treatments for a wide range of human diseases including cancers, neurological disease, fevers and immune system dysfunction. Very few of the 350 species in this genus have been extensively studied, but there is traditional and ethnobotanical evidence of efficacy of Scutellaria species from around the world. The objective of the current study was to compare the phytochemical diversity in three species of Scutellaria from vastly different geographical locations and ecosystems. Metabolomic analysis revealed that the Chinese species, Scutellaria baicalensis had 1388 compounds that were not present in extracts of the other species. The North American Scutellaria lateriflora had a spectrum of 1261 unique compounds while the Central and South American species Scutellaria racemosa had 1733 unique phytochemicals. Equally interesting was the conserved phytochemistry. The neurotransmitters, melatonin and serotonin, were found in all three Scutellaria species. The neuroprotectant wogonin was also found in all 3 species of Scutellaria along with the flavonoids, baicalin, baicalein, and scutellarin. Wogonin was found at the highest levels in Scutellaria racemosa, a plant with traditional indigenous use as a narcotic. Recent studies with animal models indicate neuroprotective capacityin extracts from this species. The presence and conservation of neurologically active phytochemicals across plants from different geographical locations and ecosystems may provide new opportunities for studies of their potential role in plant adaptation and plant development.
39 Indole-3-butyric acid as a signal in early events of arbuscular mycorrhizal associations
Jutta Ludwig-Müller, Dorothee Thiel, Kerstin Jentschel, Frank Rehn, Mike Güther
Institut für Botanik, Technische Universität Dresden, 01062 Dresden, GERMANY
Email: [email protected]
Plant hormones are suitable candidates to function as continuous signals between roots and arbuscular mycorrhizal (AM) fungi during the establishment of symbiosis. Auxins might play a role during early events of an arbuscular mycorrhizal association with respect to changes in root morphology and gene expression. As examples several different plant-AM fungus systems will be compared. Inoculation of Zea mays with Glomus intraradices resulted in the significant increase in the percentage of lateral roots during early stages of colonization which coincided with an increase in the levels of the auxin indole-3-butyric acid (IBA). Addition of TFIBA, an inhibitor of IBA-induced root growth and lateral root induction, to roots inoculated with AM-fungi reduced the formation of fine roots and the amount of endogenous free IBA as well as the percentage of colonization. The increase in IBA levels was accompanied by increased enzymatic synthesis of IBA. In the model legume Medicago truncatula IBA was also induced during AM. Transcripts from Medicago truncatula roots differentially induced by IBA and AM were identified by microarray analysis. A small set of genes was simultaneously regulated by both factors. We have validated the expression levels of several transcripts by RT-PCR and found up-regulation of a leghemoglobin by IBA and AM and down-regulation of a high-affinity nitrate transporter by both factors. A model will be presented which summarizes the possible effects of plant hormones, especially auxins, during AM symbiosis.
40 A role for phospholipase A in auxin gene regulation and auxin responses. The receptor may not be TIR1
Günther F. E. Scherer1, Steffen Rietz2, Chalie Assefa-Fantaye1, Marc Zahn1, Esther Oppermann1, Marie Shishova3, André Holk1, Judy Callis4, Alan M. Jones5
1 Universität Hannover, Inst. Zierpflanzenbau und Gehölzwissenschaften, Abt. Molekulare Ertragsphysiologie, Herrenhäuser Str. 2, D-30419 Hannover, Germany 2 MPI Köln, Carl v. Linnè Weg 9, D-50829 Köln 3 State University St. Petersburg, Dept. Plant Physiology & Biochemistry, Russia 4 University of California, Department of Molecular and Cellular Biology, Davis, CA 95616, USA 5 University of North Carolina, Departments of Biology and Pharmacology, Chapel Hill, NC 27516, USA
Email: [email protected]
The only plant cytosolic phospholipase A form is the iPLA2 or patatin-related PLA around which our work centers. Auxin increases phospholipase A activity within 2 min (Paul et al. (1998) Plant J. 16: 601-611) and phospholipase A inhibitors, ETYA and HELSS, inhibited PLA activation and elongation growth of etiolated Arabidopsis hypoctyls (Holk et al. (2002) Plant Physiol. 130: 90- 101). To identify the mode of action, rapid auxin-regulated gene expression was tested for sensitivity to PLA2 inhibitors using seedlings harbouring the synthetic auxin-responsive reporter DR5::GUS. ETYA and HELSS inhibited auxin-induced increases in GUS activity, the steady-state level of the corresponding GUS mRNA and the mRNAs encoded by auxin-activated genes (IAA1, IAA5, and ARF19). Auxin regulation of the steady-state level of Aux/IAA proteins is mediated by the auxin receptor, E3 ubiquitin ligase, TIR1. The velocity of the auxin-induced decrease of an IAA1-luciferase fusion protein was unaltered by ETYA and HELSS during the first 20 minutes when biosynthesis of IAA1-luciferase was prevented by cycloheximide addition. In kinetics of auxin- induced degradation IAA1-luciferase measured without cycloheximide these inhibitors blocked the auxin-induced decrease in steady-state levels. When two phospholipase A genes were over- expressed in the DR5-GUS background the sensitivity of DR5 promoter to auxin was greatly increased. The results here suggest that phospholipase A mediates auxin-regulated gene transcription via a receptor other than TIR1 and may act upstream of TIR1. Knockout lines for the PLA genes AtPLA I, AtPLA IVA, and AtPLA IVC were isolated and found to be damaged in typical auxin-related functions. The knockout for AtPLA I is expressed in vascular tissue in stems, roots and leaf veins, additionally in pollen and trichomes. The corresponding knockout plants are defect in gravitropism, phototropism, nutation and root tip coiling. The slower response to laterally applied auxin of the AtPLA I knockout suggests a regulatory function of PLA I of auxin transport. The AtPLA IVA is expressed strongly in the root and the knockout for AtPLA IVA is defect in side root formation suggesting a function in auxin- stimulated side root formation. The gene AtPLA IVC is expressed in the gynaecium. With ABA treatment expression of PLA IVC is enhanced in roots and in veins in the shoot. The knockout shows a defect in the response of the root to phosphate starvation. The main root growth is not repressed but side roots are, contrary to the wild type response. Phosphate starvation affects auxin transport which may be disturbed in the pla IVC mutant. The hyocotyl is elongated in these knockouts pointing out a relevance to cell elongation for PLA IVC. Taken together, the phenotypes of PLA knockout plants point out their function in auxin signalling. PLA is involved in the regulation of early auxin genes. The receptor, however, may not be TIR1.
41 Evolutionary history of the domain architecture of plant formins
Michal Grunt, Fatima Cvrckova
Department of Plant Physiology, Faculty of Sciences, Charles University, Vinicna 5, CZ 128 43 Praha 2, Czech Republic
Email: [email protected]
This work has been supported by the GACR 204/05/0268 grant. Part of author´s salaries has been funded by the MSM LC06004 (MG) and 0021620858 (FC) projects.
Formins (FH2 proteins) are ancient actin-binding proteins believed to participate in actin filament nucleation. They are defined by the hallmark FH2 domain, usually preceded by a Pro-rich FH1 region. In addition, metazoan, fungal and Dictyostelium formins often contain a N-terminal GTPase-binding domain mediating interaction with Rho GTPases. In angiosperms, two groups of formins can be defined on the basis of FH2 domain sequence; each group also exhibits typical domain architecture. Class I formins are usually transmembrane proteins, while Class II formins often possess a PTEN-related domain that might also mediate membrane association. Thus, formins are good candidates for a link between the actin cytoskeleton and the surface structures of the plant cell. We have performed a thorough analysis of over 100 plant FH2 protein sequences, as well as more than 120 formins encoded by fully sequenced non-plant genomes representing the metazoans, fungi, amoebae, chromalveolates and excavates. The characteristic plant Class I and Class II formins are indeed both universal for the plant kingdom. Moreover, we found a novel domain combination including a RhoGAP domain, present in some algal, moss and lycophyte formins (but absent in angiosperms), suggesting an ancient formin-mediated functional association between Rho GTPases and the actin cytoskeleton.
42 Phospholipid-based signalling - 'seeing is believing'
Teun Munnik
Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, Netherlands
Email: [email protected]
Phospholipids are essential molecules contributing to the structural definition of cell membranes and participating in the regulation of cellular processes as signaling molecules and reservoirs of lipid messengers. While the bigger pools of phospholipids are involved in membrane structure, those involved in cell signalling are usually very small and consequently have been failed to be picked-up for years. Especially polyphosphoinositides (PPI) and phosphatidic acid (PA) have been emerging as signalling molecules. Over the last few years, we and others have shown that a number of pathways involved in their metabolism, are activated in response to a wide variety of biotic- and abiotic stresses (see reviews). In general, these activations are fast (seconds to minutes) and the lipid responses small and transient, exhibiting rapid turnover. ‘How’ these pathways are activated, ‘where’ in the cell or plant this takes place, and ‘what’ the functional significance of these activations are, is still a big mystery. To start addressing these questions, a number of opportunities are being explored in my laboratory. These include, i) Arabidopsis T-DNA insertion lines, ii) GFP-based lipid biosensors and iii) proteomic approaches to identify PA targets. Some recent developments in these areas will be discussed.
REFERENCES 1. Testerink C, Dekker HL, Lim Z-Y, Johns MK, Holmes AB, de Koster CG, Ktistakis NT, Munnik T. (2004) Isolation and identification of phosphatidic acid targets from plants. Plant J 39: 527–536 2. Van Leeuwen W, Okresz L, Bögre L, Munnik T. (2004) Learning the lipid language of plant signalling. Trends Plant Sci 9: 378-384 3. Testerink C, and Munnik T. (2005) Phosphatidic acid - a multifunctional stress-signalling lipid in plants. Trends Plant Sci 10: 368-375 4. Bargmann BOR, Laxalt AM, ter Riet B, Schouten E, van Leeuwen, W, Dekker HL, de Koster CG, Haring MA and Munnik T. (2006) LePLDb1 activation and re-localization in suspension-cultured tomato cells treated with xylanase. Plant J 45: 358-368 5. Van Schooten B, Testerink C. and Munnik T. (2006) Signalling diacylglycerol pyrophosphate, a new phosphatidic acid metabolite. Biochim Biophys Acta 1761: 151-159 6. Vermeer JEM, van Leeuwen W, Tobeña-Santamaria R, Laxalt AM, Jones DR, Divecha N, Gadella Jr. TWJ and Munnik T. (2006) Visualisation of PtdIns3P dynamics in living plant cells. Plant J 47: 687-700 7. Zonia L, Munnik T. (2006). Cracking the green paradigm: functional coding of phosphoinositide signals in plant stress responses. Subcell Biochem 39: 207-237 8. Bargmann BO, Munnik T. (2006) The role of phospholipase D in plant stress responses. Curr Opin Plant Biol 9: 515-522 9. Zonia LE, Müller M, Munnik T. (2006) Cell volume oscillations in the pollen tube apical region are an integral component of the biomechanics of Nicotiana tabacum pollen tube growth. Cell Biochem Biophys 46: 209-232 10. Zonia LE, Munnik T. (2007) Life under pressure: Hydrostatic pressure in cell growth and function. Trends Plant Sci 12: 90-97 11. Kooijman EE, Tieleman DP, Testerink C, Munnik T, Rijkers DTS, Burger KNJ, de Kruijff B (2007) An electrostatic/hydrogen bond switch as basis for the specific interaction of phosphatidic acid with proteins. J Biol Chem, In press 12. Van Leeuwen W, Vermeer JEM, Gadella Jr. TWJ, Munnik T. (2007) Visualisation of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings. Plant J, Re-submitted
43 The role of human neurotransmitters in plant cell division
Murch J Susan1, Saxena K Praveen2
1 Chemistry, IK Barber School of Arts and Sciences, University of British Columbia Okanagan, Kelowna, British Columbia, Canada V1V 1V7 2 Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
Email: [email protected]
Each individual plant cell has the potential to become a whole plant through a process of dedifferentiation, redifferentiation and tissue regeneration. It has been hypothesized that this process is induced by alterations in the balance of two classes of plant growth regulating compounds viz. auxin and cytokinin but the exact mechanisms by which these compounds induce plant cell division is not known. In previous research, we have shown that mediation of endogenous neurotransmitter metabolism impaired plant regenerative functions but exogenous application of human neurohormones did not alter plant growth. In this communication, we show for the first time, that exogenous application of the human neurohormone serotonin induced three different developmental pathways in isolated tobacco mesophyll cells: (1) changes in cell structure including formation of pearl-necklace and torpedo-like cells, (2) low-frequency cell division, and (3) high frequency cell division followed by callogenesis when combined with auxin. These data provide the first evidence that serotonin acts as a growth regulator in plant cells and that synthetic auxin induces serotonin-melatonin production, potentially as a precursor to the indoleamine biosynthetic pathway.
44 Jasmonates as inducers of Ca2+ signals in the nucleus and the cytosol of plant cells
Axel Mithöfer1, Agnes Walter1, Christian Mazars2, Raoul Ranjeva2, Wilhelm Boland1
1 MPI for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, GERMANY 2 Signaux et Messages Cellulaires chez les Vegataux, UMR CNRS/UPS 5546, 31326 Castanet- Tolosan, FRANCE
Email: [email protected]
Jasmonates representing a group of oxylipin phytohormones are shown here to differentially induce changes in intracellular Ca2+ concentrations in two distinct compartments of plant cells, the cytosol and the nucleus. Based on the Aequorin technique, a structure-activity analysis revealed that jasmonates and related compounds fall into three distinct classes: (1) compounds inducing Ca2+ changes in both the cytosol and the nucleus (2) compounds inactive on either compartment or (3) compounds acting selectively on the nucleus.
REFERENCES
Wasternack, C, Hause B (2002) Progr Nucleic Acid Res 72: 165–221 Mithöfer A, Maitrejean M, Boland W (2005) J Plant Growth Regul 23: 170-178 Mithöfer A, Mazars C (2002) Biol Proced Online 4: 105-118
45 Plasma mebrane NADPH oxidases (NOX) in plants – beyond ROS signalling.
Viktor Zarsky1,2, Martin Potocky1, Radek Bezvoda2
1 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 135, 160 00 Prague 6, Czech Republic 2 Department of Plant Physiology, Faculty of Science, Charles University, Viničná 5 12844 Prague 2, Czech Republic
Email: [email protected]
This work is supported by MSMT LC06034 a MSM0021620858
Our recent results show that tip-localised ROS produced by a NOX enzyme are needed to sustain the normal rate of pollen tube growth. As in root hairs and growing root cells the same phenomenon is observed, it is likely that this activity is a general mechanism related to plant cells expansion. The regulation of NOX activity is also in plants related to the Rac/Rop GTPases activity and calcium signalling; and signalling relay involving ROS is well documented in plants. Here we will discuss possible significance of NOX activity in electrogenic processes at the growing plant cell domain.
REFERENCE
Potocky M, Jones MA, Bezvoda R, Smirnoff N, Zarsky V. (2007) Ractive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol, In press
46 The role for PEX11 and dynamin-related proteins in Arabidopsis peroxisome proliferation
Xinchun Zhang1, Travis Orth1, Sigrun Reumann2, Jilian Fan1, Kyaw Aung1, Dirk Wenzel3, Sheng Quan1, Jianping Hu1
1 MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA. 2 Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, University of Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany. 3 Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, D- 37077 Göttingen, Germany.
Email: [email protected]
Plant peroxisomes are highly dynamic organelles that play pivotal roles in development and in stress response. To establish a model for peroxisome proliferation in plants, which is largely unknown, we have taken forward and reverse genetic and proteomic approaches using Arabidopsis thaliana. Except for Pex11p, which in yeast is involved in peroxisome proliferation with an unknown mechanism, the Arabidopsis genome does not contain obvious sequence homologues to most proteins that operate in yeast to control peroxisome proliferation. The five-member Arabidopsis PEX11 protein family is composed of three subfamilies: PEX11a, PEX11b, and PEX11c to PEX11e, all of which target to peroxisomes, as demonstrated by fluorescence microscopy and immunobiochemical analysis. Overexpression of At PEX11 genes in Arabidopsis increased peroxisome elongation and number, whereas reduction in gene expression lowered peroxisome abundance. PEX11c and PEX11e partially complemented the growth phenotype of the S. cerevisiae pex11 null mutant on oleic acid. Our results suggested that the Arabidopsis PEX11 proteins promote peroxisome proliferation with some functional specificity between subfamilies (Orth et al. 2007 Plant Cell). Using genetic screens and proteomic analysis of the peroxisome membrane, we also identified a subset of the dynamin-related large GTPases that mediates the division of both peroxisomes and mitochondria. These data indicated that despite their distinct evolutionary paths, peroxisomes and mitochondria may use the same set of dynamin-related proteins (DRPs) for division. Additional forward genetic screens and proteomic experiments are conducted in the lab to uncover more plant-specific components of the peroxisome proliferation machinery.
REFERENCE
Orth T, Reumann S, Zhang X, Fan J, Wenzel D, Quan S, Hu J (2007). The PEROXIN11 protein family controls peroxisome proliferation in Arabidopsis. Plant Cell 19, published online Jan 12
47 Myosin and actin function in directing mitochondria movement in living pollen tubes of Picea wilsonii
Maozhong Zheng, Jinxing Lin
Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
Email: [email protected]
Mitochondria are dynamic organelles providing subcellular spatial energy as needed and serving as fundamental elements in intracellular signalings in plant cells. However the nature of the mitochondria-cytoskeleton interactions in pollen tubes has not been explored. By using time lapse confocal microscopy, total internal reflection fluorescence microscopy (TIRFM) and spinning-disk confocal microscopy (SDCM), we investigated the effects of cytoskeletal inhibitors on the transportation and positioning of mitochondria in living pollen tubes. It was revealed that the actin filament disrupting drug latrunculin B (LATB), the myosin ATPase inhibitor, 2, 3-butanedione 2-monoxime (BDM) and the actin filament stabilizing drug jasplakinolide (Jas), apparently inhibited mitochondrial motility, while microtubule disrupting drug taxol and oryzalin showed slight effects, demonstrating that intact actin cytoskeleton is required for active mitochondrial movement. Two-dimensional (2-D) trajectory and velocity of individual mitochondrion was obtained to characterize the mitochondrial movement. It was showed that mitochondria of Picea wilsonii pollen tubes underwent three classes of linear movements: rapid movement (> 5.0 μm/s instantaneous velocities), slow movement (<5.0 μm/s instantaneous velocities) and mixed movement (ranging from 0.16 to 10.35 μm/s instantaneous velocities). Jas treatment abolished mixed mitochondrial movement and rapid mitochondrial movement, while rapid movement and slow movement were not found in BDM-treated pollen tubes. Taxol treatment increased the frequency of positioning and velocities of mixed mitochondrial movement. Oryzalin treatment caused curve mitochondrial trajectories with similar velocities compared to the control pollen tubes. Taken together, these findings suggested that actin cables provided tracks for mitochondrial slow movement which are powered by myosin, on the other hand, actin cables also served as “conveyor belts” to drive mitochondrial mixed movements via actin polymerization. Therefore, microtubule dynamics regulated mitochondrial positioning, velocities and trajectories via affecting the actin filament dynamics, rigidity and flexibility.
48 Actomyosin-mediated gravisensing and early transduction events in gravistimulated cut snapdragon spikes
Zhaoqi Zhang1,2, Haya Friedman1, Shimon Meir1, Abraham H. Halevy3*, Sonia Philosoph-Hadas1
1 ARO, The Volcani Center, Department of Postharvest Science of Fresh Produce, Bet-Dagan 50250, ISRAEL 2 College of Horticulture, South China Agricultural University, 510642 Guangzhou, CHINA 3 The Hebrew University of Jerusalem, Faculty of Agriculture, The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Rehovot 76100, ISRAEL
Email: [email protected]
* Dedicated to the memory of Prof. Abraham H. Halevy, from the Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel, who had actively participated in this research.
Horizontal placement of snapdragon (Antirrhinum majus L.) flowering shoots initiates an upward gravitropic bending via the chain reaction of gravity perception, signal transduction and growth response (Philosoph-Hadas et al. 1996, Friedman et al. 1998, 2003). Our previous studies (Friedman et al. 2003) have demonstrated that the actin cytoskeleton within the cells of snapdragon (Antirrhinum majus L.) spikes is necessary for normal amyloplast displacement upon gravistimulation. Pharmacological disruption of the actin cytoskeleton with cytochalasin D (CD), demonstrated in cortical and endodermal cells, and with latrunculin B (Lat B), demonstrated in cortical cells, delayed the displacement of amyloplasts and resulted in a significant inhibition of the gravitropic bending. In the present study we have investigated the involvement of myosin in addition to actin in the different phases of the gravitropic response of snapdragon spikes. Using indirect immunofluorescence double-labeling of actin and myosin, we have demonstrated that no organization changes in actin filaments occurred in cortical and endodermal cells of the stem bending zone during gravistimulation. These results suggest that actin depolymerization is not required for amyloplast sedimentation. The amyloplasts in the endodermis were found to be surrounded by actin and myosin, and seem to be attached to the actin filaments via the motor protein, myosin. This suggests the involvement of myosin in amyloplast translocation. This suggestion was supported by the findings showing that pulsing spikes with the myosin inhibitor, 2,3-butanedione-2-monoxime (BDM), inhibited the gravity-induced amyloplast displacement in the endodermis. Indeed, the BDM treatment altered characteristic distribution patterns of myosin-like proteins in the cortex and disrupted the normal organization of the actin network and microtubules. This further indicates that myosin functions in the normal actin network organization. Both BDM and CD inhibited lateral auxin transport and stem bending. It seems therefore, that the inhibitors which affect amyloplast displacement also inhibit the subsequent event of lateral auxin transport leading to inhibition of stem bending. Taken together, our results suggest that the acto-myosin system mediates displacement of amyloplasts, which under normal conditions possibly move along the actin filaments, using myosin as a motor protein, to reorient their position following gravistimulation.
REFERENCES
Friedman H, Meir S, Rosenberger I, Halevy AH, Kaufman PB, Philosoph-Hadas S (1998) Plant Physiol 118: 483-392 Friedman H, Vos JW, Hepler PK, Meir S, Halevy AH, Philosoph-Hadas S (2003) Planta 216: 1034-1042 Philosoph-Hadas S, Meir S, Rosenberger I, Halevy AH (1996) Plant Physiol 110: 301-310
49 Temporary changes in gravity conditions affect oxygen influx at root level
S. Mugnai1, E. Azzarello1, E. Masi1, C. Pandolfi1, A. Hlavacka1,2, B.Voigt1,2, F. Baluska1,2, D. Volkmann2, S. Mancuso1
1 LINV, University of Florence, Viale delle idee 30, 50019 Sesto F.no (FI), ITALY 2 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY
Email: [email protected]
Oxygen influx shows a gravity-regulated asymmetry in the transition zone (TZ) of root apices on ground when root orientation varies from vertical to horizontal. In details, oxygen influx increased only on the upper side of TZ, remaining stable on the lower one, since 18 ± 2 sec after changing the root position. Considering that the tilting procedure took around 15 s, the first O2 signal can be hypothesized to appear just few seconds after gravistimulation. This rapid change in the oxygen flux into root apices is by far the fastest ever reported plant response to gravity. To analyze this phenomenon in a very low gravity situation, an experiment has been set up on a parabolic flight, which permits a sequence of normal, hyper- and microgravity conditions. During the flight, oxygen flushes in roots of Zea mays seedlings have been constantly monitored by selective microelectrodes and a respirometer. A clear and distinct signal in oxygen fluxes has been detected only in the apex zone, starting just 2.0 ± 0.5 s after the imposition of microgravity conditions, while no significant changes have been monitored neither in normal nor in hypergravity conditions. The significance of these results on the nature of the graviperception will be discussed.
50 Chemical factors inducing leaf-movement in Leguminosae and carnivorous plants
Minoru Ueda
Department of Chemistry, Tohoku University, Sendai 980-8578, JAPAN
Email: [email protected]
Chemical aspects of two different types of plant leaf movements, the circadian leaf movement known as nyctinasty and trap leaf-closure of Dionaea muscipula, will be discussed. Nyctinastic leaf movement is induced by the swelling and shrinking of motor cells in the pulvinus, joint-like thickening located at the base of the petiole. A flux of potassium ions across the plasma membrane of the motor cells is followed by massive water flux, which results in swelling or shrinking of these cells. An issue of great interest is the regulation of the opening and closing of the potassium channels involved in nyctinastic leaf movement. Each of nyctinastic plants of five different genuses so far examined contained a pair of factors, one of which induces leaf closure and another induces leaf opening. The relative contents of the closing and opening factors changed correlating with the nyctinastic leaf movement. Use of fluorescence-labeled and photoaffinity labeled factors revealed that the factors bind to motor cells and that the membrane fraction of the pulvini contained two potential receptor proteins which can bind to the factor. Venus’s flytrap (Dionaea muscipula) is known as representative insectivorous plant. This plant traps the insects by its large leaves called trap, and digested them between the traps by digestive enzymes. Interestingly, there observed some “memory” in the leaf-closure of Dionaea. The rapid closure of the traps requires twice stimuli within thirty seconds on their sensory hairs which exist on the internal surface of the trap leaves. Only one stimulus never induces leaf closure. This phenomenon suggested that Dionaea memorizes the first stimuli on the sensory hair. We found that Dionaea has endogeneous chemical factor which induce the closure of traps without stimuli. This plant “memory” can be explained by the stepwise accumulation of secreted chemical factor.
REFERENCE
Ueda M, Nakamura Y (2006) Natural Product Reports 23: 548-557
51 Vesicular trafficking as a mechanism of abiotic stress tolerance in plants
Arnaldo L. Schapire1, Boris Voigt2, Abel Rosado1, Jan Jasik3, Stefano Mancuso2, Victoriano Valpuesta1, Frantisek Baluska3, Miguel A. Botella1
1 LBBV, University of Málaga, Campus Teatinos S/N, 29071 Málaga, SPAIN 2 LINV, University of Florence, Viale delle idee 30, 50019 Sesto f.no(FI), ITALY 3 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY
Email: [email protected]
Synaptotagmins are a family of transmembrane proteins that function as transducers of Ca2+ signaling in membrane fusion events. All members of the synaptotagmin gene family span membranes once, have short lumenal domains and long cytoplasmic regions containing two C2 domains connected by a short linker. There are 16 known vertebrate synaptotagmins. Detailed biochemical and in vivo studies of the best characterized isoform, synaptotagmin1 (syt 1), have provided compelling evidence that it functions as a calcium sensor for fast neurotransmitter release at synapses. In Arabidopsis there are six synaptotagmin-like genes (sytA-F) with unknown functions. In search for genes that were essential for salt stress tolerance, we screened a T-DNA population at high NaCl concentration. We found that loss-of-function of synaptotagmin A (SytA) in Arabidopsis produces hypersensitivity to sodium but only at low Ca2+ concentration. We analyzed the phospholipid binding properties of SytA. These studies revealed that only C2A binds phospholipids in a Ca2+ dependent manner, while C2B shows phospholipid binding independently of Ca2+. These results, combined with SytA localization in the plasma membrane, suggest that Ca2+ dependent membrane trafficking mediated by SytA is important for plant survival under abiotic stress conditions.
52 A plasmodesmata associated β-1,3-glucanase in Arabidopsis regulates plasmodesmata function
Amit Levy, Michael Erlanger, Michal Ben-Cohen, Dana Guenoune-Gelbart and Bernard L. Epel
Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Email: [email protected]
Plasmodesmata (Pd), plasma-membrane-lined channels that connect plant cells, are not static organelles, but rather show a high degree of plasticity and can change in a transient manner from 'closed' to 'open' to 'dilated'. The dynamic properties of Pd play an important role in regulating the direct cell-to-cell transport of molecules between cells, in providing a cell-to-cell passageway for plant viruses and in the organization and functioning of symplasmic domains. Two different mechanisms are assumed to produce these focused changes in the tunnels. The first model suggests that conductivity changes due to alterations in plasmodesmal structure motivated by plasmodesmal associated cytoskeleton proteins actin, myosin and centrin. The second model suggests that changes in the wall sheath surrounding the Pd, mediated by callose (β-1,3-glucan) synthesis and hydrolysis, cause changes to Pd structure that alter its conductivity. Recently we have identified the first β-1,3-glucanase Arabidopsis enzyme that is associated to the Pd complex, termed AtBG-pap (plasmodesmal associated protein) (Levy et al. 2007). When fused to GFP, this previously identified GPI anchored protein localizes to the ER and the plasma membrane, where it appears in a punctuate pattern that co-localizes with callose present around Pd. In T-DNA insertion mutants that do not transcribe AtBG-pap, GFP cell-to-cell movement between epidermal cells is reduced and callose levels around Pd are elevated. Many plant β-1,3-glucanases are "Pathogenesis Related" (PR) proteins, and are induced in response to microbial pathogen infection. Measuring the RNA levels of AtBG-pap following infection with cucumber mosaic virus (CMV) and Pseudomonas syringae pv. tomato showed no significant increase of AtBG-pap transcription levels, suggesting that AtBG-pap is not a PR protein and is not involved in virus cell-to-cell spread. Physiological measurements of 20 days old AtBG_pap mutant plants suggest that growth of these mutants is inhibited. Germination of the mutant seeds is severely delayed, and totally inhibited in ~50% of the seeds. AtBG-pap RNA levels were shown to be induced during germination just prior to testa and endosperm rupture. These results suggest that callose degradation by AtBG-pap at Pd is required for the regulation of germination, possibly by the release of seed dormancy and the activation of symplasmic connection between cells.
REFERENCE
Levy A, Erlanger M, Rosenthal M, Epel BL (2007) Plant J 49: 669–682
53 Cellular polarization for membrane dynamics in interaction of barley with pathogenic Blumeria graminis
Ralph Hueckelhoven, Christina Opalski, Holger Schultheiss, Ruth Eichmann, Qianli An
Technical University of Munich, Chair of Phytopathology Lustus-Liebig University of Giessen, Institute of Phytopathology
Email: [email protected]
When parasitic Blumeria graminis, the grass powdery mildew fungus, attempts to penetrate into barley epidermal cells, the plant reacts by cellular reorganization. The cytoplasm polarizes, the filamentous actin cytoskeleton focuses, and the nucleus migrates to the site of attack. Consequently dynamic reorganisation of the endomembrane system takes place, and endocytotic multivesicular bodies (MVBs) form. MVBs either target the vacuole in a lysosomal-like pathway or they are redirected to fuse with the plasma membrane and to release their vesicular cargo into the apolast. MVB-based secretion is involved in formation of cell wall appositions in which B. graminis is restricted. However, both actin reorganization and MVB formation are also involved in compatibility leading to accommodation of fungal feeding structures, haustoria, in intact cells. This might indicate corruption of cellular defence mechanisms for generation of the haustorial complex. Accordingly, barley RHO-like proteins, which is involved in actin organization, and presumably defensive NADPH oxidase are required for successful penetration of B. graminis into epidermal cells of barley.
54 Syntaxin SYP121 is involved in a number of pathogen defence mechanisms
Hans Thordal-Christensen1, Andrea Lenk1, Angela Feechan1, Boris Voigt2, Carsten Pedersen1, Mads Eggert Nielsen1,3, Mats Andersson1, Ziguo Zhang1, Frantisek Baluska2
1 Dept. of Agricultural Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark 2 Institute of Cellular & Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany 3 Carlsberg Research Laboratory, DK-2500 Copenhagen, Denmark
Email: [email protected]
Plant disease resistance is the result of the collective activity of separate defence mechanisms. We have previously discovered that the syntaxin gene SYP121 (PEN1) in Arabidopsis is required for penetration resistance1,2. SYP121 is necessary for vesicle trafficking leading to formation of papillae, which are local cell wall appositions functioning as barriers against fungal penetration. Based on the use of a functional GFP-SYP121 fusion, we demonstrate that SYP121 in addition to its plasma membrane localization, also is found in endosomal compartments involved in polarized secretion. Pharmacological analyses have demonstrated the involvement of endocytosis and endosomal secretion in the SYP121-dependent penetration resistance. The most closely related syntaxin gene, SYP122, is not required for penetration resistance. Meanwhile, the phenotype of the syp121 syp122 syntaxin double mutant have shown that these genes act as negative regulators of several signalling pathways leading to pathogen defence-related programme cell death. The syp121 syp122 plant exhibits a lesion mimic phenotype, which we by introducing knock-out mutations have shown partly to be due to an active SA signalling pathway3. Introducing knock-out mutations in a number of other well-known defence pathways have unravelled that several of these also contribute to the lesion mimic phenotype. The fact that several defence pathways are activated in the syntaxin double mutant syp121 syp122 has allowed us to study genetically to what extend these pathways are parallel. Re-mutagenesis of syp121 syp122 has led to isolation of a large number of triple mutants, which at varying degree rescues the lesion mimic phenotype. The third mutations have occurred in SUPPRESSOR OF SYNTAXIN-RELATED DEATH (SSD) genes. While a number of these have been positionally cloned, many of them have been placed in signalling pathways using high through- put genetics based on examining hundreds of F2 populations of crosses between triple mutants. Often combination of two ssd mutations lead to an enhanced suppression of the lesion mimic phenotype, indicating that the SSD genes control parallel signalling pathways, each contributing to the lesion mimic phenotype of the syp121 syp122 double mutant. In summary, our observations have helped us to draw a map of signalling pathways that are active in syp121 syp122.
REFERENCES
1 Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu JL, Hückelhoven R, Stein M, Freialdenhoven A, Somerville SC, Schulze-Lefert P (2003) Nature 425: 973-977 2 Assaad FF, Qiu JL, Youngs H, Ehrhardt D, Zimmerli L, Kalde M, Wanner G, Peck SC, Edwards H, Ramonell K, Somerville CR, Thordal-Christensen H (2004) Mol Biol Cell 15: 5118–5129 3 Zhang Z, Feechan A, Pedersen C, Newman M-A, Qiu JL, Olesen KL, Thordal-Christensen H (2007) Plant J 49: 302–312
55 Mining iron for host defense and pathogen virulence
Yangdou Wei1, Guosheng Liu1, David Greenshields1, Gopalan Selvaraj2
1 Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK, S7N 5E2, Canada 2 Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada
Email: [email protected]
Iron (Fe) is a ubiquitous redox-active element essential to both pathogenic microorganisms and their hosts. In the plant immune response, the formation of localized cell wall appositions, the oxidative burst and the production of pathogenesis-related proteins are hallmarks of plant defense reactions. Using a wheat-powdery mildew pathosystem, we have shown that Fe is a central mediator linking these three phenomena. Upon powdery mildew attack, Fe in leaf epidermis redistributes to the apoplast, which leads to Fe deficiency in the cytosol of attacked cells. The accumulated apoplastic Fe mediates the oxidative burst, which further stimulates Fe efflux and intracellular Fe deficiency. H2O2 and Fe deficiency induce expression of defense-related genes while suppressing the expression of Fe storage-related genes. Our work identifies Fe as an underlying factor regulating cereal defenses, and establishes links between disease-related Fe homeostasis in plants and animals. Fungal pathogens have evolved at least two pathways for Fe uptake from plant hosts regulated by siderophore-assisted Fe mobilization and reductive Fe assimilation systems. To examine the relative contribution of the reductive and siderophore pathways of Fe uptake, we created mutants disrupted at the ferroxidase gene Fet3 (∆fet3) or the siderophore biosynthetic gene SidA (∆sidA) from the head blight pathogen Fusarium graminearum. Targeted disruption of the Fet3 gene has no effect on virulence, whereas the SidA gene is an essential virulence attribute in the wheat-F. graminearum pathosystem. Together, these data show how pathogenic fungi compete with the host for Fe and how the host uses Fe to counteract this threat, providing insights on developing novel strategies for plant disease control.
56
Plant Physiology & Electrophysiology
57 Action potential in charophytes
Mary Jane Beilby
School of Physics, Biophysics, The University of NSW, NSW 2052, Sydney, AUSTRALIA
Email: [email protected]
The plant action potential (AP) has been studied for more than half a century. The experimental system was initially provided by the large charophyte cells, which allowed insertion of multiple electrodes and manipulation of cell compartments. The early experiments were modelled on the Hodgkin and Huxley (1952) (HH) voltage clamp technique developed for the squid axon. The HH analysis identified sodium ion inflow and potassium ion outflow as the as the depolarising and repolarising phases, respectively, of the nerve AP. The plant AP was also modelled in terms of voltage-gated opposing ion flows. The return to the resting potential difference (PD) was, indeed, due to the outflow of potassium ions. However, the depolarising agent was found to be the outflow of chloride ions with involvement of the calcium ions in activation of the chloride channels (Hope and Findlay, 1964). Later the patch clamp technique characterised the chloride ion channels, but the source of the calcium increase in the cytoplasmic space remained unclear (Thiel et al., 1993). Further, using tonoplast-free cells, Japanese researchers obtained APs without chloride ions in the perfusion medium in the charophyte cells, suggesting that calcium could be the depolarising ion under some circumstances (Shimmen and Tazawa, 1980). At the turn of the century, the paradigm of the charophyte AP shifted to include several chemical reactions, second messenger activated channel and calcium ion liberation from internal stores. The threshold voltage pulse mobilizes the second messenger inositol-1,4,5,-triphosphate (IP3) from its membrane-bound precursor phosphatidyl inositol 4,5-biphosphate (PIP2). IP3 has to reach critical concentration to stimulate calcium concentration rise in the cytoplasm (Biskup et all, 1999; Wacke et al. 2003). Many aspects of this new model await further clarification. The role of AP in plant movements is well documented in higher plants. The charophytes, on the other hand, are a good system to study the involvement of the AP in wound signalling (Shimmen, 2002) and turgor regulation (Beilby and Shepherd, 1996; 2006), which will be discussed in more detail.
REFERENCES
Beilby MJ, Shepherd VA (2006) Plant Cell Environ 29: 764–777 Beilby MJ, Shepherd VA (1996) Plant Cell Environ 19: 837–847 Biskup B, Gradmann D, Thiel G (1993) FEBS Letts 453: 72-76 Hodgkin AL, Huxley AF (1952) J Physiol 117: 500-544 Hope AB, Findlay GP (1964) Plant Cell Physiol 5: 377–379 Shimmen T (2002) Plant Cell Physiol 43: 1575–1584 Shimmen T and Tazawa M (1980) J. Membr Biol 55: 223–232 Thiel G, Homann U, Gradmann D (1993) J Membr Biol 134: 53–56 Wacke M, Thiel G, Hutt M-T (2003) J Membr Biol 191: 179-192
58 Electrophysiology of Venus flytrap (Dionaea muscipula Ellis)
Alexander G. Volkov1, Tejumade Adesina1, Emil Jovanov2
1Department of Chemistry and Biochemistry, Oakwood College, 7000 Adventist Blvd., Huntsville, AL 35896, USA 2 Electrical and Computer Engineering Dept., University of Alabama in Huntsville, Huntsville, AL 35899, USA
Email: [email protected]
Electrical signaling and rapid closure of the carnivorous plant Dionaea muscipula Ellis (Venus flytrap) have been attracting the attention of biophysicist and electrophysiologists since the nineteenth century [1,2]. When an insect touches the trigger hairs of the Venus flytrap, mechanosensors on these trigger hairs generate an electrical signal that acts as an action potential which activate the motor cells. Six trigger hairs protruding from the upper leaf epidermis act as mechanosensors, with three of the trigger hairs located in the center of each half of the lamina. The exact mechanism of Venus flytrap closure is still unknown. Moreover, a traditionally used slow data acquisition systems cannot capture plant electrical signals with frequencies higher than half of the sampling frequency. Using an ultra-fast data acquisition system with measurements in real time, we found that action potentials in the Venus flytrap have an average speed of 10 m/s with a duration time of about 1.5 ms and are fast enough to induce the closure of the leaves by the motor cells. A few minutes after closing of the Venus flytrap, electrical signaling was also detected in the lower part of the leaf of the Venus flytrap in the form of graded potentials with amplitudes of 20 mV or less. In terms of electrophysiology, Venus flytrap responses can be represented as the following sequence: stimulus perception, signal transmission, and induction of response. We discovered that the electrical impulse between a midrib and a lobe allows the Venus flytrap leaf to close by activating motor cells without mechanical stimulation of trigger hairs. The average closing time of Venus flytraps by electrical stimulation of motor cells is 0.3 s, which is the same as mechanically induced closing by a small piece of a gelatin or cotton thread. Our results demonstrate that electrical stimulation can be used to study mechanisms of fast activity in motor cells of the plant kingdom.
REFERENCES
1 Ksenzhek OS, Volkov AG (1998) Plant Energetics, Academic Press, San Diego 2 Volkov AG (Ed.) (2006) Plant Electrophysiology, Springer, Berlin, New York
59 Effects of acetylcholine on the blue-light response of dark-grown Arabidopsis seedlings
Mary A Bisson1, Roger R Lew2, Amanda Diamond1
1 Department of Biological Sciences, University at Buffalo, Buffalo NY USA 2 Biology Department, York University, Toronto, Ontario, Canada
Email: [email protected]
An early response of etiolated (dark-grown) Arabidopsis to light is a transient depolarization. It has been proposed (Lewis & Spalding, 1998) that this depolarization is due to activation of a Cl- channel in a ligand-gated channel family, and blocking channel function prevents the inhibition of growth that is part of the greening response. Based on previous experiments with Chara that showed a chloride channel activated by acetylcholine (ACh) (Gong & Bisson, 2002), we explored the possibility that the light-stimulated Arabidopsis channel is similarly activated by ACh. Since the Chara experiments indicated that the binding site for ACh is cytoplasmic, we microinjected ACh into the cytoplasm of hypocotyl cells of etiolated plants, and determined the effect on the light-induced depolarization. We found, instead of activation, that microinjection of ACh inhibited the depolarization due to light. Because nicotine potentiated the effect of ACh in Chara, we microinjected nicotine with ACh in Arabidopsis. Instead of potentiating the effect, nicotine restored the depolarization, although it significantly delayed it. To test the physiological significance of these effects, we treated etiolated Arabidopsis plants with ACh with and without nicotine. Treatment decreased growth in the dark somewhat, but eliminated the inhibitory effect of light. In fact, plants treated with ACh consistently grew longer after brief exposure to light than those in constant darkness. We explore models for the action of ACh on the greening process.
REFERENCES
Gong X, Bisson M (2002) J Membr Biol 188: 107-113 Lewis B, Spalding E (1998) J Membr Biol 162: 81-90
60 Photoperiodic adaptation by systemic control of growth and rates and planes of cell division via systemic electrophysiological communication from the cellular to the organismic level
Marco Vervliet-Scheebaum, Johannes Normann, Justyna Veit, Edgar Wagner
Albert-Ludwigs University, Institute of Biology, Schänzlestrasse 1, D-79104 Freiburg, Germany
Email: [email protected]
Vegetative growth and the transition to reproductive growth involve continuous communication between all plant organ systems and their response to the networking of internal and external signals. Time-lapse photography is clear evidence of such adaptative behaviour to environmental signals resulting in a precise pattern of rhythmic phenomena. Rhythmic integration of the whole plant involves modulation of turgor pressure via stretch-activated ion channels and concomitant changes in membrane potentials, potentially leading to action and/or variation potentials. As evident on the cellular level, induction of polarity, the basis for changes in rates and planes of cell division, involves latent changes in patterning of plasmamembrane electrochemistry, which eventually becomes stabilised by structural polarity involving cytoskeleton elements. It is proposed that the dynamic electrochemical activity is continuously integrating internal and external signalling for developmental adaptations in a changing environment.
61 Mesophyll cells are the driving force for light- and acid- induced leaf blade expansion of Pisum sativum var. argenteum
Rainer Stahlberg, Robert E. Cleland, Elizabeth Van Volkenburgh
University of Washington, Seattle, WA USA
Email: [email protected]
Dicot leaves have a laminate structure of four cell layers: the two epidermal layers surrounding the palisade and spongy mesophyll tissues. There has been persisting uncertainty as to which of these layers is driving the expansion of leaf blades. To solve this problem we made use of the Argenteum mutant of pea, where viable epidermal layers can be easily removed from the leaflets. Removal of the main vein or just one epidermis did not alter the growth rate of excised leaflet strips, but removal of the second epidermis caused a rapid increase in the growth rate. Long-term experiments confirmed that the light-response of isolated mesophyll strips excedes that of the complete-leaflet strips by 50 %, while isolated epidermis strips expand only when pulled by an external force. Both isolated mesophyll and epidermal tissues undergo rapid elongation in response to a change in solution pH from 6.0 to 4.5. Previous experiments comparing the ability of isolated epidermis and mesophyll layers to pump protons in response to light found the response of the mesophyll to be larger (Stahlberg & VanVolkenburgh 1999). These results support the idea that the mesophyll layers drive and control the rate of leaf expansion.
62 The linear phase of sucrose uptake concentration curve in sink organs is largely mediated by fluid phase endocytosis
Ed Etxeberria1, Diego Pozueta1, Pedro Gonzalez1, Javier Pozueta2
1 University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA 2 Agrobioteknologia Instituta, Consejo Superior de Investigaciones Cientificas and Nafarroako Jauralitza, Mutiloako etorbidea zembaki gabe, 31192 Mutiloabeti, Nafarroa, Spain
Email: [email protected]
Biochemical studies have demonstrated that sucrose uptake kinetics into sink cells consist of multiple components collectively characterized by a bi-phasic curve. Whereas the hyperbolic phase at low external sugar concentration is believed to represent a high-affinity, membrane- bound, carrier-mediated component, the linear non-saturable phase at higher concentrations denotes facilitated diffusion presumably mediated by a sucrose binding protein. Previous observations that FPE in celery parenchyma was only induced at high external mannitol prompted us to re-examine the possible role of FPE and membrane-carrier transport within both phases of the characteristic concentration uptake curve. At low external concentration (5 mM), sucrose uptake into turnip (Brassica campestris) hypocotyl discs was inhibited by the sucrose carrier inhibitor phloridzin (2 mM) but unaffected by the endocytic inhibitor latrunculin-B (10 μM). When sucrose concentration was increased to 100 mM, transport was significantly reduced by both phloridzin and latrunculin-B. Uptake of the endocytic marker Alexa-488 was strongly inhibited by latrunculin-B at both external sucrose concentrations with no effect noted in the presence of phloridzin. Analyses of the data and of Alexa-488/sucrose ‘specific activity’ revealed that the characteristic linear phase of sucrose uptake concentration curves is largely mediated by fluid phase endocytosis. Time-lapse photography using confocal laser scanning microscopy captured the rapid fusion of a ~5 μm vesicle with the vacuole supporting earlier suggestions that most of the endocytic uptake occurs through a clathrin-independent micropinosome system. In other photographs, the inclusion of multivesicular bodies within the vacuole was evident. The presence of multivesicular bodies (containing fluorescent vesicles) strongly suggest the existence of a retrograde vesicle transport system from the vacuole capable of reconciling vacuolar volume and constituting the energy- dependent phase that concentrate solutes within.
63 Cytokinin oxidase/dehydrogenase activity in oat xylem sap
Klára Hoyerová1, Václav Motyka1, Blanka Sýkorová1, Ivana Raimanová2, Marie Trčková2, Petre I. Dobrev1, Miroslav Kamínek1
1 IEB, AS CR, Rozvojová 263, 165 02 Prague 6, CZECH REPUBLIC 2 Crop Research Institute, Drnovská 507, 161 00 Prague 6, CZECH REPUBLIC
Email: [email protected]
This research was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (IAA600380507 and IAA600380701).
Cytokinins affect plant growth and development by stimulating cell division and differentiation. Modulation of flux of cytokinins from roots to shoots via xylem flow in response to environmental signals has been repeatedly reported. The concentration of cytokinins in apoplast can be potentially modulated by extracellular cytokinin metabolism. We found the cytokinin oxidase/dehydrogenase (CKX) activity in xylem sap of oat (Avena sativa L.) plants. The enzyme exhibited pH optimum at 8.5 and its activity was associated with glycosylated protein. Since the pH of root-sourced xylem sap is much lower (6.1) the activity of the CKX leaving the roots is suppressed protecting the co- transported cytokinins from degradation. The potential role of CKX in control of the cytokinin concentration in xylem sap in response to environmental signals was tested by the exposure of - 12 d old plants for 48 h to nutrient solutions differing in NO3 concentration (16-1000 µM). The - flux of the root-sourced CKX activity was increased with the increasing NO3 supply up to 7-fold correlating well with the increasing flux of cytokinins [trans-zeatin riboside, trans-zeatin and N6- (2-isopentenyl)adenine]. The flux of cis-isomers of zeatin was, with exception of cis-zeatin O-glucoside, - not affected by the exogenous NO3 indicating different regulation of biosynthesis of trans- and cis-zeatins by nitrate
64
ECOLOGY
65 The role of volatiles in plant-to-plant communication
Mark C. Mescher, Consuelo M. De Moraes.
Department of Entomology, Pennsylvania State University, University Park PA, USA 16801.
Email: [email protected]
Plant volatiles have long been known to mediate many important interactions between plants and insects, but their importance in interactions among plants has been much debated. We discuss two recent projects dealing with volatile mediated plant-to-plant communication. The first demonstrates that seedlings of the parasitic dodder plant Cuscuta pentagona use volatile cues to locate host plants and to distinguish between more and less preferred hosts. Several individual compounds present in volatile blend of the preferred host tomato (Lycopersicon esculentum) are shown to be attractive to C. pentagona seedlings, while one compound present in the non-host wheat (Triticum aestivum) is shown to be repellent. The second project shows that volatiles released by herbivore-wounded leaves of hybrid poplar (Populus deltoides x nigra) prime defenses in adjacent leaves on the same plant that have little or no vascular connection to the wounded leaves. Undamaged leaves exposed to volatiles from wounded leaves on the same stem had elevated defensive responses to feeding by gypsy moth larvae (Lymantria dispar) compared to leaves that did not receive volatiles. While previous research has focused on signaling between plants, self- signaling via volatiles is consistent with the short distances over which plant response to airborne cues has been observed to occur, suggesting that within-plant signaling may greater ecological significance than previously realized.
66 Allelochemicals as a signaling molecules in the negative plant-plant interaction
Renata Bogatek, Krystyna Oracz, Agnieszka Gniazdowska
DPP, Warsaw University of Life Sciences – SGGW, Nowoursynowska 159, 02-776 Warsaw, POLAND
Email: [email protected]
Allelopathy phenomenon is defined as the influence of one plant on another through chemicals (allelochemicals) released into the environment. The action of allelochemicals in target plant is diverse and affects a large number of biochemical reactions resulting in the modifications of variety physiological processes. Sunflower (Helianthus annuus L.) actively influences the growth of surrounding plants due to its strong allelopathic potential. We investigated mode of action of sunflower allelochemicals during germination of mustard (Sinapis alba L.) seeds. Inhibition of germination was associated with alterations in reserve (lipids, proteins) mobilization and energy (ATP) generation in the catabolic phase of germination (Kupidlowska et al. 2006). Additionally, sunflower allelopathic compounds induced oxidative stress manifested as enlarged production and accumulation of reactive oxygen species (ROS) (Oracz et al. 2007). It correlated well with loss of membrane integrity (Bogatek et al. 2006). Therefore we suggested that in allelopathy stress ROS (H2O2) may act as signaling molecules leading to disturbances in the balance of phytohormones crucial for seed germination. ABA concentration in seeds increased after exposition to sunflower allelochemicals, in the contrast, ethylene emission was strongly repressed (Gniazdowska et al. 2007). Low ethylene concentration, resulting from inhibition of key enzymes activities of ethylene biosynthesis may enhance seed sensitivity to ABA. The alteration in phytohormones level leads to decreasing metabolic activity of the embryo and blocking seed germination as well as growth of young seedlings. The putative relationship between ROS and phytohormones in allelopathy interaction will be discussed.
REFERENCES
Kupidłowska E, Gniazdowska A, Stępień J, Corbineau F, Vinel D, Skoczowski A, Janeczko A, Bogatek R (2006) J Chem Ecol 32: 2569-2583 Oracz K, Bailly C, Gniazdowska A, Come D, Corbineau F, Bogatek R. (2007) J Chem Ecol 33: 251-264 Bogatek R, Gniazdowska A, Zakrzewska W, Oracz K, Gawroński SW (2006) Biol Plant 50: 156-158 Gniazdowska A, Oracz K, Bogatek R (2007) Allelopathy J 19: 215-226
67 Chemical communication between roots and shoots in tomatoes
De Oliveira RF, Zsögön A, Peres LEP
Department of Biological Sciences. Escola Superior de Agricultura Luiz de Queiroz – LCB/USP, Av. Pádua Dias, 11 CP. 09 CEP 13418-900 Piracicaba - SP. Brazil
Email: [email protected]
Abscisic acid (ABA) is not only synthesized in leaves, but also in roots and it is conventionally accepted that root-sourced ABA plays a key role upon water deficit, triggering stomatal closure in the leaves. Here, we used the ABA-deficient mutant notabilis (not) in Lycopersicon esculentum, its isogenic cultivar Lukullus (Luk) and a naturally desiccation-resistant wild relative L. pennellii (pen) to study the relative importance of leaf and root-derived ABA on stomatal closure. We conducted a series of graftings with these genotypes in all possible shoot/rootstock combinations and then imposed water stress on the plants. Measurements of stomatal conductance, transpiration and water potential were performed. Thesuccess of grafts was minimal when not was the scion or pen was the rootstock. In graftings involving a not shoot, stomatal conductance and transpiration were reduced during water stress and the recovery period if pen or Luk was used as rootstock rather than not itself. Conversely, low stomatal conductance was also observed in pen even when the rootstock was not. The not/not graftings attained the permanent wilt point in 5 days whereas not/pen survived without irrigation for 21 days. These results suggest that the genotype of the shoot determines stomatal activity under normal irrigation and that under dehydration and the subsequentrecovery the control is given by a root-derived substance, which appears to be in a higher dose in L. pennellii. This opens interesting perspectives for the basic and applied aspects of water stress resistance in plants.
68 Neurotoxicity of aluminium: parallelism between plants and animals (including men)
Charlotte Poschenrieder1, Montserrat Amenós1, Snezhanka Doncheva2, Juan Barceló1
1 Lab. Fisiología Vegetal, Facultad de Biociencias, Universidad Autónoma de Barcelona, SPAIN 2 Bulgarian Acad. Sci., Inst. Plant Physiol., BU-113 Sofia, BULGARIA
Email: [email protected]
Supported by the Spanish Government DGICYT BFU2004-02237-CO2-01 Project PLANTIONREG
Although epidemiological studies in men are still inconclusive, it is well established that aluminium (Al), in available form, is extremely toxic to all organisms. The neurotoxic effects in animals and men are well documented. High tissue Al concentrations have been found in patients with dialytic encephalopathy, amyotrophic lateral sclerosis or Alzheimer disease. In plants, Al mainly affects root growth and development. A stunted root system with reduced capacity to explore the soil for water and nutrients is the main visible symptom of the Al toxicity syndrome. Besides these obvious differences in the outcome of Al toxicity, there are striking similarities in the mechanisms of Al toxicity in both plant and animals, including men. This presentation will give a comprehensive overview on the basic mechanisms by which Al may cause neurotoxicity in both animals and plants. At the present stage of knowledge, it is getting clear that specific cells are the primary targets of Al toxicity in both humans and plants: astrocytes in the animal or human brain and transition zone cells in roots, the “brain-like cells” (Baluška et al. 2004, Illeš et al. 2006) of plants. Special attention will be paid to Al interactions with the plasma membrane, to Al-induced oxidative stress, and to the glutamate metabolism at these primary sites of toxicity. Perception and transmission of the Al signal and its consequences for adaptative root growth in plants will be discussed.
REFERENCES
Baluška F, Mancuso S, Volkmann D, Barlow P (2004) Biologia 59: 7-19 Illeš P, Schlicht M, Pavlovkin J, Lichtscheidl I, Baluška F, Ovecka M (2006) J Exp Bot 57: 4201- 4213
69 Common cellular mechanisms of endosymbiotic root infection
Ton Timmers1, Mireille Chabaud1, Andrea Genre2, Paola Bonfante2, Joëlle Fournier1, Björn Sieberer1, David Barker1
1 Lab. of Plant-Microbe Interactions, UMR INRA-CNRS, 31326 Castanet Tolosan Cedex, France 2 Dept. of Plant Biology, University of Turin and IPP-CNR, Turin, Italy
Email: [email protected]
The mutual beneficial relationships between plants and arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria known as rhizobia, are highly important both from an agricultural and ecological point of view. Plants exchange photosynthate products for phosphate in the first and nitrate in the latter, and provide a safe niche for their microbial partner. The AM association is wide-spread, while the rhizobial symbiosis is limited to leguminous plants. During both interactions the microbes invade internal root tissues developing specific intracellular symbiotic structures called arbuscules in the AM association and symbiosomes in nodulation. In legumes, the entry of both AM and rhizobial symbionts appears to be controlled by a common signal transduction pathway concerning a small number of plant genes (DMI1, -2, -3 and their homologues). The initial entry into root tissue is intracellular involving the formation of a host membrane/cell wall interface which physically separates the microbe from the host cell cytoplasm. During nodulation, invasion takes place through curled root hairs and a subsequently formed plant-derived structure, the so-called infection thread. For the passage through root cortical cells the infection thread makes use of a predefined way consisting of cytoplasmic bridges named pre-infection threads. AM fungi penetrate directly root epidermal cell surfaces and pass through a recently identified structure named the pre-penetration apparatus (Genre et al. 2005). Thus, in both symbioses the plant appears to be the principal partner in control of initial infection and infection progression. Comparative ongoing studies on both endosymbiotic associations in our laboratory will be presented in relation to the strategies developed by plants to control beneficial microbe entry into host tissues.
REFERENCE
Genre A, Chabaud M, Timmers T, Bonfante P, Barker DG (2005) Plant Cell 17: 3489-3499
70 Piriformospora indica: A cultivable symbiotic fungus with multiple biotechnological applications: Molecular analysis of its interaction with Arabidopsis thaliana
Ralf Oelmüller
Institute of General Botany and Plant Physiology, Friedrich-Schiller-University Jena, Dornburger Str. 159, 07743 Jena, Germany
Email: [email protected]
Piriformospora indica is a wide-host root-colonizing fungus which allows the plants to grow under extreme physical and nutrient-limiting conditions. The fungus promotes growth and seed production and confers resistance against biotic and abiotic stresses (1). We study the molecular basis of the interaction between P. indica and the model plant Arabidopsis thaliana (cf. 2, 3) Based on mutant screens, expression profiling, proteomics as well as biochemical techniques, we could identify plant components, which are required for this beneficial plant/microbe interaction. Components involved in recognition, early signalling events and maintenance of the symbiotic interaction will be discussed. In a second screen, we identified Arabidopsis mutants in which growth and development is inhibited rather than promoted by the fungus. Apparently, only a few components in plants need to be manipulated to convert a beneficial into a pathogenic interaction.
REFERENCES
(1) Peskan-Berghöfer T, Shahollari B, Giong PH, Hehl S, Markert C, Blanke V, Kost G, Varma A, Oelmüller R (2004) Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant-microbe interactions and involves early plant protein modifications in the endoplasmic reticulum and at the plasma membrane. Physiol Plant 122: 465-477 (2) Sherameti I, Shahollari B, Venus Y, Altschmied L, Varma A, Oelmüller R. The endophytic fungus Piriformospora indica stimulates the expression of nitrate reductase and the starch- degrading enzyme glucan-water dikinase in tobacco and Arabidopsis roots through a homeodomain transcription factor that binds to a conserved motif in their promoters. J Biol Chem 280: 26241-26247 (3) Shahollari B, Vadassery J, Varma A, Oelmüller R. (2007) A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana. Plant J 50: 1-13
71 Ion channel-forming compounds in caterpillar regurgitate: A way to manipulate the plant plasma membrane potential during herbivory?
Heiko Maischak1, 2, Pavel Grigoriev1,3, Wilhelm Boland1, Axel Mithöfer1
1 MPI for ChemicalEcology, Hans-Knöll-Str. 8, D-07745 Jena, Germany 2 GSF National Research Center for Environment and Health, Institute of Biochemical Plant Pathology (BIOP), D-85764 Neuherberg, Germany 3 Institute of Cell Biophysics, Russian Academy of Science, Pushino 142290, Russian Federation
Email: [email protected]
When insects feed on plants, they introduce oral secretions (OS) into the plant tissue. These OS contain several molecules that are known to be involved in the induction of plant defence reactions and subsequent processes. OS was analyzed with regard to their membrane activities using the black lipid membrane (BLM) technique. Transmembrane ion fluxes were generated by OS of eight different lepidopteran larvae, which all displayed comparable ion channel-forming properties in artificial membranes. These currents were characterized by long lasting opening times and high conductivities. The OS from Spodoptera exigua exhibited channels with a preference of cations over anions. OS also induced a transient increase of the cytosolic calcium concentration in soybean cells, which was determined by the aequorin technique. Other compounds of the OS, fatty acid- amino acid conjugates (FACs), also interfere with BLMs. But unlike OS, they do not form long lasting channels. Since ion fluxes and depolarization are early responses upon insect feeding, OS-derived components may directly be involved and interact with the plant membranes. The focus of ongoing work lies on the purification and subsequent identification of the substance(s) responsible for the channel-formation.
REFERENCE
Maischak H, Grigoriev P, Vogel H, Boland W, Mithöfer A (2007) FEBS Letts 581: 898–904
72 Cutinized and suberized plant/environment interfaces: structure, biosynthesis and function
Lukas Schreiber
Institute of Cellular and Molecular Botany, Department of Ecophysiology, University of Bonn, Kirschallee 1, 53115 Bonn, Germany
Email: [email protected]
As a prerequisite for colonisation of the mainland, plants developed lipophilic biopolymers forming the interface between the plant and the surrounding air and soil environment. Leaf surfaces are covered by cuticles and waxes, stem and root interfaces are formed by suberized cell walls. As main function lipophilic interfaces form efficient transport barriers protecting land-living plants from uncontrolled water loss and at the same time they protect living plant tissue from infection by pathogens. Various aspects of our ongoing research related to the structure, biosynthesis and function of cutinized and suberized plant/environment interfaces will be presented and discussed.
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74 Endocytic uptake and traffic of sucrose linked to both starch and cellulose biosynthesis are processes specifically triggered by sucrose that require the synthesis de novo of proteins
Edurne Baroja-Fernández1, Francisco José Muñoz1, Ed Etxeberria2, Pedro González2, Ignacio Ezquer1, Javier Pozueta-Romero1
1 Instituto de Agrobiotecnología, Consejo Superior de Investigaciones Científicas, Universidad Pública de Navarra, Gobierno de Navarra. Mutiloako etorbidea z/g, 31192 Mutiloabeti, Nafarroa, Spain. 2 University of Florida, Citrus Research Center, 700 Experiment Station Road, Lake Alfred, FL.
Email: [email protected]
We have recently established that an important pool of sucrose linked to starch biosynthesis in heterotrophic cells is taken up by endocytosis (Etxeberria et al. 2005 Plant Cell Physiol. 46, 474- 481; Baroja-Fernandez et al. 2006 Plant Cell Physiol. 47, 447-456). Whether this mechanism is also involved in the sucrose-cellulose conversion process was investigated by comparing the rates of cellulose accumulation in sycamore cells cultured in the presence or absence of the endocytic inhibitors wortmannin-A, 2-4(4-morpholynyl-)8-phenyl-4H-1 benzopyran-4-1 (LY294002) and latrunculin B. These analyses revealed that sucrose-cellulose conversion involves two phases, the second of which being 35% sensitive to the effect of endocytic inhibitors. Whether endocytic uptake, traffic and metabolism of sucrose requires the de novo synthesis of proteins was investigated by comparing the rates of accumulation of the endocytic marker lucifer yellow, sucrose and starch in sycamore cells in the presence or absence of the transcriptional inhibitor cordycepine and the translational elongation inhibitor cycloheximide. These analyses revealed that the two compounds exerted a strong inhibitory effect on the accumulation of lucifer yellow, sucrose and starch. The stimulatory effect of sucrose in the endocytic uptake of external solutes could not be replaced by the non-metabolizable sucrose analogues palatinose and turanose. The overall results (a) provide a first indication that the endocytic uptake of sucrose linked to both starch and cellulose biosynthesis requires de novo synthesis of proteins and (b) further strengthen that the endocytic uptake of external solutes is very specifically triggered by sucrose.
75 Multiple isoforms of phospholipase D are involved in the regulation of plant cell morphogenesis
Radek Bezvoda1, Martin Potocký2,3, Olga Valentová3, Viktor Žárský1,2
1 Department of Plant Physiology, Charles University, Vinicna 5, 12844 Prague 2, Czech Republic 2 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 265, 165 02 Prague 6, Czech Republic 3 Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technická 5, 166 28 Prague 6, Czech Republic
Email: [email protected]
Support from LC06034 project of Czech Ministry of Education is acknowledged.
Phospholipase D (PLD) cleaves structural phospholipids, namely phosphatidylcholine, producing second messenger phosphatidic acid (PA). PLD and PA play crucial role in many signal transduction pathways across eukaryotic kingdom. In animal and yeast cells, PLD was implicated in the regulation of vesicular trafficking and dynamics of actin cytoskeleton. In plants, PLD research is mainly focused on its role in responses to various stresses and involvement of PLD/PA in the mechanisms controlling cell polarity is only partially characterised. Here we present data showing that multiple PLDs are required for polar growth of tobacco pollen tubes. We cloned five partial PLD cDNAs from tobacco pollen tubes and BY2 cells covering all major PLD subfamilies. RT-PCR analysis suggested differential expression of studied cDNAs. In order to functionally characterize distinct PLD isoforms, we used gene specific knock-down mediated by antisense oligonucleotides. The suppression of NtPLDbeta1 and NtPLDdelta lead to lower growth rates, whereas exogenously applied PA restored normal growth, thus confirming the importance of PLD signaling for polar growth of pollen tube and raising the question of downstream targets of PA. Visualization of actin cytoskeleton indicated the involvement of PLD/PA signalling in cytoskeletal dynamics.
76 Plant γ-tubulin is essential for noncentrosomal microtubule nucleation from microtubules and membranes associated dispersed sites
Pavla Binarová1, Jirina Prochazková1, Vera Cenklová2, Ondrej Plihal1, Jana Hendrychová1, Jindrich Volc1
1 Institute of Microbiology ASCR, Videnska 1083, Prague, 142 20 Czech Republic 2 Institute of Experimental Botany, ASCR, 772 00 Olomouc, Czech Republic
Email: [email protected]
Supported by grants GACR 204/07/1169, MSMT LC 545, IAA 500200719.
Microtubules (MTs) nucleated independent of defined microtubule organizing centres such as centrosomes or spindle pole bodies have been only recently shown to play an important role in designing cytoskeleton architecture. γ-Tubulin is required for MT nucleation at defined microtubule organizing centres but its role in nucleation of noncentrosomal MTs is much less understood. In higher plants where all somatic and gametic cells are acentrosomal, there are several microtubular arrays organized during cell cycle progression from undefined dispersed sites. Well characterized γ-tubulin ring complexes that are essential for centrosomal MT nucleation in animal cells have not yet been identified in plants. Rather we found the presence of heterogeneous protein complexes of γ-tubulin in cytoplasm, in association with membranes and MTs. Large γ-tubulin complexes were active in microtubule nucleation. To further analyze the role of γ-tubulin, we conditionally downregulated γ-tubulin by inducible expression of RNAi constructs in Arabidopsis thaliana. After induction of RNAi γ-tubulin was gradually depleted from all known cellular locations including the microsomal and the microtubular fraction. We found that γ-tubulin as a component of cortical nucleation templates guides cortical MTs. The regrowth of MTs from perinuclear membrane rich region after drug depolymerization was delayed in cells with reduced γ-tubulin levels. Similarly, immunodepletion of γ-tubulin from A. thaliana extracts strongly compromised the in vitro polymerization of MTs. Almost complete RNAi depletion of γ-tubulin led to the absence of microtubules. In summary, we showed that γ- tubulinu is essential for MTs nucleation from dispersed sites in acentrosomal plant cells. Further characterization of γ-tubulin forms and their protein interactions is under progress.
77 Glutamate and ethanol deplete F-actin and inhibit vesicle recycling at “plant synapses” in root apices
Christian Burbach, Markus Schlicht, Diedrik Menzel, František Baluška
IZMB & LINV, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY
Email: [email protected]
L-Glutamate is a well-known neurotransmitter in brain and it has also dramatic impacts on plant root apices (Filleur et al. 2005, Walch-Liu et al. 2006). Specifically, the primary root apex is sensitive to L-Glutamate, which does not affect apices of young lateral root primordia (Walch- Liu et al. 2006). Plants also express glutamate-like receptor family proteins (GLRs) gated by glutamate and glycine (Dubos et al. 2003, Gilliham et al. 2006). Glutamate gated GLRs emerge to act in plants, similarly like in animals, as calcium channels (Demidchik et al. 2004, Kang et al. 2006, Qi et al. 2006) involved in the response of plants to sensoric stimuli and to stress from the environment (Kim et al. 2001, Sivaguru et al. 2003, Kang et al. 2004, Meyerhoff et al. 2005). Genetic evidence suggest, that GLR are essential for the organization and functioning of primary root apices (Li et al. 2006). Here we have analyzed effects of glutamate on the actin cytoskeleton and vesicle trafficking in primary root apices of Arabidopsis and maize. Our data reveal, that the most sensitive subcellular domains are the cell end-poles, which represent what we have defined as plant synapses (Baluška et al. 2005). Especially in the transition zone (Verbelen et al. 2006), F-actin gets depleted and vesicle trafficking inhibited at plant synapses in primary root apices. Surprisingly, similar effects have been scored also with ethanol at concentrations even lower as those (Offenhäuser et al. 2006) which has been recently reported to affect F-actin at mouse brain synapses (Offenhäuser et al. 2006, Sordella and Van Aelst 2006). In the future, we will study the behavior and performance of roots challenged with exogenous glutamate and ethanol.
REFERENCES
Baluška F, Volkmann D, Menzel D (2005) Trends Plant Sci 10: 106-111 Demidchik V, Essah PA, Tester M (2004) Planta 219: 167-175 Dubos C, Huggins D, Grant GH, Knight MR, Campbell MM (2003) Plant J 35: 800-810 Filleus S, Walch-Liu P, Gan Y, Forde BG (2005) Biochem Soc Trans 33: 283-286 Gilliham M, Cambell M, Dubos C, Becker D, Davenport R (2006) In: Communication in Plants: Neuronal Aspects of Plant Life, F Baluška, S Mancuso, D Volkmann (eds), Springer-Verlag, 187-204 Kang J, Mehta S, Turano F (2004) Plant Cell Physiol 45: 1380-1389 Kang S et al. (2006) Mol Cells 21: 418-427 Kim SA et al. (2001) Plant Cell Physiol 42: 74-84 Li J et al. (2006) Plant Cell 18: 340-349 Meyerhoff O et al. (2005) Planta 222: 418-427 Offenhäuser N et al. (2006) Cell 127: 213-226 Qi Z, Stephens NR, Spalding EP (2006) Plant Physiol 142: 963-971 Sivaguru S, Pike S, Gassmann W, Baskin TI (2003) Plant Cell Physiol 44: 667-675 Sordella R, Van Aelst L (2006) Cell 127: 37-39 Verbelen J-P, De Cnodder T, Le J, Vissenberg K, Baluška F (2006) Plant Signal Behav 1: 296-304 Walch-Liu P, Liu LH, Remans T, Tester M, Forde BG (2006) Plant Cell Physiol 47: 1045-1057
78 γ-Tubulin and its role in Arabidopsis development
Věra Cenklová1, Anna Doskočilová2, Beata Petrovská1, Barbora Gallová1, Olga Kofroňová2, Oldřich Benada2, Pavla Binarová2
1 Institute of Experimental Botany, ASCR, v.v.i., Sokolovská 6, 77200 Olomouc, Czech Republic 2 Institute of Microbiology, ASCR, v.v.i., Vídeňská 1083, 14220 Prague, Czech Republic
Email: [email protected]
Supported by grants GACR 204/07/1169, MSMT LC06034 (V.C.), MSMT LC 545, IAA 500200719 and 204/05/H023 GACR (P.B.).
We found that inducible RNAi depletion of γ-tubulin led to serious distortions of development in A. thaliana seedlings. Cells with decreased levels of γ-tubulin could progress through mitosis, but late mitotic events and cytokinesis were strongly affected. Particularly, we observed that polar distribution of γ-tubulin during late mitosis was disturbed and the phragmoplast formation failed. In contrast to the control cells where anaphase spindles were rearranged into the phragmoplast, long anaphase spindles persisted between separated nuclei in RNAi cells. The cell plate formation sites were often misaligned. These discrepancies in late mitosis and cytokinesis often resulted in bi- or multi-nuclear cells and disruption of regular cell files and some morphogenic changes were observed. Strict developmental pattern of stomata was disrupted and clusters of two to four stomata were observed in RNAi expressing plants with reduced γ-tubulin levels. In addition to the stomata clustering, the cytokinetic defects of guard cells were found when γ-tubulin was severely depleted. Dorsoventral polarity during leaf development was disturbed in seedlings with reduced γ-tubulin levels. Ectopic root hairs formation was observed in cells with randomized microtubules, anisotropic growth of root hairs was disturbed, formation of two growth axes was often observed. We suggest that some functions of γ-tubulin that are important for cytokinesis, cell specification and polar growth might be microtubule independent and require further analysis.
79 Physiological basis for altered responsiveness to auxin and light in modern corn hybrids – role of auxin-binding proteins
Mária Čudejková1, Jan Humplík1, David Zalabák1, Aleš Pěnčík2, Jakub Rolčík2, Liz Van Volkenburgh3 & Martin Fellner1,4
1 Department of Cell Biology and Genetics, Palacky University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic 2 Laboratory of Growth Regulators, Institute of Experimental Botany ASCR, v.v.i. and Palacky University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic 3 Department of Biology, University of Washington, Seattle, WA, USA 4 Institute of Experimental Botany ASCR, v.v.i., Šlechtitelů 11, 783 71, Olomouc, Czech Republic
Email: [email protected]
The work was supported by grant from the Ministry of Education of the Czech Republic to MF (grant no. 1P05ME792), and by Pioneer Hi-Bred, Intl., USA, Austria and the Czech Republic
Modern varieties of corn (Zea mays L.) developing erect leaves have been selected for their ability to maintain production in dense planting. We showed earlier that on the whole plant level, and at the cellular and molecular levels, the modern hybrid 3394 is less sensitive to exogenous auxin than two older hybrids 307 and 3306. Others confirmed our results since there is a decline in response to auxin over the decades of varieties release. We also showed that the levels of endogenous free IAA in 307 and 3394 were similar. The modern hybrid 3394 growing in the dark was also less sensitive to exogenous auxin than other two older hybrids 3366 and 317. Also, excised mesocotyl segments of 3394 were less responsive to NAA than segments of the older varieties. An additional modern hybrid, Benecia developing erect leaves, showed less sensitivity to exogenous auxin than older hybrids PR39A37 and PR39G83 with less erect leaves for leaf angle development and expression of ABP4 (auxin- binding protein 4). Interestingly, the three hybrids did not differ in the level of endogenous free IAA in etiolated mesocotyls. We published recently that growth of 3394 seedlings is less inhibited than growth of older hybrids by red (R) and far-red light (FR). Here we found that 3394 mesocotyl is also less responsive than all the tested older hybrids to the inhibitory effect of blue light (BL). In contrast to R or FR, BL in our experimental conditions promoted elongation of coleoptile, and the stimulatory effect was much stronger in 3394 than in the older varieties. Interestingly, under BL, coleoptile elongation of the modern variety 3394 was inhibited by NAA significantly more than growth of coleoptile in 307. To understand more the role of maize ABPs in growth and development, we analyzed maize single mutants abp1 and abp4, and the double mutant abp1abp4. Mutations in ABP1 and ABP4 genes caused changes in development of leaf angle. In comparison with the corresponding wild-type (WT), abp1 and abp4 developed more and less erect leaves, respectively. Interestingly, etiolated WT, abp1, and abp4 seedlings exhibited similar responses to exogenous auxin for coleptile, mesocotyl, and root growth. However, mesocotyls of double mutant abp1abp4 were distinctly less sensitive to the inhibitory effect of exogenous auxin. Analysis of endogenous auxin in etiolated mesocotyls revealed that all the abp mutants contain significantly greater levels of free IAA. Our results support the existence of interaction between auxin and light in regulation of growth and development of young corn seedlings. The results further indicate that ABP1 and ABP4 are involved in mesocotyl growth and leaf angle development, but also suggest ABP redundancy in maize. Finally, our data support the hypothesis that modern corn hybrids developing erect leaves are less responsive to exogenous auxin. Specific function of blue light in development of maize seedlings is discussed.
80 Possible interaction between blue light and anion and water channels during plant responses to abiotic stresses
Martin Fellner1,2, Katerina Cermákova1, Emeric Sagot1, Ekaterina Ossipova1, Jana Zaoralová1, Renata Hlouskova1, Kristyna Pospisilova1, Vladka Hlavackova3, Ondra Novak3
1 Department of Cell Biology and Genetics, Palacky University in Olomouc, Czech Republic 2 Institute of Experimental Botany ASCR, v.v.i. Olomouc, Czech Republic 3 Laboratory of Growth Regulators, Institute of Experimental Botany ASCR, v.v.i. and Palacky University in Olomouc, Czech Republic
Email: [email protected]
The work was supported by grant from the Ministry of Education of the Czech Republic (grant no. MSM6198959215) and by grants from Academy of Sciences of the Czech Republic to MF (grant no. I036 and grant no.18102) .
We previously reported that spontaneous mutant 7B-1 in tomato is resistant to osmotic and salt stress in seed germination specifically under blue light (BL). We showed that in tomato wild-type (WT), BL strongly inhibits seed germination, but essentially less than in 7B-1. Relative to the WT, 7B-1 seedlings develop longer hypocotyl in WL and BL. The long-term objective of our work is to determine whether 7B-1 gene is involved in blue light signaling, and what is the role of 7B-1 product in plant tolerance to stresses. Here we report that NPPB, an anion-channel blocker, can intensify the inhibitory effect of BL on germination in tomato, when applied on WT seeds cultured under BL. Interestingly, germination of 7B-1 seeds in BL is almost completely resistant to NPPB. Aquaporins could represent a prerequisite for strategies against osmotic stress. Like NPPB, HgCl2, an inhibitor of aquaporin, under BL powerfully inhibits seed germination in WT, but not in 7B-1. Differently from 7B-1, mutations cry1-1 and cry1-2 do not result in the resistance of seed germination to mannitol, and do not alter sensitivity of seed germination to the inhibitory effect of NPPB or HgCl2. On the other hand, defects in two Arabidopsis genes coding for anion channels result in increased sensitivity of seed germination to mannitol, specifically under BL. Our results suggest that functional anion-channels and/or aquaporins may be involved in ability of seeds to tolerate osmotic stress. Data also indicate that BL is involved in the process. 7B-1 mutant seems to have some traits of plants less responsive to biotic stress. We found that in the dark and BL hypocotyls of the 7B-1 mutant are less sensitive than WT to the inhibitory effect of Pseudomonas syringae phytotoxin coronatine added to the culture medium. Interestingly, the resistance was associated with the fact that in contrast to the WT plants, level of endogenous salicylic acid (SA) and jasmonic acid (JA) in 7B-1 hypocotyls could not be altered by BL. Our results suggest that BL plays a role in plant tolerance to abiotic stresses, and that anion channels and/or aquaporins may be involved in the process. Pleiotropic effect of the mutation suggests that 7B-1 gene product can function as an upstream element in light signaling pathway(s). We hypothesize that specifically in BL, 7B-1 mutation enhances activity of anion and/or water channels, which can help the mutant seeds to tolerate osmotic stress. Analysis of recently obtained cDNA microarray data is in progress, and cloning of 7B-1 gene will follow.
81 Acetylcholine signalling targets “plant synapses”
Silvio Henze, Markus Schlicht, Diedrik Menzel, František Baluška
IZMB & LINV, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY
Email: [email protected]
The classical neurotransmitter acetylcholine (ACh) is well-known for propagating action potentials across neuronal synapses (Phillis 2005). At the neuromuscular synapse, the entire process of signal transmission, including vesicular release ofACh, its diffusion across the synaptic cleft, reversible binding with nicotinic ACh receptor, and finally the enzymatic hydrolysis of ACh by acetylcholinesterase (AChE) takes only a few milliseconds. In plant tissues, ACh is an abundant molecule, which increases its endogenous concentrations under stress situation (Tretyn 1991). Plants express AChE, which is inhibited by neostigmine bromide, a specific inhibitor of the animal AChE (Sagane et al. 2005). Older studies already revealed that exogenous ACh stimulates plant cell elongation (Evans 1972). Moreover, endogenous ACh levels are sensitive to light and oscillate (Tretyn and Tretyn 1990). It has also been shown, that ACh-hydrolyzing activity in maize is essential for the root graviresponse (Momonoki 1997, Momonoki et al. 2000). Here we report, that experimental manipulations of ACh levels exerts specific actions on root apices of Arabidopsis and maize. The F-actin-enriched cross-walls in the root transition zone, which we have previously defined as “plant synapses” (Baluška et al. 2005), emerge as the most sensitive intracellular domains. Excess of ACh, induced either by addition of exogenous ACh or by inhibition of the AChE, has a dramatic impact on spatial control of cell division planes, F-actin assembly, endocytosis and vesicle recycling activities, as well as on the overall architecture of the plant synapse.
REFERENCES
Baluška F, Volkmann D, Menzel D (2005) Trends Plant Sci 10: 106-111 Evans ML (1972) Plant Physiol 50: 414-416 Momonoki YS (1997) Plant Physiol 114: 47-53 Momonoki YS et al. (2000) Plant Prod Sci 3: 17-23 Phillis JW (2005) Crit Rev Neurobiol 17: 161-217 Tretyn A (1991) Bot Rev 57: 34-73 Tretyn A, Tretyn M (1990) Chronobiologia 17: 45-52 Sagane Y et al. (2005) Plant Physiol 138: 1359-1371
82 Involvement of ABA in the generation and propagation of electrical signal upon local burning demonstrated on wild-type and ABA-deficient tomato mutant plants
Vladimíra Hlaváčková 1,*, Petr Ilík1, Ondřej Novák2, Jan Nauš1
1 Laboratory of Biophysics, Department of Experimental Physics, Palacký University tř. Svobody 26, 771 46 Olomouc, Czech Republic 2 Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71 Olomouc, Czech Republic
*Email: [email protected]
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant No. MSM 6198959215).
An important role of electrical signals in short-term long-distance (systemic) plant responses to local wounding has been recently established (Koziolek et al. 2004, Hlaváčková et al. 2006). Simultaneously, a rapid action of plant hormones (abscisic and jasmonic acids) in systemic tobacco responses upon local stress has been shown (Hlaváčová et al. 2006, Hlaváčková and Nauš 2007). However, little is known about the interactions of these signals (electrical and chemical) and about the mechanisms whereby they mediate the systemic responses. We examined short-term (up to 1 h) local and systemic electrical responses of wild-type (WT) and abscisic acid (ABA)-deficient tomato mutant (sitiens) plants to local burning (12s) of an upper leaf. A lower endogenous concentration of ABA (about one third of WT) in leaves located below the burned one was detected in untreated and also burned sitiens plants compared to the WT tomato. The electrical recordings obtained from the wounded leaf and three others situated below the burned one (in basipetal direction) revealed significant differences between both variants. Firstly, the amplitude of electrical signal of WT (50-60 mV) plants was twice as high as that of sitiens (20-35 mV) plants. Secondly, the way of electrical signal propagation seemed to be influenced by ABA. While in the WT plants the amplitude and propagating velocity of electrical signal decreased with increasing distance from the site of burning (indicating the variation potential, VP), it did not hold for sitiens plants. Although the 5th leaf in the sitiens plants was closer (27 cm) to the burned site (6th leaf), the VP wave was delayed as compared to that of the more distant 4th (29 cm) and 3rd (31 cm) leaves. Taken into account the angle position of measured leaves on sitiens tomato plants, the VP wave propagated faster along the leaf trace of burned leaf (vascular bundles coming from the burned leaf) down the stem in tomato sitiens plants. Comparing amplitudes and propagation velocities of electrical potential changes in WT and sitiens mutant tomato plants, our results suggest a participation of ABA in the electrical signal generation and propagation in tomato plants after local wounding. Thus, an interaction of both, electrical and chemical signals, in rapid systemic plant stress responses is plausible.
REFERENCES
Hlaváčková V, Krchňák P, Nauš J, Novák O, Špundová M, Strnad M (2006) Planta 225: 235-244 Hlaváčková V, Nauš J (2007) Plant Signal Behav 2: In press Koziolek Ch, Grams TEE, Schreiber U, Matyssek R, Fromm J (2004) New Phytol 161: 715-722
83 Photoelectrical reaction of pumpkin plants and its interaction with action potential induced by electrical and thermal stimuli
Waldemar Karcz, Zbigniew Burdach, Renata Kurtyka
University of Silesia, Department of Plant Physiology, ul. Jagiellonska 28, PL-40032 Katowice, POLAND
Email: [email protected]
Many studies have shown that plants possess numerous mechanisms which enable them to perceive, transduce and respond to a variety of environmental signals. Light signals are amongst the most important environmental factors that regulate plant growth and development. The gol of this study was to determine the interaction of light-induced changes in electlical potential of pumpkin plants with the action potentials evoked by electrical and thermal stimuli. The experiments were carried out with 14-16 day-old pumpkin plants (Cucurbita pepo L.) grown in Hoagland`s medium under incandescent and luminescent light. The measurements of electrical potential difference were done with a non-invasive, surface-contact electrodes. The electrical reactions of pumpkin were induced by electrical (square current pulses), thermal (burning) and light (400 W m-2) stimuli. The action potential (AP) generated by electrical pulses in the lower part of the hypocotyl was transmitted and propagated with decrement to cotyledons and leaves. These voltage transient changes had a shape of single peak and fulfilled all-or-none law. The action potential triggered by thermal stimulus (local burning of cotyledons) differed in terms of amplitude and duration as compared to electrically induced AP. The irradiation of the plants with white light caused an electrical response of a specific nature which did not resemble the APs induced by electrical and thermal stimuli. The light-induced response comprised two phases: a relatively fast hyperpolarization followed by a slower depolarization. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity as compared to intracellular recordings. The amplitude and time course of depolarization phase depended on the duration of irradiation. When pumpkin plants were stimulated simultaneously with both light and electrical or thermal stimuli an additive effect was observed. The results are discussed taking into account the ionic basis of both light-induced electrical response and action potential triggered by electrical or thermal stimuli.
84 Functional adaptation of suspension-cultured tobacco BY-2 cells to the osmotic stress
Anna Kasprowicz1, Przemysław Wojtaszek1,2
1 Department of Molecular and Cellular Biology, Adam Mickiewicz University, Mi?dzychodzka 5, 60-371 Pozna?, Poland 2 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Pozna?, Poland
Email: [email protected]
This research is supported by the Ministry of Science and Higher Education grant PBZ-KBN- 110/P04/2004 to P.W.
Plant cells are continuously exposed to changing osmotic conditions, dependent on the versatile water accessibility. Long-term osmotic stress evokes activation of adaptive mechanisms, leading to changes in e.g. cellular metabolism. As a consequence, the cell is able to survive the stress. However, biochemical adaptations are not the only ones. Another group constitute mechanical adaptations, and the continuum between cell wall, plasma membrane and actin cytoskeleton is the major player here. The wall-protoplast interactions are particularly important for mechanical stabilization of the cells subjected to changing environmental conditions. They prevent either bursting of the protoplast under iso- and hypotonic conditions, or collapsing of the plasmolysing protoplast in hypertonic environment. The cytoskeleton plays an important role at the protoplast side in controlling cell shape and mediating intracellular signalling. Both microtubules and actin filaments might be anchored at the plasma membrane and further in the surrounding cell walls. This anchoring could be crucial for the proper functioning of cytoskeletal networks. The very special case of the cell’s response to stress is the adaptation to such conditions that are lethal to non-adapted cells. We have adapted the suspension-cultured tobacco BY-2 cells to extreme osmotic stress conditions evoked by high levels of ionic (NaCl, KCl) and nonionic (mannitol, sorbitol, polyethylene glycol) agents. The concentrations of these agents were chosen in such a way as to cause similar changes of the water potential. Nonionic and ionic osmotica act in different manner and result in specific responses of adapted cells. Ionic agents increase adhesive properties of the cells, and formation of cell aggregates. On the other hand, nonionic agents stimulate strictly positioned cell divisions and thus induce formation of cell files. Surprisingly, analyses of actin and tubulin cytoskeletons in adapted cells and non-adapted, unstressed, cells reveal no significant changes. However, tobacco BY-2 suspension cells exposed to short-term osmotic stress could cope with it in a cytoskeleton-dependent manner. Such cells reveal disruption of fine networks of cortical microfilaments and microtubules, and, most probably, formation of thicker cables. Changes in the actin cytoskeleton occur at membrane zones detached from cell walls – protoplast’s regions especially subjected to mechanical stress. It seems that upon prolonged exposure to osmotic stress conditions adaptive, alterations in cell wall composition will occur. This will probably change anchoring of the cytoskeleton in the walls and further modify functioning of the whole cell wall-plasma membrane-cytoskeleton continuum. In that way, cell’s mechanical balance restoration will be ensured and, in consequence, cell will be able to resist osmotic pressure and divide in severe stress conditions.
85 Signal transduction from elicitation with N-acetylchitooligosaccharide to biophoton generation
Kimihiko Kato, Chizuko Kageyama, Hiroyuki Iyozumi, Hidehiro Inagaki, Hideki Nukui
Shizuoka Agriculture and Forestry Research Center, 678-1, Tomioka, Iwata, Shizuoka-Ken, Japan
Email: [email protected]
Biophotons are ultraweak light emissions from biochemical reactions in a living body. Elicitor- responsive photon emissions (ERPE) increase in suspension-cultured rice (Oryza sativa L.) cells when elicited by N-acetylchitooligosaccharide. Biochemical analyses were undertaken to clarify the emission mechanism of ERPE. Exogenously applied phosphatidic acid (PA), the second messenger leading to the reactive oxygen species (ROS) generation in the signal transduction of disease response, raised photon emissions in rice cells. Comparisons of photon emissions from PA and ERPE regarding time courses, spectral compositions, and the inhibition ratios of several inhibitors, as well as a loss- and gain-of-function assay using the protein synthesis inhibitor cycloheximide and PA, showed the possibility that ERPE were generated through PA, an intermediate of phospholipid signaling. The effects of protein phosphorylation (K252a) and the 2+ Ca signaling inhibitors (EGTA and LaCl3), caused ERPE to decrease. It is clear that ERPE are regulated by Ca2+ signaling and protein phosphorylation. ERPE were suppressed when cells were pretreated with ROS-generating inhibitors: pyrocatechol-3,5-disulfonic acid disodium salt (Tiron); diphenylene iodonium (DPI); and salicylhydroxamic acid (SHAM). Conversely, exogenously applied ROS (superoxide and hydrogen peroxide) was able to induce photon emissions. ERPE are closely associated with the ROS-generating system. In addition, we found that the pattern of ERPE is almost identical to that of hydrogen peroxide generation. ERPE were inhibited with the pretreatment of NO scavenger, cPTIO. Interestingly, exogenously applied NO did not induce biophotons, but suppressed ERPE dose-dependently when applied together with N-acetylchito- oligosaccharide. It appears that NO plays a role of controlling ERPE through interacting with the ROS-generating system.
86 Alterations of actin cytoskeleton - the signalisation highway - in root hairs of GFP-FABD2 Arabidopsis thaliana treated with Pb2+
Krzeslowska Magdalena1, Wozny Adam1, Samardakiewicz Slawomir2, Napieralska Anna1
1 LGB, Adam Mickiewicz University, Umultowska 89, 61- 614 Poznań, Poland 2 LECM, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań, Poland
Email: [email protected]
We are grateful to Prof. Diedrik Menzel (University of Bonn, Germany) for generous gift of the GFP-FABD2 Arabidopsis thaliana line.
Our earlier studies show that in response to lead tip growing Funaria hygrometrica protonemata formed cell wall thickenings (CWT) localized at the apex. The thickenings were built mainly from pectins able to bind Pb2+ and callose. In fact, they accumulated large amounts of this metal (Krzeslowska et al. in press). It seemed to be probable that such reaction was the result of alterations in actin cytoskeleton caused by lead. We supposed moreover that it might be a typical one for tip growing cells. This hypothesis was verified in root hairs of GFP- FABD2 Arabidopsis thaliana. GFP- FABD2 A. thaliana incubated 10 days in vitro on MS medium, were treated with 16 μM Pb by 24h, applied as an aqueous solution of PbCl2. Control material was incubated for the same amount of time on distilled water. Afterwards, in vivo studies of the actin filaments (MFs) and cell wall (CW) structure and composition in the control and in lead treated root hairs, were carried out. Callose was detected by aniline blue and pectins by ruthenium red. All observations were carried out in fluorescence microscope Axiovert 200M and laser scanning confocal microscope LSM 510 (Carl Zeiss, Jena, Germany). Treated with lead root hairs often formed cell wall thickenings (CWT), localized mainly at the tip of the cell. Preliminary studies of their composition showed that they contained first of all pectins and callose. Microfilament bundles in growing control root hair run parallel to the onger axis of the cell and they do not reach the tip. Opposite to this, in lead treated material MFs bundles were much more thick than in control and reached the tip of the root hair which often was swollen. In this region MFs array was not parallel to the longer axis of the cell and were running in various directions. In root hairs where CWT appeared the number of MF bundles was lower. Some of them were running just under the CWT, often parallel to its edges. Other MF bundles were arrayed in various directions. If the CWT occurred in subapical or lateral cell walls, one or a few MF bundles, running more or less parallel to the longer axis of the cell in this region, curved and run directly to CWT. Tip growing cells showed similar response to lead: swollen tips and formation of CWT. CW formation during cell elongation requires both proper transport of vesicles from GA to growing tip and recycling of cell wall compounds via endocytotic pathway. Both processes are strongly dependent on actin cytoskeleton (Ovecka et al. 2005). The results of our studies shown, however, that in root hairs of A. thaliana treated with lead the array of MFs is markedly altered, especially in the tip region of the cell. Thus we conclude that its disturbation was probably one of the main reason of CWT formation in lead treated plant cells. Furthermore, disturbations of cell wall – cytoskeleton continuum strongly suggests some alteration(s) in signalisation within stressed plant cells.
REFERENCES Ovecka M, Lang I, Baluska F, Ismail A, Illes P, Lichtscheidl IK (2005) Protoplasma 226: 39-54
87 Exocyst subunit Sec8 of A.thaliana is essential for proper seed coat mucilage development
Ivan Kulich1, Rex Cole2, John Fowler2, Fatima Cvrcková1, Viktor Žárský1
1 Department of Plant Physiology of Faculty of Natural Sciences, Charles University, Vinicna 5, 12844 Prague 2, CZECH REPUBLIC 2 Botany&Plant Pathology, Oregon State University, 2082 Cordley Hall, Corvallis, OREGON, USA
Email: [email protected]
This work was supported by MSMT ME841
Exocyst, a large protein complex of 8 subunits, has been shown to be required for proper post- Golgi vesicle targeting at the plasma membrane. Its orthologs can be found in most of eukaryotes. In Arabidopsis, defects of exocyst subunits Sec8 and Exo70 show a drastic phenotype, most visible at pollen-tubes and root hair defects. Post-Golgi secretion is required for both polarized growth and secretion. Here we show a new phenotype of sec8 mutants affecting seed coat mucilage. Seed coat is missing in plants lacking Sec8 protein and is significantly smaller with truncated Sec8. These data support an idea, that seed coat mucilage can be used as a marker of exocytosis in Arabidopsis thaliana.
REFERENCES Cole et al. (2005) Plant Physiol 138: 2005-2018 Synek et al. (2006) Plant J 46: 54-72
88 Structural and functional modification of WMC proteins caused by nitric oxide
Agnieszka Łapa1, Przemysław Wojtaszek1,2
1. Institute of Bioorganic Chemistry, Polish Academy of Sciences; Z. Noskowskiego 12/14, 61 704 Poznań, POLAND 2. Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University; Międzychodzka 5, 60-371, Poznań, POLAND
Email: [email protected]
This research is supported by the Ministry of Science and Higher Education grant PBZ-KBN- 110/P04/2004 to P.W.
In recent years, nitric oxide was reported to be involved in many physiological processes in plants - in fact it is regarded as an important signaling molecule in the plant world (1). NO can modulate the functioning of proteins, either through binding to transition metal ions (such as Fe+2 in heme groups in guanylate cyclase (2)) or via modification of amino acid residues. Among the latter modifications, the key roles play S-nitrosylation of cysteine thiols and nitration of thyrosine residues. Both modifications can affect the structure and the activity of proteins.As a consequence, S-nitrosylation can be treated as an nitric oxide-dependent signaling modification, involved in the mechanism for redox-based regulation of signal transduction pathway (3). There were two aims of this work. First, to identify proteins of the cell wall-plasma membrane - cytoskeleton continuum (WMC) potentially modified by NO. Second, to investigate the effects exerted by various NO donors and modulators of NO activity on the functioning of WMC continuum as a whole and the actin cytoskeleton as part of it. Nitric oxide was localized in Arabidopsis thaliana (in suspension-cultured cells and seedlings) of utilizing DAF-2-FM diacetate (4). For the analyses of protein modifications, MALDI TOF mass spectrometry was used. The proteins were identified using specific antibodies and western blot analysis. These studies were complemented with the microscopic studies of the organization of the actin cytoskeleton in roots of Arabidopsis thaliana seedlings and in vivo immunolocalization of S-nitrosotiols (SNO) and nitrotyrosines in cell suspension culture.
REFERENCES
(1) Wendehenne D, Durner J, Klessig DF (2004) Curr Opin Plant Biol 7: 449-455 (2) Denninger JW, Marletta MA (1999) Biochim Biophys Acta 1411: 334-350 (3) Stamler JS, Lamas S, Fang FC (2001) Cell 106: 675–683 (4) Balcerczyk A, Soszynski M, Bartosz G (2005) Free Radic Biol Med 39: 327-335
89 Abnormal cell wall formation as a reaction to abiotic stress
Irene K. Lichtscheidl1, Ingeborg P. Lang1, Julia Rumé-Strobl1, Sabine Müller2, Alexander Lasselsberger1, Michael Volgger1, Beatrix Riedel1, Robert Kartusch3
1 Department of Cell Imaging and Ultrastructure Research, University of Vienna. Althanstrasse 14, A-1090 Wien, Austria 2 Department of Plant Biology, Carnegie Institution of Washington, Stanford University. 260 Panama Street, Stanford, CA 94305 3 Department of Ecophysiology and Functional Anatomy of Plants, University of Vienna. Althanstrasse 14, A-1090 Wien, Austria
Email: [email protected]
In growing plant cells, cell wall deposition is a highly organized process that is completed when cell expansion comes to an end. The formation of additional callose is assumed to be a general defense reaction which can be elicited either by physical stress, e.g. mechanical perturbations (Foissner et al. 1996), or by organic and anorganic chemical agents. It requires the plasma membrane bound enzyme 1,3-ß-glucan synthase which polymerizes the callose from glucose within the cytoplasm, wherefore normally the participation of organelles is ruled out. And actually there are only few cases where the involvement of the Golgi apparatus is discussed as well (reviewed by Kauss 1996). We induced abnormal callose synthesis in differentiated onion inner epidermal cells by mechanical stress (puncturing of the cell by a micro-needle) and by incubating onion cells in solutions of copper sulphate (Kartusch 2003), and we stimulated aberrant cell wall thickening and branching in growing root hairs of Triticum aestivum. We describe changes of the cytoskeleton and of the motility of the organelles and the nucleus.
REFERENCES
Foissner I, Lichtscheidl IK, Wasteneys GO (1996) Cell Motil Cytoskel 35: 35-48 Kartusch R (2003) Protoplasma 220: 219-225 Kauss H (1996) In Membranes: Specialized functions in plants. Smallwood M, Knox JP, Bowles DJ, eds. Bios Scientific Publishers.
90 Crawling roots: animal-like behaviour in plants
Michal Martinka1, Markus Schlicht2, Alexander Lux1, Dieter Volkmann2, František Baluška2
1 Department of Plant Physiology, Comenius University, Mlynska dolina B2, SK-842 15 Bratislava, SLOVAKIA 2 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY
Email: [email protected]
This study was financial supported by grant COST 0004-06 from APVV.
Exploratory root movements closely resemble behaviour of lower animals (Darwin 1880) due to their co-ordinated bendings in two different zones: the transition and elongation region (Wolverton et al. 2000). This allows invasively growing root apices to be highly flexible in avoiding obstacles as well as dangerous soil patches due to limited amount of water or increased amounts of toxic metals. Roots of parasitic plants actively detect and grow towards root apices of their host plants and to colonize them using haustorial hairs which penetrate into the transition zone of pray roots (Tomilov et al. 2005). Moreover, roots growing down along gravity vector and hitting mechanical obstacle start “to probe the shape“ of this mechanical obstacle and use the first possibility to grow down the gravity vector (Massa and Gilroy 2003). Very similar behavior can be documented by growing roots up of a slope when ethylene signalling proved to be essential to accomplish worm-like crowling of roots searching for weak sites in the substratum (Hahn et al. 2006). In order to understand this complex animal-like behavior of roots, we have performed a series of experiments using both intact and decapped maize (Zea mays, cv. Careca) roots. Scored behaviour of roots implicate gravity sensing in decapped roots (Mancuso et al. 2006) and document that root bendings in the transition zone and elongation region are highly coordinated to perform the worm-like crawling movements. Root cap removing is perturbing this coordinated behavior of two bending domains, suggesting that the intact root apex is essential for this coordinated root behavior. Removing of the root cap even promotes the root growth (see also Mancuso et al. 2006) but these roots grow straight and are impaired in their abilities to grow down the gravity vector which is an inherent part of their crawling movement, allowing them to analyze the substrate properties as well as to avoid dangerous environments. In future, we will use this new experimental system to challenge growing roots, both of wild-type as well as relevant mutant lines, treated with drugs and neurobiologically active substances to analyze their roles in animal-like behaviour of roots.
REFERENCES
Darwin C (1880) The Power of Movements in Plants, John Murray Hahn A, Firn R, Edelmann HG (2006) Signal Transduct 6: 449-455 Mancuso S, Barlow PW, Volkmann D, Baluška F (2006) Plant Signal Behav 1: 52-58 Massa GD, Gilroy S (2003) Plant J 33: 435-445 Tomilov AA, Tomilova NB, Abdallah I, Yoder J (2005) Plant Physiol 128: 1469-1480 Wolverton C, Mullen JL, Ishikawa H, Evans ML (2000) Plant Cell Environm 23: 1275-1280
91 Detecting electrical network activity in root apex by multielectrode arrays (MEAs)
Masi E1, Ciszak M2, Montina A2, Malachovska V1, Mugnai S1, Azzarello E1, Pandolfi C1, Renna L1, Stefano G1, Voigt B1, Hlavacka A1, Arecchi FT2, Mancuso S1
1 LINV Laboratorio Internazionale di Neurobiologia Vegetale - Dipartimento di Ortoflorofrutticoltura - University of Florence - viale delle Idee 30 - 50019 Sesto Fiorentino (Florence) - Italy 2 INOA Istituto Nazionale di Ottica Applicata – Largo E. Fermi 6 – 50125 Florence – Italy
Email: [email protected]
All processes of living organisms examined with suitable and sufficiently sensitive measuring techniques generate electric fields that must be regarded as one of the most universal properties of living organisms. Many studies have demonstrated that bioelectrochemical signals exist in plants at all levels of evolution (action potentials or excitation waves). AP are possible mechanisms for intercellular and intracellular communication in the presence of environment changes. Plants respond to environmental stimuli and excitation can be dispersed throughout the entire plant, travelling from the top of the stem to the root and from the root to the top of the stem. Though excitation waves appear strongly after stimulation, a basic electrical activity can be found in the whole plant. The theoretical description of the electrical activity and the propagating model through single cells is still not understood. Simultaneous multisite recording is a prerequisite to understand the nature of electrical phenomena. For extracellular recording from electrogenic cells pursuing these goals, substrate integrated, planar microelectrode arrays (MEAs) have been developed to monitor spikes and local field potentials. A typical setup for MEA recording is based on metal microelectrodes fabricated on a planar chip, discrete-element preamplifiers located close to the MEA device and a multi-wire cable that conducts the pre- amplified analog signals to a data acquisition card. Here we report for the first time recordings of single-unit spike activity with MEAs in acute slice of Zea mays L. root apex. Field potentials were recorded simultaneously from 60 electrodes (30 μm diameter) with high spatial and temporal resolution. This new technique allowed us to map functionally discrete regions of the root and to observe the space-time relationships and the spontaneous, synchronous electrical activity of the root apex. The nature of spike shapes has been studied on each MEA electrodes (200 μm interelectrode spacing). We conclude that extracellular recording of independent single-unit spike activity with MEAs is indeed suitable to monitor electrical network activity in root apex, making MEAs an exceptionally useful tool for the assessment of fast network dynamics in plants.
92 Bioorganic study of nyctinasty on genus Albizzia using molecular probes
Yoko Nakamura1, Akira Matsubara1, Ryoji Miyatake1, Minoru Ueda1
1 Tohoku University, Aoba-ku, Sendai 980-8578, JAPAN
Email: [email protected]
Most leguminous plants close their leaves in the evening, as if to sleep, and open them in the morning according to the circadian rhythm controlled by a biological clock. Nyctinastic plants have a pair of endogenous bioactive chemical factors that control leaf movement. Potassium β- D-glucopyranosyl-12- hydroxyjasmonate and cis-p-coumaroylagmatine were isolated as leaf- closing factor (LCF) and leaf-opening factor (LOF) of leguminous plants bolonging to genus Albizzia, respectively. Our studies focus on the mechanism of leaf movement using these chemical factors as molecular probes. We developed molecular probes consisting of modified leaf-movement factors of Albizzia plants in order to identify their target cells. We conducted a double fluorescence-labeling study using FITC-labeled LCF and rhodamine-labeled LOF. Interestingly, both of the probes bound to the same motor cells called extensor cells in the pulvini. Therefore, the motor cell with a set of receptors for leaf-movement factors is located on the extensor side of pulvini. Since extensor cells are difined as cells that increase their turgor during opening, and decrease their turgor during closing, the leaf-movement factor must facilitate a decrease or increase in the turgor of extensor cells. In Albizzia plants, the trigger for leaf-movement might be related to the change in turgor of extrensor cells. We also synthesized a pair of enantiomers of FITC-labeled LCF, and used them for fluorescence studies. Comparing the results, FITC-labeled LCF of natural stereochemistry bound to the extensor cells of Albizzia plants, whereas its enantiomer could not bind to it. The results demonstrated the involvement of a receptor in the extensor cell, which recognizes the stereochemistry of jasmonate-type LOF.
REFERENCES
Nakamura Y, Miyatake R, Matsubara A, Kiyota H, Ueda M (2006) Tetrahedron 62: 8805-8813 Nakamura Y, Matsubara A, Okada M, Kumagai T, Ueda M (2006) Chem Lett 35: 744-745
93 Use of an extracellular oxygen vibrating microelectrode system to detect rapid changes in oxygen fluxes in electrotropically-stimulated maize roots
Pandolfi C, Mugnai S, Azzarello E, Masi E, Mancuso S
LINV – International Lab for Plant Neurobiology - Department of Horticulture - Polo Scientifico, University of Florence, Viale delle idee 30, 50019 Sesto Fiorentino (FI), Italy
Email: [email protected]
It is well known that the direction of growth of certain plant cells or organs can be modified by an applied electric field. This phenomenon, known as electrotropism has been reported in fungi (McGillavray and Gow, 1986) and algae (Brower and Giddings, 1980) as well as in the pollen tubes (Marsh and Beams, 1945), roots (Ishikawa and Evans 1990; Wolverton et al. 2000), and shoots (Schrank 1959) of higher plants. The correlation between electrical changes and gravitropic curvature suggests the possibility that the curvature of roots in an electric field results from electrical changes within the root that mimic those caused by gravistimulation. This possibility is strengthened by reports that root electrotropism is suppressed by inhibitors of auxin transport (Moore et al. 1987). We examined the effects of electrotropism in solutions of low electrolyte concentration using primary roots of maize (Zea mays L., variety Merit). When submerged in oxygenated solution across which an electric field was applied, the roots curved rapidly and strongly toward the positive electrode (anode). The strength of the electrotropic response increased and the latent period decreased with increasing field strength. At a field strength of 1.5 volts per centimetre the latent period was few minutes and we were able to analyse changes in oxygen fluxes outside the root thanks to the vibrating probe. The experimental measurement of ion or gaseous molecules fluxes in roots is fundamental when discriminating normal physiological function from abnormal or stressed states. We took measures in three basic anatomical parts of root apex: meristematic zone, transition zone, and elongation zone (Verbelen et al. 2006). The goals of the research described in this poster are (a) to determine the changes of oxygen fluxes in different zones of the root related to the electrotropic curvature in maize roots, investigating the early phase of the electrotropic response, from the apply of the current to the visible bending, and (b) to determine the role and the effects of pharmacological manipulation in the electrotropic response and their changes on oxygen fluxes.
REFERENCES
Brower DL, Giddings TH (1980) The effects of applied electric fields on Micrasterias. II. The distributions of cytoplasmic and plasma membrane components. J Cell Sci 42: 279-290 Ishikawa H and Evans ML (1990) Electrotropism of maizerRoots. Role of the root cap and relationship to gravitropism. Plant Physiol 94, 913-918 Marsh G, Beams HW (1945) The orientation of pollen tubes of Vinca in the electric current. J Cell Comp Physiol 25: 195-204 McGillavray AM, Gow NAR (1986) Applied electrical fields polarize the growth of mycelial fungi. J Gen Microbiol 132: 2515-2525 Moore R, Fondren WM, Marcum H (1987) Characterization of root agravitropism induced by genetic, chemical, and developmental constraints. Am J Bot 74: 329-336 Schrank AR (1959) Electronasty and electrotropism. In E Bunning, ed, Physiology of Movements, Encyclopedia of Plant Physiology, New Series, Vol 17. Springer, New York, pp 148-163 Verbelen J-P, De Cnodder T, Le J, Vissenberg K, Baluška F (2006) The root apex of Arabidopsis thaliana consists of four distinct zones of cellular activities: meristematic zone, transition zone, fast elongation zone, and growth terminating zone. Plant Signal Behav 1: 296-304 Wolverton C, Mullen JL, Ishikawa H, Evans ML. (2000) Two distinct regions of response drive differential growth in Vigna root electrotropism. Plant Cell Environ 11: 1275-1280
94 In situ visualization of nanoparticles internalisation into plant tissues as new systems for treatment delivery
González-Melendi P1,6, Corredor E1, Coronado MJ1, Fernández-Pacheco R2, Marquina C3, Ibarra MR2,3, Rubiales D4, Pérez-de-Luque A5, Testillano PS1, Risueño MC1
1 Biological Research Center, CIB, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain 2 Instituto de Nanociencia de Aragón, University of Zaragoza, Zaragoza, Spain 3 Instituto de Ciencia de Materiales de Aragón-Departamento de Física de la Materia Condensada, CSIC-University of Zaragoza, Zaragoza, Spain 4 Instituto de Agricultura Sostenible, CSIC, Córdoba, Spain 5 IFAPA-CICE (Junta de Andalucía), Área de Mejora y Biotecnología, Córdoba, Spain 6 Present address: Centro de Biotecnología y Genómica de Plantas. UPM-INIA, Madrid, Spain
Email: [email protected]
The great potential of nanoparticles as delivery systems to be directed to specific targets in living beings has been first explored for medical uses. In agriculture, nanotechnology applications can also have a broad range of uses in particular to tackle infections with nanosystems tagged to pesticides or other substances for efficient and local treatments, thus reducing the dose of chemicals released to the environment. In order to explore the benefits of nanotechnology applications in agriculture, the first level is to achieve the penetration, movement and targeting of the nanoparticles through the plant at specific sites. In this context, the precise localization of the particles in the plant tissues and in the different subcellular compartments is pivotal. We have performed preliminary assays with carbon coated magnetic nanoparticles in plants, the magnetic core allowing allocation of the nanoparticles in the site of interest (affected tissues) using small magnets. In this work, a number of tools for the detection and analysis of magnetic nanoparticles introduced into plants have been evaluated, by using different techniques and levels of observation, ranging from conventional light microscopy to confocal and electron microscopy. We have inoculated in vitro growing plants with a ferrofluid composed of carbon-coated magnetic nanoparticles. Tissue samples were then collected, fixed, cut and observed with different processing techniques to detect the presence of nanoparticles at the above-mentioned microscopy levels. These techniques include conventional light microscopy, fluorescence microscopy, confocal scanning laser microscopy and electron microscopy, combined with different fixation and/or embedding processes. The results showed that the nanoparticles can be visualised by reflection on a confocal microscope; inferred as dark areas in an autofluorescent background (either natural or induced), and as a punctuate pattern on the light microscope, further identified as clusters of nanoparticles on the electron microscope due to their iron core. Our first data showed the presence of nanoparticles both in the extracellular space and within some cells. Further work is needed to evaluate how the nanoparticles penetrate and are transported within the plants, and the mechanism(s) of intracellular internalisation to explore the potential of nanoparticles as smart treatment delivery systems in plants.
González-Melendi P, Fernández-Pacheco R, Coronado MJ, Corredor E, Testillano PS, Risueño MC, Marquina C, Ibarra MR, Rubiales D, Pérez-de-Luque A. Nanoparticles as smart treatment delivery systems in plants: first report of penetration inside living plants and assessment of different techniques for their visualisation. New Phytol.(submitted). Supported by project NanoAgro-200540F0041 funded by the Spanish Research Council, CSIC.
95 Subtilisin-like proteases of Arabidopsis thaliana involved in the regulation of plant development
Ewelina Rodakowska1, Marta Derba1, Przemysław Wojtaszek1,2
1 Department of Molecular and Cellular Biology, Adam Mickiewicz University, Mi?dzychodzka 5, 60-371 Poznan, Poland 2 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
Email: [email protected]
Subtilisin-like proteases are serine endopeptidases with catalytic triad of aspartate, histidine and serine. Eucaryotic subtilases belong to two families of subtilisin-like proteases: kexins and pyrolysins, based on amino acid sequence similarity. Kexins, called proprotein convertases, are well known in mammals to play pivotal role in generation of bioactive molecules: polypeptide hormones, growth and neurotrophic factors, receptors, adhesion molecules and other proteases through highly specific proteolytic cleavage. In recent years, a growing body of evidence indicates that regulation of many aspects of plants growth and development depends not only on classical phytohormones but also on peptide signaling. Although many of these peptides and their precursor are being identified, there is still no direct evidence for generating bioactive peptide by plant protease. To gain knowledge about possible roles of subtilases in the regulation of plant growth and development, we have chosen two subtilase genes At5g19660 and At5g59810 from among 56 in Arabidopsis thaliana genome. For functional analysis of At5g59810, we decided to utilize the insertion mutants as well as transgenic plants overexpressing this gene under control of CaMV promoter, and a line with promoter::GUS construct. Phenotypical changes, immunolocalization and GUS expression analysis will be presented. On the basis of these evidences, we suggest that At5g59810 could be an enzyme involved in the generation of signal peptides regulating plant development. Second subtilisin At5g19660 is an ortholog of S1P/SKI-1, a conservative animal type of subtilases. These are transmembrane proteins located in endoplasmic reticulum/Golgi apparatus. In animals, they catalyze proteolysis of transmembrane precursors of transcription factors, and enable release of active molecules from the endomembrane system. In this communication, bioinformatic data of At5g19660 protein and their potential substrates will be presented. Additionally, we will show evidence for the developmental role of this protease based on the analysis of Arabidopsis thaliana insertion mutants.
96 Cell-type specific disruption and recovery of the cytoskeleton in Arabidopsis epidermal root cells upon heat shock stress
Jens Müller1, Diedrik Menzel1, Jozef Šamaj1,2
1 Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D – 53115 Bonn, Germany 2 Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O.Box 39 A, SK – 950 07 Nitra, Slovak Republic
Email: [email protected]
The cytoskeleton in plant cells plays an important role in controlling cell shape and mediating intracellular signalling. However, almost nothing is known about the reactions of cytoskeletal elements to heat stress, which represents one of the major environmental challenges for plants. Here we show that living epidermal root cells of Arabidopsis thaliana could cope with short-term heat shock stress showing disruption and subsequent recovery of microtubules and actin microfilaments in a time-dependent manner. Time-lapse imaging revealed a very dynamic behaviour of both cytoskeletal elements including transient depolymerization/disassembly upon heat shock (40-41°C) followed by full recovery at room temperature (20°C) within 1-3 hours. Reaction of microtubules, but not actin filaments, to heat shock was dependent on cell type and developmental stage. On the other hand, recovery of actin filaments but not microtubules from heat shock stress was dependent on the same parameters. The relevance of this adaptive cytoskeletal behaviour to intracellular signalling is discussed.
97 Interactions between auxin and retinoid-like signaling in plants
Markus Schlicht1, Olga Šamajová1, Doreen Schachtschabel2, Irene Lichtscheidl3, Diedrik Menzel1, Wilhelm Boland2, František Baluška1
1 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY 2 Max-Planck-Institut für Chemische Ökologie, Hans-Knöll-Str. 8, D-07745 Jena, GERMANY 3 Cell Physiology and Scientific Film, University of Vienna, Althanstrasse 14, A-1090 Vienna, AUSTRIA
Email: [email protected]
The C18- apocarotenoid D’orenone, a precursor of the trisporic acid signalling molecules acting as pheromones in soil fungi (zygomycetes, Schachtschabel et al. 2007), exerts extremely rapid effects on roots of higher plants, ranging from the monocot Zea mays up to the dicot Arabidopsis thaliana. Most sensitive are tip-growing root hairs, which stop their tip growth within a few minutes of exposure to D’orenone. The actin cytoskeleton is rapidly remodelled, involving F- actin depolymerization in root hairs. We have shown this in vivo using the transgenic GFP-ABD2 actin-reporter line of Arabidopsis (Voigt et al. 2005) and in situ using a polyclonal maize actin antibody on Steedman's wax sections taken from maize root apices (Baluška et al. 1997). D’orenone rapidly depolymerizes F-actin and disintegrates the vesicle-rich ‘clear zone’ at the very tip of growing roots hairs. Vacuoles protrude up to the very tips of the root hairs as growth 2+ . is ceasing. Labelling of Arabidopsis roots with the cell permeable Ca dye Fluo3-AM and the O2 - sensitive dye NBT revealed, that a few minutes after the D’orenone treatment the tip-focused ROS and cytoplasmic Ca2+-gradient disappear. Intriguingly, D’orenone exposed roots display an activation of the auxin response reporter DR5rev::GFP specifically in the root tip. A similar phenomenon was monitored for the phosphatidylinositol-3-OH kinase inhibitor wortmannin (Jaillais et al. 2006). Like wortmannin, D’orenone treatment affects both PIN2 abundance and subcellular location. Nearly the complete PIN2–GFP signal, which is normally observed in epidermal cells and cells of the lateral root cap in root tips, vanished and the PIN2–GFP signal started to be expressed strongly in the transition zone cells. PIN2 polarity was still maintained at the plasma membrane, but PIN2–GFP also accumulated within vesicular compartments and at the tonoplast of vacuoles. Double treatment with D’orenone and the general secretion inhibitor brefeldin A (BFA) revealed that PIN2-GFP- positive BFA-induced compartments start to appear only after long treatment periods of more than 2 h instead of usual 30 minutes. Importantly, external addition of auxin rescues all aspects of the D´orenone induced phenotype, i.e. on the levels of root hair formation, root growth, and root graviresponse. External auxin also makes the roots more resistant to additionally applied D’orenone. All this implicates that D’orenone is a very active biological molecule possibly affecting either the PIN2- dependent auxin efflux or auxin signalling; relevant for both the auxin dependent root hair tip- growth (Lee and Cho 2006) and root growth (Blilou et al. 2005). D’orenone might also function as an important component of the myxomycete – plant communication. Finally, the most attractive scenario would be that endogenous D’orenone-like substances exist in plants and act as a new, hitherto unknown, class of plant hormones.
REFERENCES
Baluška F., Vitha S., Barlow P.W., Volkmann D. (1997) Eur J Cell Biol 72: 113-121 Blilou I et al. (2005) Nature 433: 39-44 Jaillais, Fobis-Loisy I, Miege C, Rollin C, Gaude T (2006) Nature 443: 106-109 Lee SH, Cho HT (2006) Plant Cell 18: 1604-1616 Schachtschabel D, Boland W (2007) J Org Chem 72: 1366-1372 Voigt B, Timmers ACJ, Šamaj, Müller J, Baluška F, Menzel D (2005) Eur J Cell Biol 84: 595-608
98 History of polar auxin transport - the evolutionary aspects
Petr Skůpa1,2, Pavel Křeček1,2, Jan Petrášek1,2, Eva Zažímalová1,2
1Institute of Experimental Botany, the Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic 2Department of Plant Physiology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic
Email: [email protected]
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic, project no. LC06034 and by the Grant Agency of the Academy of Sciences of the Czech Republic, project no. KJB600380604.
Polar auxin transport (PAT) is one of the fundamental processes in the life of higher plants. The cell-to-cell active transport of auxin molecules underlies their uneven and complex spatio- temporal distribution within plant body. The proper function of PAT is crucial for many, often very diverse plant developmental processes and/or situations, and it delimits basic processes such as embryogenesis, polarity maintenance and growth responses to environment. PAT in higher plants is the process with very complex regulation. When studying PAT, plant models representing individual stages in the course of plant phylogeny may help to assess early development of individual traits in auxin transport. The understanding of the evolution of PAT may contribute to decipher various strategies in its regulation in higher plants and - in general - it would give a better insight into the basis of the whole process. From physical-chemical reasons, the efflux of auxin from cells is the crucial step in PAT. PIN proteins from Arabidopsis thaliana were shown to play a rate-limiting role in catalyzing the auxin efflux from cells and their asymmetrical/polar cellular localization determines the direction of cell-to-cell auxin flow. Therefore, we have collected known sequences coding for the homologues of auxin efflux carriers of PIN family and resulting data were related to available information on the distribution of PAT, apical and polarized growth and other PAT- related characteristics during plant evolution. We have also outlined the next possible steps in data-mining strategies related to studies of co-evolution of PINs together with various forms of auxin transport, and some possible implications of PAT for land plant evolution.
99 Glutamate-induced changes in electrical potential, circumnutations and stem growth in Helianthus annuus L.
Maria Stolarz, Halina Dziubinska, Elzbieta Krol, Andrzej Kurenda, Maciej Krupa, Tadeusz Zawadzki, Kazimierz Trebacz
Department of Biophysics, Institute of Biology, Maria Curie-Sklodowska University, Akademicka 19, PL-20-033 Lublin, Poland
Email: [email protected]
Supported by the grant from the Vice-Rector of Maria Curie-Skłodowska University
In sensitive sejsmonastic and carnivorous plants, action potential is an essential factor that evokes rapid movement of plant organ e.g. leaf or flay-trap. In ordinary plants, APs can also participate in the regulation of plant pollination, fertilization, respiration, photosynthesis, growth and gene expression. Recently it was shown that in plants the glutamate receptor is involved in electrical response and light and growth signalling. Here, the effect of glutamate on membrane potential, stem movement and growth was studied in three-week-old sunflowers. Extracellular and intracellular electrical potential measurements were carried out. Time-lapse photography from a top and side view camera was used for stem movement and growth study. Two drops of millimolar glutamate solution were injected into the lowest part of the sunflower stem. Injection of glutamate solution resulted in a series of APs lasting several dozens of minutes. The evoked APs propagated from the injection site along the stem and were able to enter the petiole. Some were initiated in the upper part of the stem and propagated downwards. The AP series were often accompanied by variation potential. Local application of glutamate resulted in a decreased rate of circumnutation, which reached its minimum in the third hour after the glutamate application. The preliminary experiments with application of the time-lapse photography technique did not show any significant growth inhibition following glutamate injection. The overall results provide a first indication that in higher plants the injection of glutamate solution evokes propagating series of APs and inhibits the endogenous stem movement.
100 Defects in light receptors affect boron-regulated growth in Arabidopsis seedlings*
Zdeněk Svoboda1, Tomáš Kocábek2, Martin Fellner1,3
1 Department of Cell Biology and Genetics, Palacky University in Olomouc, Šlechtitelů 11, 783 71, Olomouc, Czech Republic 2 Biology Centre of the ASCR, v.v.i., Institute of Plant Molecular Biology, Branišovská 1160/31, 370 05, České Budějovice, Czech Republic 3 Institute of Experimental Botany ASCR, v.v.i., Šlechtitelů 11, 783 71, Olomouc, Czech Republic
Email: [email protected]
The work was supported by grant No. KJB 600510503 from the Grant Agency of the Academy of Science of the Czech Republic
Boron (B) is essential microelement in all vascular plants. Among others, it plays an important role in cell wall synthesis. The physiological role of B in plants is depicted as that of a transducer in several processes initiated by light, gravity, and some plant hormones. Information concerning the role of boron in plant growth and development during photomorphogenesis is very poor. It has been previously observed (Rölfe et al. unpublished results)that under red (RL), but not in blue light (BL) boron can stimulate hypocotyl growth, and that the element effect also depends on light intensity. Here, we studied effects of elevated boron concentrations on Arabidopsis growth and development in in vitro conditions with respect to light quality signal. Analysis of mutants with defects in light perception could suggest interaction between boron and light signaling pathways during plant growth and development. In this genetic approach we also investigated boron and light effects on growth of mutants with defects in genes involved in synthesis of plant cell wall components, especially cellulose. For the analyses, we used photomorphogenic mutants cry1 (hy4), cry2, and hy2, and cell wall mutants rsw1-1 and rsw1-10. We found that hypocotyl elongation in all Arabidopsis ecotypes tested was stimulated by boron at concentrations from 2 to 3 mM H3BO3, but inhibited at higher boron concentrations. We revealed that hypocotyl of cry1 mutant was not essentially stimulated by boron in BL or RL, and even not in dark. The data suggest that functional photoreceptor CRY1 is positively involved in boron-induced stimulation of hypocotyl growth. In contrast, cry2 plants grown in the dark, or under BL or RL showed WT responses to boron supplemented in the culture medium. Under normal conditions, etiolated rsw1-10 seedlings develop very short hypocotyl. We found that in rsw1-10 mutant H3BO3 highly stimulates hypocotyl elongation even at the concentration (10mM) extremely toxic for control plants. The stimulation was associated with strong reduction of BOR1 expression in mutant hypocotyl. The positive effect of boron on hypocotyl growth was most intensive in dark, but it was also essential in RL and BL, i.e. light quality only reduces growth amplitude. In contrast to rsw1-10, mutation rsw1-1 did not affect hypocotyl responsiveness to high boron concentrations. Results of our experiments led to several important conclusions. First, we revealed that boron at relatively high concentrations could stimulate hypocotyl elongation not only in red light, but also in blue light and in the dark. Other results suggest that in RL and BL, functional photoreceptors in Arabidopsis can maintain high capacity of boron to stimulate hypocotyl elongation. Analyses of mutants with defects in cell wall synthesis revealed that mutation rsw1-10 results in reduction in primary root and hypocotyl sensitivity to toxic effects of high boron concentrations. Differential expression of BOR1 in the mutant and wild- type plants supports the existence of mechanism by which plants can tolerate toxic effects of high boron concentrations on plant growth
101 Programmed cell death as an integrated plant cell response to stress treatments which induce changes of developmental programmes
Coronado MJ, Chakrabarti N, Cortés-Eslava J, Rodríguez-Huete A, Risueno MC, Testillano PS
Biological Research Center, CIB, CSIC. Ramiro de Maeztu 9, 28040 Madrid, Spain
Email: [email protected]
Supported by projects granted by Spanish MEC BFU2005-01094 and AGL2005-05104
The immature pollen grain, at the stage of vacuolate microspore can be switched, upon stress, from their normal pollen development programme to the embryogenesis pathway. Pollen embryogenesis is of much interest for basic studies and for applied research, being the best and more used tool to obtain double haploids. This process occurs by the reprogramming of microspores upon an abiotic stress treatment, followed by embryogenesis. The effectiveness of pollen cultures varies among species and essays. On occasions, many of the cells do not progress after the stress treatment. This could be due to a different response after stress, cell death events or different signal transduction pathways. The programmed cell death (PCD) pathways are not well defined in plants. In barley (Hordeum vulgare L.), an agronomically interesting species, pollen embryogenesis is induced by a starvation treatment in isolated microspore cultures. Different stages of pollen embryogenesis cultures were analyzed: during and after the stress treatment. Lines of in vitro suspension cells in barley were also developed as a convenient/simpler model system to evaluate the cell response to the inductive treatment and the occurrence of PCD events. The same conditions of the microspore cultures were reproduced on the suspension cells and various PCD markers were evaluated. A study on the characterization of PCD has been undertaken. Ultrastructural, cytochemical and inmunocytochemical analysis showed structural changes during stress treatment in the cultures similar to those established in animal cell apoptosis. After 24 hour of stress treatment different cytoplasmic and nuclear changes were found in the microspores. Results showed an increase of the number of vacuoles during this starvation treatment and a segregation of the cytoplasm after longer treatments. DAPI (fluorocrome specific of DNA) staining of the cultures showed disorganized nuclei with small fluorescent inclusions, similar to the apoptotics bodies in animal cells. The apoptotic features found in the stress-treated in vitro systems were compared with another stress-induced PCD system of plant cycling cells (1, 2) and with the developmental PCD process of tapetal cells, a male germ-derived cell line. In these systems, cytoplasmic release of the cytochrome C, DNA fragmentation, chromatin condensation, RNP segregation and nuclear lobulation were observed. Active cleaved-caspase 3 antigen was detected by Western blot, immunofluorescence and immunogold labelling in the cytoplasm of the treated cells and at specific developmental stages in the tapetum. Enzimatic activity of caspases was detected. Cleaved-caspase 3-like protein has been also localized by inmunofluorescence and immunogold labelling during stress treatment in pollen embryogenesis. All of these changes were not found when microspores and cell suspensions were put in nutrient medium. These results during pollen embryogenesis pathway are indicating a defined PCD assopciated with this developmental process. The knowledge of this process would allow us to influence it with drug treatments to increase the survival of the cultures in the early stages and the efficiency of the system in double-haploid production.
REFERENCES
1. Testillano PS, Coronado MJ, Cortès-Eslava J, Risueño MC (2006) Defined apoptotic-like nuclear changes and expression of caspase 3-like proteins accompany the stress-induced PCD in plant cells. Proc. EMBO Workshop on The Functional Organization of the Cell Nucleus, Prague, Czech Republic 2. Coronado MJ, Chakrabarti N, Cortès-Eslava J, Risueño MC, Testillano PS.(2006) Stress-induced PCD in plant cells involves apoptotic nuclear domain rearrangements and expression of caspase 3- like proteins. Proc. 48th Symposium of Histochemistry of Cell Damage and Death, Stresa, Italy
102
Arabidopsis synaptotagmin A is enriched at cortical ER domains and is implicated in salt stress tolerance
Boris Voigt1, Arnaldo L. Schapire2, Miguel A. Botella2, Stefano Mancuso1, František Baluška3
1 LINV, University of Florence, Viale delle idee 30, 50019 Sesto f.no(FI), ITALY 2 LBBV, University of Málaga, Campus Teatinos S/N, 29071 Málaga, SPAIN 3 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY
Email: [email protected]
Sequence analysis of various animal and plant genomes revealed the presence of synaptotagmin genes in all animals and land plants, but there is no evidence of synaptotagmin genes in unicellular organisms or those with simple forms of multicellularity. In the Arabidopsis genome we find six members, SytA to SytF, which belong to this protein family. They show the same domain pattern like their animal counterparts. They possess a N-terminal transmembrane sequence, which is followed by a linker of different length and two distinct C2 domains, C2A and C2B. Here, we show the ubiquitous expression of SytA in Arabidopsis and its localization via transient transformation of tobacco leaves and stable transformation of Arabidopsis seedlings in distinct cortical ER domains localized at plasma membrane – cell wall adhesion sites. Furthermore, a T-DNA SytA loss-of-function mutant shows response to salt stress through inhibited root growth and aberrant growth of root hairs. These findings suggest possible role(s) of SytA in vesicle- and calcium-mediated salt stress tolerance.
103 Darkness and blue light illuminations control endocytosis and vesicle recycling at the plasma membrane of plant cells
Yinglang Wan1, Ulrich Kubitschek2, Rujin Chen3, Dieter Volkmann1, František Baluška1
1 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY 2 Institut für Physikalische und Theoretische Chemie, Wegelerstr 12, D-53115 Bonn, Germany 3 Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA
Email: [email protected]
The blue light signal is one of the most important environmental signals for plants. It causes phototropism of plant organs, stomatal opening, and chloroplast movements. In the last decades, several members of blue light receptors have been discovered in plants. Phototropin1, the essential photoreceptor of the blue light mediated phototropism, has been considered as an important factor in almost all kinds of blue light responses. In our previous studies (in preparation), we showed that the blue light initiates endocytotic translocation of PHOT1. The level (speed and rate) of the internalization of PHOT1 can reflect the intensity of blue light signals. In this study, using the PIN2::GFP artificial protein, dynamic natures of both styryl dye FM4-64 and PIN2 have been studied under darkness and controlled blue light illuminations. Blue light signals increased the rate of FM4-64 internalization into cytoplasm, changed the localization of PIN2::GFP from vacuole to the plasma membrane, and increased the trapping of both components within Brefeldin A-induced endosomal compartments. We conclude that the blue light signals controls the localization of putative auxin exporter PIN2, and affects homeostasis of the plasma membrane via endocytosis and vesicle recycling. Endocytosis and the endocytic network of plant cells may play roles in the blue light signal transduction.
104 The subcellular localization and blue-light-induced movement of Phototropin 1-GFP in etiolated seedlings of Arabidopsis thaliana
Yinglang Wan1, William Eisinger2, David Ehrhardt3, Ulrich Kubitschek4, Frantisek Baluska1, Winslow Briggs3
1 IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, GERMANY 2 Department Biology, Santa Clara University, Santa Clara, CA 95053, USA 3 Department Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305, USA 4 Institut für Physikalische und Theoretische Chemie, Wegelerstr 12, D-53115 Bonn, Germany
Email: [email protected]
Phototropin 1 (phot1) is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and likely solar tracking in response to blue light. Following earlier work with PHOT1::GFP (Sakamoto and Briggs 2002), we investigated the pattern of cellular and subcellular localization of phot1 in etiolated seedlings of Arabidopsis thalinana. The Phot1::GFP fusion protein is expressed strongly in the abaxial tissues of the cotyledons and in the elongating regions of the hypocotyl. It is moderately expressed in the shoot/root transition zone and the root near the apex. The plasma membranes of mesophyll cells near the colyledon margin appear labeled uniformly except for strongly labeled cell plate-like structures. The pattern of labeling of individual cell types varies with cell type and developmental stage. Label is undetectable in the root epidermis, root cap, and root apical meristem. Blue-light treatment causes PHOT1::GFP, initially relatively evenly distributed at the plasma membrane, to become reorganized into a distinct mosaic with strongly labeled punctate areas and other areas completely devoid of label, a phenomenon best observed in cortical cells in the hypocotyl elongation region. Concomitant with or following this reorganization, PHOT1::GFP moves into the cytoplasm in all cell types investigated. It disappears from the cytoplasm after several hours in darkness. Neither its appearance in the cytoplasm nor its eventual disappearance in darkness is prevented by the translation inhibitor cycloheximide, although the latter process is retarded. We hypothesize that this relocalization modulates blue light-activated signal transduction.
REFERENCE
Sakamoto K, Briggs WR (2002) Plant Cell 14: 1723-1735
105
LIST OF PARTICIPANTS
106 Elisa Azzarello Denmark Dep. Horticulture - University of Florence, Phone: +45 35283484 Viale delle idee 30 Email: [email protected] 50019 Sesto Fiorentino (FI) Italy Pavla Binarova Phone: +390554574060 Institute of Microbiology Email: [email protected] Videnska 1083 142 00 Praha4 František Baluška Czech Republic Institute of Cellular and Molecular Botany Phone: 420 2410602130 (IZMB) Email: [email protected] Kirschallee 1 D-53115 Bonn Mary Bisson Germany University at Buffalo Phone: +49 228 73 4761 Dept. Biological Sciences Email: [email protected] Cooke Hall 109 14260 Buffalo Peter Barlow United States of America School of Biological Sciences Phone: 1 716 645 2363 x. 102 University of Bristol Email: [email protected] BS8 1UG Bristol United Kingdom Renata Bogatek Phone: +44 117 928 7475 University of Life Sciences - SGGW, Dept. of Email: [email protected] Plant Physiology 02-776 Warsaw Edurne Baroja-Fernandez Poland Instituto de Agrobiotecnologia Phone: +48 22 59 325 20 CSIC / Universidad Publica de Navarra Email: [email protected] Carretera de Mutilva s/n 31192 Mutilva Baja (Navarra) Wilhelm Boland Spain Max Planck Institute for Chemical Ecology Phone: + 34 948 168030 Hans-Knöll-Straße 8 Email: [email protected] 07745 Jena Germany Mary Jane Beilby Phone: 0049-3641-571200 The University of New South Wales Email: [email protected] NSW 2052 Sydney Zbigniew Burdach Australia University of Silesia, Faculty of Biology, Phone: 61 2 9385 5463 Department of Plant Physiology, Jagiellonska Email: [email protected] 28 40-032 Katowice Radek Bezvoda Poland Katedra fyziologie rostlin UK PřF Phone: +48322009466 Viničná 5 Email: [email protected] 12844 Praha 2 Czech Republic Lenka Burketová Phone: +420 22195 1685 IEB ASCR, Na Karlovce 1a Email: [email protected] 16000 Praha Czech Republic Patrick Bienert Phone: +420224310108 Faculty of Life Sciences Email: [email protected] Copenhagen University Thorvaldsensvej 40 Francisco Calvo Garzon 1871 Frederiksberg C Departamento de Filosofía, Ed Luis Vives
107 Campus de Espinardo Vinicna 5 Universidad de Murcia 12844 Praha 2 30100 Murcia Czech Republic Spain Phone: 00420-221 951 685 Phone: 34968363460 Email: [email protected] Email: [email protected] Consuelo De-Moraes Věra Cenklová 539 ASI Building, Center for Chemical Institute of Experimental Botany, Public Ecology , Penn State University Research Institute, Academy of Sciences of 16802 State College the Czech Republic, Sokolovská 6 United States of America CZ-77200 Olomouc Phone: (814)863-2867 Czech Republic Email: [email protected] Phone: +420 58 520 5862 Email: [email protected] Anna Doskocilova videnska 1083 François Chaumont 14220 Praha 4 - Krc Croix du Sud 5-15 Czech Republic B-1348 Louvain-la-Neuve Phone: 00420737944572 Belgium Email: [email protected] Phone: +32-10-478485 Email: [email protected] Edita Drdová Institute of Experimental Botany, Academy Milada Ciamporova od Sciences of the Czech Republic, Institute of Botany, Slovak Academy of Rozvojova 263 Sciences, Dúbravská cesta 14 CZ-16502 Prague 845 23 Bratislava Czech Republic Slovakia (Slovak Republic) Phone: +420225106449 Phone: 00421-2-594 26 114 Email: [email protected] Email: [email protected] Halina Dziubinska Marzena Ciszak Department of Biophysics, Institut of C.N.R.-Istituto Nazionale di Ottica Biology, Maria Curie-Sklodowska University, Applicata, L.go E. Fermi 6 str. Akademicka 19 50125 Florence 20-033 LUBLIN Italy Poland Phone: +39-055-2308208 Phone: (081)5375012 Email: [email protected] Email: [email protected]
Ian Cole Wagner Edgar University of British Columbia Okanagan Institute for Biology II, University of 3333 University Way Freiburg, Schaenzlestr. 1 V1V1V7 Kelowna D-79104 Freiburg Canada Germany Phone: 250-807-7874 Phone: +49-761 203 2637 Email: [email protected] Email: [email protected] freiburg.de Mária Čudejková Department of Cell Biology and Genetics, Ed Etxeberria Slechtitelu 11 University of Florida 783 71 Olomouc-Holice Citrus Research Center Czech Republic 700 Experiment Station Road Phone: +420608426219 33850 Lake Alfred Email: [email protected] United States of America Phone: 863-956-1151 Fatima Cvrckova Email: [email protected] Katedra fyziologie rostlin PrF UK 108 Ignacio Ezquer Palacký University, tř. Svobody 26 Agrobiotechnology Institute · Campus de 77146 Olomouc Arrosadía · Czech Republic 31192 Mutilva Baja Phone: +420585634179 Spain Email: [email protected] Phone: +34 600722548 Email: [email protected] Klara Hoyerova IEB, v.v.i., Rozvojova 263 Martin Fellner 16502 Praha 6 Palacky University in Olomouc, Dept. Cell Czech Republic Biology and Genetics, Šlechtitelů 11 Phone: +420225106436 78371 Olomouc Email: [email protected] Czech Republic Phone: +420-585 634 905 Jianping Hu Email: [email protected] MSU-Plant Research Lab 106 Plant Biology Bldg Matyáš Fendrych 48824 East Lansing Institute of Experimental Botany, Academy United States of America od Sciences of the Czech Republic, Phone: 517-432-4620 Rozvojova 263 Email: [email protected] CZ-16502 Prague Czech Republic Ralph Hueckelhoven Phone: +420225106458 Chair of Phytopathology, Am Hochanger 2, Email: [email protected] 85350 Freising Germany Paul Galland Phone: +49 8161713682 Fachbereich Biologie Email: [email protected] Philipps-Universität Marburg Karl-von-Frisch Str. 8 Thomas Jahn D-35032 Marburg Thorvaldsensvej 40 Germany Faculty of Life Sciences Phone: +49-6421-2822061 Copenhagen University Email: [email protected] 1871 Frederiksberg C Denmark Michal Grunt Phone: +45 38253484 Vinicna 5 Email: [email protected] 12842 Praha Czech Republic Waldemar Karcz Phone: +420221951685 University of Silesia, Dept. of Plant Email: [email protected] Physiology, Str. Jagiellonska 28, 40-032 Katowice, Poland Jana Hendrychová 40-032 Katowice Vídeňská 1083 Poland 14220 Praha 4 Phone: (032) 2009 460 Czech Republic Email: [email protected] Phone: +420296442357 Email: [email protected] Anna Kasprowicz Department of Molecular and Cellular Andrej Hlavacka Biology LINV Adam Mickiewicz University University of Florence Międzychodzka 5 50019 Sesto f.no (FI) 60-371 Poznań Italy Poland Phone: +390554574001 Phone: +48618292736 Email: [email protected] Email: [email protected]
Vladimira Hlavackova Kimihiko Kato 109 678-1, Tomioka, Iwata, Shizuoka-Ken, Japan 1090 Wien 438-0803 Iwata Austria Japan Phone: 0043-664-432 55 44 Phone: 81-538-36-1580 Email: [email protected] Email: [email protected] Jinxing Lin Tomáš Kocábek Institute of Botany, Chinese Academy of Biology Centre of the Academy of Sciences Sciences of the Czech Rep. 100093 Beijing 37005 Ceske Budejovice China Czech Republic Phone: 0086-10-62836211 Phone: +420387775543 Email: [email protected] Email: [email protected] Frank Ludewig Ladislav Kováč University of Cologne, Botanical Institute Dept. of Biochemistry Gyrhofstr. 15 Faculty of Natural Sciences 50931 Cologne Comenius University Germany Mlynska dolina CH1 Phone: +49 221 470 6596 84215 Bratislava Email: [email protected] Slovakia Phone: +421 2 602 96 402 Jutta Ludwig-Müller Email: [email protected] Technische Universität Dresden Institut für Botanik Magdalena Krzeslowska 01062 Dresden Adam Mickiewicz University, Lab. of General Germany Botany ul. Umultowska 89, Phone: 49 351 46333939 61-614 Poznań Email: [email protected] Poland Phone: +48 (61) 8295603 Schreiber Lukas Email: [email protected] Institute of Cellular and Molecular Botany (IZMB), Department of Ecophysiology Ivan Kulich University of Bonn Katedra fyziologie rostlin PřF UK, Viničná 5, Kirschallee 1 12844 Prague 2 53115 Bonn Czech Republic Germany Phone: 00420739053652 Phone: ++49 +228 73 4687 Email: [email protected] Email: [email protected]
Agnieszka Lapa Heiko Maischak IInstitute of Bioorganic Chemistry, Polish MPI Chemical Ecology, Dept. Bioorganic Academy of Sciences,Z. Noskowskiego 12/14 Chemistry 61-704 Poznań Hans-Knöll-Str.8 Poland 07745 Jena Phone: (+48 61) 852-85-03 (169) Germany Email: [email protected] Phone: +49 3641 571 Email: [email protected] Amit Levy Department of plant sciences, Tel Aviv Stefano Mancuso University LINV - University of Florence 69978 Tel Aviv Viale delle idee 30 Israel 50019 Sesto Fiorentino (FI) Phone: 972-3-6408598 Italy Email: [email protected] Phone: ++390554574063 Email: [email protected] Irene Lichtscheidl Althanstrasse 14 Jan Martinec 110 Institute of Experimental Botany LINV - University of Florence Rozvojova 263 Viale delle idee 30 165 02 Praha 6 50019 sesto fiorentino Czech Republic Italy Phone: +420225106416 Phone: 00390554574047 Email: [email protected] Email: [email protected]
Michal Martinka Teun Munnik Department of Plant Physiology Department of Plant Physiology Faculty of Natural Sciences Swammerdam Institute for Life Sciences Comenius University in Bratislava University of Amsterdam Mlynská dolina B2 Kruislaan 318 SK - 842 15 Bratislava 1098 SM Amsterdam Slovakia Netherlands Phone: +421 2 602 96 459 Phone: +31 20 525 7763 Email: [email protected] Email: [email protected]
Elisa Masi Francisco Munoz-Perez LINV - University of Florence Instituto de Agrobiotecnologia Viale delle Idee 30 CSIC/UPNA 50019 Sesto Fiorentino (Florence) Carretera de Mutilva s/n Italy 31192 Mutilva Baja (Navarra) Phone: +39 055 457 4066 Spain Email: [email protected] Phone: +34 948168030 Email: [email protected] Patrick Masson Laboratory of Genetics (room 3262) Susan Murch University of Wisconsin-Madison University of British Columbia Okanagan 425G Henry Mall 3333 University Way 53706 Madison V1V1V7 Kelowna United States of America Canada Phone: (608) 265-2312 Phone: 250-807-9566 Email: [email protected] Email: [email protected]
Mark Mescher Miriam Nadubinska 539 ASI Building, Center for Chemical Institute of Botany, Slovak Academy of Ecology , Penn State Univesity Sciences, Dubravska cesta 14 16802 University Park SK-845 23 Bratislava United States of America Slovakia (Slovak Republic) Phone: (814)865-4208 Phone: ++421-2-59 426 102 Email: [email protected] Email: [email protected]
Axel Mithöfer Yoko Nakamura MPI Chemical Ecology, Hans Knöll-Str 8 Tohoku University, Aoba-ku, Sendai 07745 Jena 9-2-704 Futsukamachi, Aoba-ku, Sendai Germany 980-8578 Sendai Phone: +49 3641 571263 Japan Email: [email protected] Phone: +81 22 795 6556 Email: nakamura- Toshiaki Mitsui [email protected] 8050 Ikarashi-2 9502181 Niigata Akihiko Nakano Japan RIKEN Discovery Research Institute Phone: +81-25-262-6641 Molecular Membrane Biology Laboratory Email: [email protected] 2-1 Hirosawa 351-0198 Wako Sergio Mugnai Japan 111 Phone: +81 48 467 9547 Email: [email protected] Beata Petrovska Institute of Experimental Botany, Public Mads Nielsen Research Institute, Academy of Sciences of Thorvaldsensvej 40 the Czech Republic, Sokolovska 6 DK-1871 Copenhagen CZ-77200 Olomouc Denmark Czech Republic Phone: +45 3528 3439 Phone: +420 58 520 5862 Email: [email protected] Email: [email protected]
Velemir Ninkovic Sonia Philosoph-Hadas Swedish University of Agricultural Science, ARO, The Volcani Center, Dept. of Department of Ecology, Box 7044 Postharvest Science SE-750 07 Uppsala 50250 Bet-Dagan Sweden Israel Phone: +46 18 672541 Phone: 972-3-9683604 Email: [email protected] Email: [email protected]
Ariel Novoplansky Ondrej Plihal Ben-Gurion University MBU AV CR, Videnska 1083 84990 Sede-Boqer Campus 14220 Praha4-Krc Israel Czech Republic Phone: +972-8-6596820 Phone: 00420721759196 Email: [email protected] Email: [email protected]
Ralf Oelmueller Charlotte Poschenrieder Friedrich-Schiller-Universitaet Jena Lab. Fisiología Vegetal, Facultad Biociencias Institut fuer Allgemeine Botanik & Universidad Autónoma de Barcelona Pflanzenphysiologie E-08193 Bellaterra 07743 Jena Spain Germany Phone: 34 935812163 Phone: ++49 3641 949231 Email: [email protected] Email: [email protected] Javier Pozueta-Romero Sakiko Okumoto Instituto de Agrobiotecnología 260 Panama St. (CSIC/UPNA/Gobierno de Navarra), Ctra. de 94305 Stanford Mutilva s/n United States of America 31192 Mutilva Baja, Navarra Phone: 650 325 1521 x363 Spain Email: [email protected] Phone: 34-948168009 Email: [email protected] Ricardo Oliveira UNIVERSIDADE DE SÃO PAULO/ESALQ Luciana Renna DEPARTAMENTO DE CIÊNCIAS BIOLÓGICAS LINV AV. PÁDUA DIAS 11 50019 Sesto Fiorentino (FI) 13418900 PIRACICABA, SP Italy Brazil Phone: +390554574001 Phone: 55 19 34294458 Email: [email protected] Email: [email protected] Maria-Carmen Risueno Camilla Pandolfi Centro de Investigaciones Biológicas, CSIC, LINV - University of Florence Ramiro de Maeztu 9 Viale delle idee 30 28040 Madrid 50019 Sesto Fiorentino (FI) Spain Italy Phone: +34 91 8373112 Phone: 0039.055.457.4060 Email: [email protected] Email: [email protected] 112 Daniel Robert Department of Biophysics, The University of School of Biological Sciences NSW University of Bristol 2057 sydney BS8 1UB BRISTOL Australia United Kingdom Phone: 02 4751 6335 Phone: 0041 117 928 7484 Email: [email protected] Email: [email protected] Petr Skůpa Ewelina Rodakowska Lab. horm. regulations in plants; IEB AS CR, ul. MIędzychodzka 5 v.v.i.; Rozvojová 263 60-371 Poznań 165 00 Praha 6 Poland Czech Republic Phone: 048618292736 Phone: +420605720378 Email: [email protected] Email: [email protected]
Jozef Samaj Mark Staves IZMB Biology Department 53115 Bonn Grand Valley State University Germany 49401 Allendale Phone: 0049 228 736800 United States of America Email: [email protected] Phone: 616-331-2473 Email: [email protected] Arnaldo Schapire Facultad de Ciencias, Campus Teatinos S/N Giovanni Stefano Av. Jorge Luis Borges Nº43 4ºC CP:29010 LINV 29071 Málaga 50019 Sesto Fiorentino (FI) Spain Italy Phone: 0034-952134264 Phone: +390554574001 Email: [email protected] Email: [email protected]
Günther Scherer Maria Stolarz Univ. Hannover, Inst. f. Zierpflanzenbau & Department of Biophysics, Institute of Gehölzwissenschaften Biology, Maria Curie-Skłodowska University Molekulare Ertragsphysiologie Akademicka 19 Herrenhäuser Str. 2 20-033 Lublin D-30419 Hannover Poland Germany Phone: 0815375955 Phone: +49-511-762-3153 Email: [email protected] Email: [email protected] Zdeněk Svoboda Markus Schlicht PřF UP v Olomouci, Katedra buněčné Kirschallee 1 biologie a genetiky, Šlechtitelů 11 53115 Bonn 783 71 Olomouc Germany Czech Republic Phone: +49228 735506 Phone: +420604677625 Email: [email protected] Email: [email protected]
Julian Schroeder Lukáš Synek University of California, San Diego Institute of Experimental Botany, Academy Division of Biological Sciences 0116 od Sciences of the Czech Republic, CA 92093-0116 La Jolla Rozvojova 263 United States of America CZ-16502 Prague Phone: (858) 534-7759 Czech Republic Email: [email protected] Phone: +420225106458 Email: [email protected] Virginia Shepherd Frank Telewski 113 W.J. Beal Botanical Garden Phone: +81-22-795-6553 412 Olds Hall Email: [email protected] Michigan State University 48824 East Lansing Elizabeth Van Volkenburgh United States of America Biology Department Phone: 001-517-355-9582 Box 35 5325 Email: [email protected] University of Washington 98195 Seattle Pilar Testillano United States of America Centro de Investigaciones Biologicas, CSIC, Phone: 206 543 6286 Ramiro de Maeztu 9 Email: [email protected] 28040 Madrid Spain Bruce Veit Phone: +34 91 8373112 AgResearch Email: [email protected] Private Bag 11008 4442 Palmerston North Hans Thordal-Christensen New Zealand Dept. of Agricultural Sciences Phone: 0064-6-351-8217 Faculty of Life Sciences Email: [email protected] University of Copenhagen DK-1871 Frederiksberg C Boris Voigt Denmark LINV, University of Florence Phone: +45 35283443 Viale delle Idee 30 Email: [email protected] 50019 Sesto Fiorentino Italy Ton Timmers Phone: +39 055 457 4001 INRA/CNRS LIPM, 24 chemin de borderouge Email: [email protected] 21326 Castanet-Tolosan France Dieter Volkmann Phone: +33(0)5 612 85051 Institute of Cellular and Molecular Botany Email: [email protected] (IZMB) Kirschallee 1 Hana Toupalová D-53115 Bonn Institute of Experimental Botany, Academy Germany od Sciences of the Czech Republic, Phone: #49 228 73 4747 Rozvojova 263 Email: [email protected] CZ-16502 Prague Czech Republic Alexander Volkov Phone: +420225106458 Department of Chemistry Email: [email protected] Oakwood College 7000 Adventist Blvd Kazimierz Trebacz 35896 Huntsville Department of Biophysics, Institute of United States of America Biology, Maria Curie-Sklodowska University, Phone: (256)7267113 Akademicka 19 Email: [email protected] 20-033 Lublin Poland Yinglang Wan Phone: 48(81)537 59 31 Kirschallee 1 Email: [email protected] 53115 Bonn Germany MInoru Ueda Phone: +49228 735534 Department of Chemistry, Faculty of Science Email: [email protected] Tohoku University 6-3 Aoba, Aramaki-aza, Aoba-ku, Sendai Yangdou Wei 980-8578 Department of Biology, University of 980-8578 Sendai Saskatchewan, 112 Science Place, Japan Saskatoon, SK S7N 5E2, Canada 114 S7N 5E2 Saskatoon Canada Phone: 1-306-966-4447 Email: [email protected]
Przemyslaw Wojtaszek Department of Molecular and Cellular Biology Adam Mickiewicz University Międzychodzka 5 60371 Poznan Poland Phone: +48-61 829 27 35 Email: [email protected]
Zheng Yan College of Life Sciences, Anhui Normal University 241000 Wuhu China Phone: 86-553-3836873 Email: [email protected]
Nan Yao Hedanqing-tang, Rm305, School of Life Science, State Key Laboratory of Biocontrol, Sun Yat-Sen University, 510275 Guangzhou China Phone: 86-20-84110827 Email: [email protected]
Viktor Zarsky Dept. of Plant Physiology, Charles Univ., Vinicna 5 CZ 128 44 Prague Czech Republic Phone: +420 22195 1685 Email: [email protected]
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Book of Abstracts
The 4th International Symposium on Plant Neurobiology
PNB2008 The 4th International Symposium on Plant Neurobiology Fukuoka, Japan (June 6-9 2008)
Book of Abstracts
ii Contents
List of Contents iii - vii
Programme viii - xiii
Abstracts for Sessions 1 to 9 1 - 81
1. Frantisek Baluska et al. 1 Plant Neurobiology from Evolutionary Perspective 2. Stefano Mancuso et al. 3 Neuroid conduction in plant 3. Tomohiro Kakizaki et al. 4 The role of plastid protein import in the regulation of nuclear gene expression 4. Ken-ichiro Shimazaki et al. 5 Stomatal opening response by blue light 5. Akiko Harada 6 Blue light-dependent calcium signaling in higher plants 6. Takayuki Hoson et al. 7 Gravity and light signaling in growth regulation of stem organs 7. Aurelien Bailly et al. 9 Regulation of auxin transport catalysts 8. Markus Schlicht et al. 10 D’orenone Blocks Polarized Tip-Growth of Root Hairs by Interfering with the PIN2-Mediated Auxin Transport Network in the Root Apex 9. De Hoff, P.L. et al. 11 The importance of rhizobial attachment for successful legume nodulation and nitrogen fixation. 10. Akihiro Suzuki et al. 13 Control of root nodulation by the R:FR ratio 11. Akie Kobayashi et al. 14 Mechanisms unique to hydrotropism in seedling roots 12. Manuel Saucedo et al. 15 Hydrotropism: root growth responses to water regulate root system architecture iii in Arabidopsis. 13. Erwan Le Deunff et al. 16 Nitrate uptake responses to AVG and ACC treatments in relation to root elongation changes 14. Takashi Yuasa 17 Plant SNF1-related kinases and stress signaling 15. Teruo Shimmen 19 Position- and substratum-sensing in rhizoid differentiation of Spirogyra 16. Koreaki Ogata et al. 20 The double water film electrode characterized the electrical properties of the gap-junction in Chara as a function of time 17. Rafik Errakhi et al. 21 Anion channel activity is necessary to induce ethylene synthesis and Programmed Cell Death in response to oxalic acid 18. Kamila Kupisz et al. 22 Effects of thermoreceptor agonists on the membrane potential in plants 19. Rainer Hedrich 23 Ligand-gated Signal Transmission in Sensory Plant Cells 20. Brault M. et al. 25 Synergism between reactive oxygen species (ROS), calcium and ABA-induced cell depolarization in Arabidopsis thaliana suspension cells 21. Hidetoshi Iida 28 Mechanosensitive Channel Condidates in Plants 22. Nobuyuki Uozumi 29 Membrane topogeneisi of voltage-dependent K channels 23. Kazuyuki Kuchitsu et al., 30 Ca2+-ROS signaling network regulating stress responses, programmed cell death and development in plants 24. Wilhelm Boland 32 Herbivore-induced early and late responses in Plant-Insect Interactions 25. Gen-ichiro Arimura et al. 33 Heivore-Elicited Events in Legumes’ Terpenoid Biosynthesis 26. Junji Takabayashi 34 Ecological Functions of Herbivore-Induced Plant Volatiles 27. Hirofumi Yoshioka 35 Molecular Mechanisms of the Radical Burst in Plant Immunity 28. Kenji Umemura 36 Disease defense response in rice plants induced by plant defense activators 29. Tomonori Kawano et al. 37 Oxidative and calcium signaling in plants exposed to UV and photochemical iv oxidants 30. Masanori Tamaoki et al. 38 Jasmonic acid and ethylene regulate selenite resistance in Arabidopsis thaliana 31. Junpei Takano et al. 39 Regulation of transporters responsible for boron transport in response to boron conditions in the environment. 32. Mary J Beilby et al. 40 Mechanisms of salt sensitivity
Abstracts for Posters
P01. Georgina Ponce Romero et al. 41 Magnetic field and root development P02. Elisa Masi et al. 42 Characteristics of the artificial electrical activity generated in plant roots under artificial changes in gravity P03. Mitsuo Kawabata 44 Effect of Mitate Conglomerate on root and shoot growth of Raphanus sativus var. radocula with special notice to Cu in cultivation medium P04. Antonin Leblanc et al. 45 Continuous GABA supply to the root affects nitrate uptake in Brassica napus seedlings P05. Hugues Renault et al. 46 Over-accumulation of GABA affects development of Arabidopsis thaliana P06. Nicolas Dumez et al. 48 Reduced glutathione (GSH) regulates nitrate uptake in winter oilseed rape (Brassica napus L. cv Capitol) P07. Takuya Hiramatsu et al. 49 A roles of salicylic acid in UV-C induced cell death signaling P08. Shinpei Etoh et al. 50 Simple and sensitive bioassays for monitoring of night time ozone in the air using model plant seedlings P09. Takashi Kadono et al. 51 Expression Analysis of Trehalose Biosynthesis Related Genes in Tomato P10. Chika Tateda et al. 52 Plant mitochondrial porin regulates defense response against bacterial pathogen and Bax-induced cell death v P11. Cun Lin et al. 53 Oxidative stress and distortion of calcium signaling by ions of group 13 elements in tobacco cells P12. Ken Yokawa 54 Computational simulation of the plant cell responses to microbial physiologically active substances: principles and ongoing approaches P13. Hidenobu Mizuki et al. 55 Specific capture of phosphoprotein by immobilized Zirconium ion affinity chromatography P14. Tadashi Okobira 57 Quantum analysis of interaction between fungal polysaccharide and single polynucleotide P15. Kazuya Uezu 58 Molecular Dynamics Studies of Side Chain Effect on the Microbial Polysaccharides Triple Helix in Aqueous Solution P16. Koreaki Ogata 59 A Dynamic Measurement Technique Characterizes the Turgor pressure Change in Characean Internodal Cells: the Hydraulic Conductivity of the Plasma Membrane is Isotropic and Independent of the External Hydrostatic Pressure P17. Yoshiumi Shinohara 61 The relationship between vegetational succession and water environmental change in a warm-temperate, volcanic peat mire in south-western Japan P18. Enen Ryu 62 Hydrological Control of River Systems P19. Ayumi Nakazono et al. 63 Effect of volcanic activity on mire vegetation in Tadewara mire, southwest Japan 20. Tomoko Kagenishi et al. 64 Inhibition of copper-induced calcium influx by prion- drived peptide in suspension-cultured tobacco cells P21. Chiaki Karaki et al. 65 Possible use of green paramecia in development of photo-controlled micro-particle transport system P22. Shunsuke Furukawa et al. 66 Development of micro-particle transport system using galvanotactically migrating green paramecium P23. Yuta Jin et al. 67 Response to the presence of glucose depending on NDHD in Synechocystis sp. PCC6803 vi Abstracts for Satelite Session
Satelite Session 1: G. Vidal et al. 68 Abscisic acid-induced anion currents activation mediated by cyclic ADP-ribose / ryanodine receptor (RyR) in Arabidopsis thaliana suspensions cells Satelite Session 2: Errakhi R et al. 70 Thaxtomin A-induced defense responses in Arabidopsis thaliana cells require an early Ca2+ influx Satelite Session 03: Tomonori Kawano et al. 71 Similarity between plant redox enzymes and copper-bound prion protein Satelite Session 04: Ken Yokawa et al. 72 Simulation of the signal transduction in artificial plant cells using NEURON: Inspired from the artificial retinal model
List of Participants 73
List of PNB2008 Organizing Committee 81 Members
vii
PNB2008 Programme
Friday, 6th of June, 2008 Reception (Beer Party)
10:00-17:00 Guests and participants from oversea will be guided from Fukuoka Airport to their hotels by student volunteers.
16:00- Registration (International hall in Kyushu University, Hakozaki Campus. Hall is opened for setting posters.)
19:00- Beer Party (Rokkakudo)
Detailed information (location) will be e-mailed to the registered participants.
Saturday, 7th of June, 2008 (1) General Topics and Cell & Molecular Biology
9:00-9:10 Opening Remark (Prof. Yuasa)
SESSION 1
9:10-9:35 Prof. BALUSKA, Frantisek Plant Neurobiology from Evolutionary Perspective
9:35-10:00 Prof. MANCUSO, Stefano Neuroid conduction in plant
10:00-10:25 Prof. INABA, Takehito The role of plastid protein import in the viii regulation of nuclear gene expression
10:25-10:40 Coffee break
SESSION 2
10:40-11:15 Prof. SHIMAZAKI, Ken-ichiro Stomatal opening response by blue light
11:15-11:40 Dr. HARADA, Akiko Blue light-dependent calcium signaling in higher plants
11:40-12:05 Prof. HOSON, Takayuki Gravity and light signaling in growth regulation of stem organs
12:05-12:30 Dr. GEISLER, Markus Regulation of auxin transport catalysts
12:30-13:30 Lunch
13:30-14:40 POSTER PRESENTATION
SESSION 3
14:40-15:05 Dr. SCHLICHT, Markus D'orenone Blocks Polarized Tip-Growth of Root Hairs by Interfering with the PIN2-Mediated Auxin Transport Network in the Root Apex
15:05-15:35 Prof. HIRSCH, Ann The importance of rhizobial attachment for (ICCERD/UoK-sponsored successful legume nodulation and nitrogen speech) fixation
15:35-16:00 Prof. SUZUKI, Akihiro Control of root nodulation by the R:FR ratio
ix
16:00-16:15 Coffee break
SESSION 4
16:15-16:50 Prof. TAKAHASHI, Hideyuki Mechanisms unique to hydrotropism in seedling roots
16:50-17:15 Prof. CASSAB, Gladys Iliana Hydrotropism: root growth responses to water regulate root system architecture in Arabidopsis
17:15-17:40 Prof. LE DEUNFF, Erwan Nitrate uptake responses to AVG and ACC treatments in relation to root elongation changes
17:40-18:00 Prof. YUASA, Takashi Plant SNF1-related protein kinases and stress signaling
18:00-19:00 Poster presentation continues
Sunday, 8th of June, 2008 (2) Electrophysiology and Long-distance signaling
SESSION 5
9:00-9:25 Prof. SHIMMEN, Teruo Position- and substratum-sensing in rhizoid differentiation of Spirogyra
9:25-9:50 Dr. OGATA, Koreaki The double water film electrode characterized the electrical properties of the gap-junction in Chara as a function of time
9:50-10:15 Prof. BOUTEAU, François Anion channel activity is necessary to induce (ICCERD/UoK-sponsored ethylene synthesis and Programmed Cell x speech) Death in response to oxalic acid
10:15-10:40 Prof. TREBACZ, Kazimierz Effects of thermoreceptor agonists on the membrane potential in plants
10:45-10:55 Coffee break
SESSION 6
10:55-11:30 Prof. HEDRICH, Rainer Ligand-gated Signal Transmission in Sensory (ICCERD/UoK-sponsored Plant Cells speech)
11:30-11:55 Prof. RONA, Jean-Pierre Synergism between reactive oxygen species (ICCERD/UoK-sponsored (ROS), calcium and ABA-induced cell speech) depolarization in Arabidopsis thaliana suspension cells
11:55-12:05 break
12:05-12:30 Prof. IIDA, Hidetoshi Mechanosensitive Channel Candidates in Plants
12:30-12:55 Prof. UOZUMI, Nobuyuki Membrane topogeneisi of voltage-dependent K channels
12:55-13:20 Prof. KUCHITSU, Kazuyuki Ca2+-ROS signaling network regulating stress responses, programmed cell death and development in plants
13:20- Lunch (Lunch box & Beer will be provided in front of bus)
-18:40 Short tour (Dazaifu shrine and Kyushu National Museum)
19:30-21:30 Conference Dinner (Recent Hotel) xi
Monday, 9th of June, 2008 (3) Sensory Biology, Chemistry and Ecology
SESSION 7
9:30-10:05 Prof. BOLAND, Wilhelm Herbivore-induced early and late responses (ICCERD/UoK-sponsored in Plant-Insect Interactions speech)
10:05-10:30 Prof. ARIMURA, Gen-ichiro Herbivore-Elicited Events in Legumes' Terpenoid Biosynthesis
10:30-11:05 Prof. TAKABAYASHI, Junji Ecological functions of herbivore-induced plant volatiles
11:05-11:20 Coffee break
SESSION 8
11:20-11:45 Prof. YOSHIOKA, Hirofumi Molecular mechanisms of the radical burst in plant immunity
11:45-12:10 Dr. UMEMURA, Kenji Disease defense response in rice plants induced by plant defense activators
12:10-12:30 Prof. KAWANO, Tomonori Oxidative and calcium signaling in plants exposed to UV and photochemical oxidants
12:30-12:55 Dr. TAMAOKI, Masanori Jasmonic acid and ethylene regulate selenite resistance in Arabidopsis thaliana
12:55-13:55 Lunch
xii
SESSION 9
Regulation of transporters responsible for 13:55-14:20 Prof. FUJIWARA, Toru boron transport in response to boron conditions in the environment
14:20-15:45 Dr. BEILBY, Mary Jane Mechanisms of salt sensitivity
15:45-15:50 Closing Remark (prof. Kawano)
17:00-21:00 Sponsored Satellite Meeting at The University of Kitakyushu and Dinner in Kitakyushu City
Tuesday, 10th of June, 2008 (4) Sponsored satellite session (at The Univ. Kitakyushu)
10:00-10:30 Prof. RONA, Jean-Pierre Abscisic acid-induced anion currents activation mediated by cyclic ADP-ribose / ryanodine receptor (RyR) in Arabidopsis thaliana suspensions cells
10:30-11:00 Prof. BOUTEAU, François Thaxtomin A-induced defense responses in Arabidopsis thaliana cells require an early Ca2+ influx
11:00-11:20 Prof. KAWANO, Tomonori Similarity between plant redox enzymes and copper-bound prion protein
11:20-11:40 YOKAWA, Ken Simulation of the signal transduction in artificial plant cells using NEURON: Inspired from the artificial retinal model
xiii 1
Plant Neurobiology from Evolutionary Perspective
Frantisek Baluska1 and Mancuso Stefano2 1 University of Bonn, Bonn, Germany, and 2 LINV, University of Florence, Florence, Italy. E-mail: [email protected]
Plant Neurobiology has a focus on communication between adjacent cells in plant tissues via synaptic mode based on endocytosis and rapid vesicle recycling, between different root and shoot apices via rapid electrical signals - action potentials, as well as for inter-organismic plant-plant, plant-fungi, plant-bacteria, plant-animals communication. The latter is part of just emerging sensory ecology. Plants are using huge battery of volatiles for their communication, as well as manipulation. Besides volatiles signalling molecules, they synthesize numerous secondary metabolites, energy-rich and neuro-active compounds which are aimed to attract and manipulate animals and humans. The current dominant view is that all this happens only as by-product of metabolism, and that plant action potentials have no signalling roles, but represent some kind of evolutionary oddity. Plant neurobiology standpoint is that all these activities serve plants for their adaptation and survival. Recently, Struik et al. (2008) questioned the validity of the plant neurobiology concept by arguing with the parsimony principle, also known as the ‘Ockham’s razor’, which suggests that the most plausible concept is that which is based on the simplest ideas and requires the lowest amount of assumptions. However, Francis Crick has commented on potential limitations of the Ockham's razor concept in biology (1988). Because biological systems are the products of (an on-going) natural selection, the mechanisms are not necessarily optimal in an obvious sense. He cautions: "While Ockham's razor is a useful tool in the physical sciences, it can be a very dangerous implement in biology. It is thus very rash to use simplicity and elegance as a guide in biological research." In addition, evolution 'weeds out', or even 'do not allows' implementation, of any energetically costly processes which have no values and benefits for organisms adaptation and survival. Neuronal processes are extremely costly – for example, to run an action potential requires a lot of ATP. Their mere presence in plants should
1 serve as evidence that they have some essential roles. It is only our narrow-minded, unflexible, and animal-centric word-view which prevent us to grasp these issues in an open-minded fashion which would allow us to reveal the true communicative nature of plants. In order to interpret correctly genetic, molecular, and physiological data, we need to understand why such an unprecedent sensory complexity is needed for sessile plants. New concepts are needed, and new questions must be asked, for advancing our still rudimentary understanding of the communicative nature of sensory plants.
Crick FHC. 1988. What Mad Pursuit: A Personal View of Scientific Discovery. New York, New York: Basic Books Struik PC, Yin X, Meinke H. 2008. Plant neurobiology and green plant intelligence: science, metaphors and nonsense. J Sci Food Agric 88:363-370.
2 2
Neuroid conduction in plant
Stefano Mancuso1, Elisa Masi 1, Marzena Ciszak2, Giovanni Stefano1, Luciana Renna1, Elisa Azzarello1, Camilla Pandolfi1, Sergio Mugnai1, Frantisek Baluska3, and Tito Arecchi4, 1 Horticulture, Università di Firenze, Firenze, Italy; 2INOA, Istituto Nazionale di Ottica Applicata, Firenze, Italy; 3Zellbiologie der Pflanzen, Rheinische Friedrich-Wilhelms-Universität , Bonn, Germany; 4 Physics, Università di Firenze, Firenze, Italy E-mail: [email protected]
The term neuroid conduction have originated with Parker (The elementary nervous system, 1919) who used it to describe the conduction of excitation in tissues of sponges, a group of animals without nervous system. Interestingly, at the time Parker wrote there was still no evidence that electrical signalling existed in sponges as this was demonstrated just 80 years after by Leys and Mackie (Nature, 1997). Nevertheless, the neuroid conduction, defined as the propagation of electrical events in the membranes of non-nervous, non-muscular cells have been demonstrated in many invertebrates as hydrozoans and tunicate, but also in the young stages of amphibian and lungfish. In carnivorous or sensitive plants as Dionea and Mimosa the spreading of the electrical signal has been described also as neuroid conduction (Mackie, 1970). Based on the results of our study we suggest that such kind of electrical transmission is a general characteristic of plants. We will show that the characteristic of the APs generated spontaneously in roots fulfil all the requirement normally associated to the neuroid conduction: a) they propagate in an all-or-none basis b) in non-nervous tissues c) going from cell to cell via plasmodesmata (in animal cells, via gap junction) and finally, d) decline rapidly in amplitude and velocity due to the flow of the current in all directions (compared with the one-directional conduction of nerves). Furthermore, the data recorded with a 60-channels Multi-Electrode-Array (MEA), revealed a vigorous and synchronised electrical activity in roots suggesting an intrinsic capacity of the cells of the root apex to generate functional electrical networks.
3 3
The role of plastid protein import in the regulation of nuclear gene expression
Tomohiro Kakizaki, Institut 1, Hideo Matsumura, Institut 2, Katsuhiro Nakayama, Institut 1, Ryohei Terauchi, Institut2, and Takehito Inaba 1 1 The 21st Century COE Program, Cryobiofrontier Research Center, Iwate University, Morioka, Japan; and 2 Iwate Biotechnology Research Center, Kitakami, Japan Email: [email protected]
Plastids, such as chloroplasts, are a highly divergent group of organelles that provide essential metabolic and signaling functions within all plant cells. It is generally believed that plastids are originated from a unicellular photosynthetic bacterium inside a eukaryotic host cell. During evolution, most of the genes encoded by the bacterial ancestor have been transferred to the host nuclear genome. Therefore, the plastid biogenesis is reliant on the expression of nuclear-encoded plastid proteins and their import into plastids. One of the key cellular processes that coordinate the plastid protein import and the nuclear gene expression is the retrograde signaling from plastids to the nucleus. However, the molecular mechanism by which plastid regulates this process remains elusive. Using ppi2 mutant lacking the Toc159 protein import receptor for photosynthetic proteins, we demonstrate that the expression of nuclear-encoded photosynthetic proteins are tightly coordinated with their import into plastids. Down-regulation of photosynthetic genes is also observed in the absence of other translocon components. Furthermore, the coordination of plastid protein import and the nuclear gene expression is likely to be mediated by a novel pathway that is distinct from GUN-ABI4. Comprehensive gene expression analysis of ppi2 mutant identifies a number of potential signaling components involved in the retrograde signaling pathway. Based on these data, we will discuss a novel mechanism that coordinates the plastid protein import and the nuclear gene expression.
4 4
Stomatal opening response by blue light
Ken-ichiro Shimazaki K, Shin-ichiro Inoue, and AtsushiTakemiya Department of Biology, Kyushu University, Ropponnmatsu 4-2-1, Fukuoka, Japan E-mail: [email protected]
Stomata open in response to blue light. Recent investigations have demonstrated that phototropins (phot1, phot2) function as blue light receptors for varius responses, including chloroplast movement, stomatal opening, leaf flattening, leaf movement, and leaf positioning. Phototropin has been discovered as a blue light receptor for phototropism. In this talk, we will present our data on the signaling in stomatal guard cells in response to blue light and functional roles of phototropins. Since blue light perceived by phototropins results in activation of the plasma membrane H+-ATPase in guard cells, we focused the signaling between phototropins and the H+-ATPase. We showed that the type 1 protein phosphatase mediates the signaling between phototropins and the H+-ATPase. Phototropins undergo autophosphorylation upon irradiation of blue light, however, physiological role of autophosphorylation remains unknown. We thus determined autophophorylation sites in phot1 by liquid chromatography tandem mass spectroscopy in vivo, and found eight phosphorylation sites of Ser and Thr residues. These located on the N-terminus, hinge region of LOV domain, kinase domain, and C-terminus. We substituted these Ser or Thr with Ala and investigated their roles after transformation of phot1 phot2 double mutant with these phot1 constructs. We indicate that phosphorylation sites in the activation loop of kinase domain have the essential role for all of the responses measured. We finally demonstrate that phot1 enhances plant growth under a weak light. The results suggest that a principal role of phototropin is an enhancement of photosynthesis under a weak light via efficient light capture.
5 5
Blue light-dependent calcium signaling in higher plants
Akiko Harada Department of Biology, Osaka Medical College, Takatsuki, Japan E-mail: [email protected]
Plants have several kinds of photoreceptors, which regulate growth and development. Recent investigations using Arabidopsis thaliana revealed that the newly found blue light receptor phototropins (phot1 and phot2) mediate plant movements and photomorphogenesis such as phototropism, chloroplast relocation, stomatal opening, rapid inhibition of hypocotyl elongation, and leaf expansion. Several physiological studies suggest that one of the intermediates in phototropin signaling is cytosolic Ca2+. Studies using phototropin mutants have demonstrated that phototropins induce an increase in cytosolic Ca2+ concentration and activate calcium-permeable channel. However, the function of Ca2+ in the phototropin-mediated signaling process is largely unknown. I will present recent findings about phototropin-mediated calcium mobilization and the involvement of calcium in blue light-dependent plant responses.
Akiko Harada and Ken-ichiro Shimazaki (2007) “Phototropins and blue light-dependent calcium signaling in higher plants” Photochemistry and Photobiology 83: 102-111
6 6
Gravity and light signaling in growth regulation of stem organs
Takayuki Hoson, Kouichi Soga, Saho Nakano, Makiko Nukada, Kazuyuki Wakabayashi Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan E-mail: [email protected]
Plants are surrounded by a variety of environmental stimuli. To respond and survive in the changeable environment, plants have to properly perceive each stimulus, and transform and transduce the perceived signal. We examined the possible signaling mechanism in suppression by hypergravity and light of stem growth. Under hypergravity conditions, elongation growth of stem organs is suppressed via a decrease in the cell wall extensibility, which is brought about by modification of the metabolism of certain matrix polysaccharides, such as xyloglucans and 1,3,1,4-β-glucans, and modification of the cell wall environment, especially pH. Such growth suppression and cell wall modifications were similarly induced by basipetal- and acropetal-hypergravity. In the basal region cellulose, instead of matrix polysaccharides, was involved in the growth suppression. Thus, the upper growing region and the basal non-growing region may independently respond to hypergravity. Growth suppression and cell wall modifications by hypergravity occurred normally in stem organs of agravitropic mutants and in decapped roots. Also, hypergravity had no effects on growth or cell wall properties in the presence of lanthanum and gadolinium ions, blockers of mechanosensitive ion channels. Taken together, these results suggest that the gravity signal is perceived directly by mechanoreceptors in each cell, independent of gravitropism, and intercellular signal propagation along stem organs is not involved in growth suppression by hypergravity. Light also suppresses elongation growth of stem organs by decreasing the cell wall extensibility. When only the basal region of azuki bean epicotyls was illuminated, elongation growth of the upper growing region was suppressed after a lag. The duration of the lag period was correlated with the distance between the growing region and illumination site, suggesting the presence of acropetal light signaling. Thus, the
7 signaling mechanism may be different between gravity and light, even if both similarly influence plant growth.
8 7
Regulation of auxin transport catalysts
Aurelien Bailly 1, Valpuri Sovero 1, Stefano Mancuso 2, and Markus Geisler 1, 1 Institute of Plant Biology, University of Zurich, Zurich, Switzerland, and 2 Department of Horticulture, University of Firenze, Sesto Fiorentino, Italy E-mail: [email protected]
Active transport of the essential signaling molecule auxin is essential for plant physiology and development. Many aspects of these are controlled by cell-to-cell or polar auxin transport, which is primarily determined by auxin efflux complexes, characterized by PIN and ABCB (PGP/MDR) auxin exporters. Both types of proteins appear to act independently but perform specific interactions that determine the specificity and direction of auxin efflux. Here, we summarize recent progress of ABCB interaction with immunophilin-like FKBP42, TWISTED DWARF1, which functions as a sensor in ABCB-mediated auxin transport. ABCB1-TWD1 interaction is disrupted by binding of synthetic and native auxin transport inhibitors, like NPA and quercetin, leading to inactivation of ABCB1. Contrary, IAA enhances ABCB1-TWD1 interaction resulting in activation of auxin transport and self-termination of its own signal. Our data suggest that a combined action of transport and regulatory components forming an auxin efflux complex is needed for the establishment and control of asymmetric auxin fluxes.
9 8
D’orenone Blocks Polarized Tip-Growth of Root Hairs by Interfering with the PIN2-Mediated Auxin Transport
Network in the Root Apex
Markus Schlicht1, Olga Šamajová1, Doreen Schachtschabel2, Stefano Mancuso3, Diedrik Menzel1, Wilhelm Boland2, František Baluška1* 1Rheinische Friedrich-Wilhelms-Universität Bonn, Zellbiologie der Pflanzen, Bonn, Germany, 2Max-Planck-Institut für chemische Ökologie, Jena, Germany, and 3University of Florence, Florence, Italy. E-mail: [email protected]
The C18-ketone ((5E,7E)-6-methyl-8-(2,6,6-trimethylcyclohex-1-enyl)octa-5,7-dien-2-one) (D’orenone) has been postulated to be an early cleavage product of β-carotene en route to trisporic acids; these act as morphogenetic factors during the sexual reproduction of zygomycetes. Here we report that D’orenone blocks the highly polarized tip growth of root hairs at causing tip-growth to stop completely within a few minutes. Importantly, external auxin restores these effects of D’orenone on root hairs. Further analysis revealed that D’orenone lowers auxin concentration in trichoblasts via PIN2-mediated auxin efflux below critical levels essential for root hair growth. D’orenone increases specifically PIN2 protein abundance without affecting PIN2 transcripts, and that the PIN2 expression domain enlarges and shifts basipetally, resulting in more active auxin transport. Final evidence for PIN2 acting as the specific target of D’orenone is the observation that this compound does not interfere with the root hair growth in roots of null mutant lines.
10 9
The importance of rhizobial attachment for successful legume nodulation and nitrogen fixation.
De Hoff, P.L.,1 Suzuki, A.,2 Fujishige, N.A.,1 and A.M. Hirsch.1 1 Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute, University of California-Los Angeles, 405 Hilgard Avenue, Los Angeles, 90095-1606, USA, 2 Department of Environmental Sciences, Faculty of Agriculture, Saga University, 1 Honjyo-machi, Saga 840-8502, Japan E-mail: [email protected]
Legume nodule development has been extensively studied, especially with regard to rhizobial nod genes and the host’s Nod Factor signal transduction pathway. However, little is known about rhizobial attachment to roots and entry into host tissues. Without attachment, rhizobia cannot colonize the root, and if the rhizobia cannot enter, they do not differentiate into nitrogen-fixing bacteroids. Previously, we showed that transferring a soybean (SBL) or pea (PSL) lectin gene into Lotus corniculatus or Medicago sativa increased the attachment of rhizobia known to nodulate soybean or alfalfa (1, 2). Although binding was dependent on the presence of nod genes that produced the cognate Nod factor, the results strongly suggested that additional factors on legume roots influence rhizobial binding. We now have introduced the PSL or the alfalfa (MsLec1) lectin genes into Arabidopsis to determine whether Rhizobium binding to the roots of the transgenic lectin plant is enhanced. For PSL, Arabidopsis was transformed 1) with the vector alone, 2) with the vector carrying PSL with a mutated sugar-binding domain, and 3) with the vector carrying an intact PSL. The PSL-transformed roots were inoculated with Rhizobium leguminosarum bv. viciae Rlv128C53/ gfp or Sinorhizobium meliloti Rm1021/gfp. For MsLec1, Arabidopsis was transformed with 1) the vector alone or 2) with a construct carrying the vector plus MsLec1. The transformed roots were inoculated with wild-type, Nod- (∆nodD1ABC), and
11 wild-type S. meliloti overexpressing the nod genes. Few Nod- S. meliloti cells bound to the roots confirming our recent results on the importance of the common nod genes for biofilm formation (3). In contrast, wild-type and Nod factor-overexpressing rhizobia attached very well to both Arabidopsis vector control roots and to roots carrying a mutated PSL. However, significantly reduced binding was observed on the transgenic Arabidopsis roots expressing either the wild-type PSL or MsLec1.
1. van Rhijn, P., Goldberg, R.B. and Hirsch, A.M. 1998. Plant Cell. 10:1233-1250.
2. van Rhijn, P., Fujishige, N.A., Lim, P.-O., and Hirsch, A.M. 2001. Plant Physiol. 125:133-144.
3. Fujishige, N.A., Lum, M.R., De Hoff, P.L., Whitelegge, J.P., Faull, K.F., and Hirsch, A.M. 2008. Mol. Microbiol. 67:504-515.
12 10
Control of root nodulation by the R:FR ratio
*Akihiro Suzuki1, Lalith Suriyagoda1, Toshiki Uchiumi2, Mikiko Abe2, Masatsugu Hashiguchi3, Ryo Akashi3, Tatsuya Sakai4, Sayaka Inada4, Shusei Sato5, Takakazu Kaneko5, Satoshi Tabata5 and Susumu Arima1 1Saga Univ., 2Kagoshima Univ., 3Miyazaki Univ., 4RIKEN, 5Kazusa DNA Res. Inst., Japan E-mail: [email protected]
Phytochrome (PHY) is a major light responsive molecule in plants, which regulates various shoot morphological functions through out the life cycle. Influence of phytochrome on root morphophysiology is less reported and the mediation on legume symbiosis is not documented. We found two EMS mutants of Lotus japonicus, 01-0017 and 01-1428, which were having elongated shoot phenotype with pale green foliage and had elongated hypocotyls when germinate under red and white light conditions. Having these evidences, subsequent sequencing analysis revealed to have mutations in the PHYB gene of both lines. Contradictorily to elongated shoots, both mutants formed lesser number of nodules per plant compared to the wild type (WT). Reciprocal and self-grafting experiments using phyB mutants showed that shoot genotype is responsible for the negative regulation of root nodulation. One possible reason for the suppression of root nodule formation in phyB mutants is due to limiting energy source (photosynthates), because PHYB is very important for development of photosynthetic organ. How about another reason? WT L. japonicus which was grown under continuous white light for 10 days was moved to high R:FR or low R:FR condition (uniform photosynthetically active radiation) and then nodulation test were carried out using Mesorhizobium loti. Surprisingly, the number of root nodules of low R:FR plants 28 days after inoculation was dramatically reduced compared with that of high R:FR plants. These results indicate that root nodule formation is one of the R:FR ratio perception reaction.
13 11
Mechanisms unique to hydrotropism in seedling roots
Akie Kobayashi, Yutaka Miyazawa, Akiko Takahashi, Tomoko Kaneyasu, Nobuharu Fujii, and Hideyuki Takahashi Graduate School of Life Sciences, Tohoku University, Sendai, Japan E-mail: [email protected]
We have demonstrated that roots of various plants display hydrotropism in response to moisture gradient, but it is interfered by gravitropic response on Earth. Interestingly, apparatus for sensing both gravity and moisture gradient appear to reside in the columella cells of the root cap. We recently showed that hydrotropic response easily overcome gravitropic response in Arabidopsis roots. Using the experimental system with Arabidopsis, we successfully isolated ahydrotropic mutants termed mizu-kussei (miz). Hydrotropic responses of miz1 and miz2 roots are impaired, whereas their gravitropic responses do not differ from that of the wild type. No morphological abnormalities are observed in miz1 and miz2 plants when compared with the wild type. These results imply that MIZ1> and MIZ2 are essential for hydrotropism but not for gravitropism. MIZ1 encodes a protein with a domain with unknown function that is highly conserved among land plant species, suggesting that MIZ1 has evolved to play an important role in the adaptation to terrestrial environment. Moreover, MIZ1 is expressed predominantly in the root columella cells, and thus we assume that this gene functions inside gravisensing cells of the roots. In Arabidopsis roots, although auxin response is required, the PIN-mediated auxin transport is unlikely required for the induction of hydrotropic response. Thus, there exist mechanisms unique to hydrotropism, which differentiate hydrotropism from gravitropism in seedling roots.
14 12
Hydrotropism: root growth responses to water regulate root system architecture in Arabidopsis.
Manuel Saucedo, Georgina Ponce, Fatima Rasgado & Gladys I. Cassab. Dep. Biol. Mol. de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Mor. 62250, México. Tel 56-22-76-60, Fax 01-777-313-99-88, E-mail: [email protected]
Roots are capable to construct perspective of their local space by sensing and regulating their growth orientation according to the environmental signals they face. By doing this, plants actively forage resources or avoid stresses from their environment. Hydrotropism allows roots to modify their growth direction in search of water overcoming their positive gravitropic response. The no hydrotropic response nhr1 mutant of Arabidopsis lacks a hydrotropic response, and shows a stronger gravitropic response than that of wild type (wt) in a medium with a water potential gradient. Local application of abscisic acid (ABA) to seeds or root tips of nhr1 increases root downward growth, indicating a critical role of ABA in tropisms. Wt roots germinated and treated with ABA in this system were strongly gravitropic, even though they had almost no starch amyloplasts in the root-cap columella cells. Hydrotropically stimulated nhr1 roots, with or without ABA, maintained starch in amyloplasts, as opposed to those of wt. Thus, starch degradation in the wt might help the root to sustain osmotic stress and carry out hydrotropism instead of reducing gravity responsiveness. We have also developed a testing system for the isolation of putative super hydrotropic response (suh) mutants in Arabidopsis. suh1 mutant roots continuously grew under water deficit for 10 days and reach the moderate water potential conditions present in the lower section of the Petri dish in contrast with wt roots, which only grew for 4 days. suh1 mutant roots also modify their root system architecture according to the position of water availability in the medium and by the local addition of ABA. Furthermore, suh1 seedlings developed a deep and highly branched root system under the stress conditions of the test medium. We conclude that ABA and water stress are critical regulators of root tropic responses.
15 13
Nitrate uptake responses to AVG and ACC treatments in relation to root elongation changes
Erwan Le Deunff, Antonin Leblanc, Julien Lecourt. Institut de Biologie Appliquée, Université de Caen Basse-Nor, CAEN, FRANCE E-mail: [email protected]
In Brassica napus seedlings changes of ethylene biosynthesis pathway by treatments with the ethylene precursor: aminocyclopropane carboxylic acid (ACC) and the ethylene biosynthesis inhibitor: aminoethoxyvinylglycine (AVG) modify elongation of exploratory root and root hair systems in a dose-dependent way. These physiological responses induced by ethylene to the root cells and their consequences in absorbing surfaces were questioned in relation to nitrate uptake and nitrate transporter genes expression. Treatments with high concentrations of ACC and AVG (10 uM) over five days revealed significant differences between root elongation, nitrate uptake capacities and nitrate transporter genes expression of BnNrt2.1 and BnNrt1.1. Although ACC increased the length and number of root hairs, the rate of N uptake and the transcript level of the nitrate transporter BnNrt2.1 were markedly reduced. In contrast, the decrease in root hairs length and number in AVG treated seedlings was over-compensated by an increase of nitrate uptake and BnNrt2.1 gene expression. These results demonstrated that root hair cells are not the only location of N absorption in the root and that BnNrt2.1 expression levels were more correlated to exploratory root system. Moreover, the changes of root elongation and nitrate uptake in AVG treated seedlings were not uniquely due to ACC synthase inhibition but certainly to an inhibition of a non-overlapping ethylene pathway. Indeed, the restoration of root elongation in AVG treated seedlings by 1mM L-glutamate suggested that AVG root elongation effects are mediated by inhibition of pyridoxal 5’-phosphate (PLP)-dependent enzymes of N metabolism instead of ACC synthase of ethylene pathway.
16 14
Plant SNF1-related kinases and stress signaling
Takashi YUASA Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka, Japan E-mail: [email protected]
Plants use complex mechanisms such as hormonal signaling, regulation of ion transport, and synthesis of osmolytes to survive under various environmental stresses. Recently, it has been reported that salinity, drought and ABA stimulate a set of plant specific protein kinases, SNF1-related kinases (SnRKs). Plant SnRK family was classified to three subgroups; 1) SnRK1, regulating nutrient metabolisms, 2) SnRK2, involving tolerance against salinity and drought, 3) SnRK3/Calcineurin B-like molecule(CBL) interacting Protein Kinase (CIPK), regulating ion transporters. SnRK2s and CIPKs are novel signal components regulating ion homeostasis and osmolyte production under osmotic stress. A SnRK2 homolog (SlSnRK2C) cDNA was isolated from tomato Micro-Tom 1). Protein levels of SlSnRK2C appeared to increase specifically during young fruits while there was no difference in mRNA leveles of SlSnRK2C among several organs of tomato. SlSnRK2C is activated in response to salt stress and chilling when SlSnRK2 is expressed transiently in Nicotiana benthamiana by agroinfiltration 2). Involvement of the fruit-specifc SnRKs on regulation of sugar metabolisms under salt stress will be discussed. A CIPK homolog (VuCIPK1) cDNA isolated from cowpea shows significant similarity to AtCIPK3 (86% amino acid identity), which is involved in K+ transport. Immunoblot with an anti-VuCIPK1 specific antibody and an anti-CBL antibody showed that immunologically CIPK- and CBL-related polypeptides were preferentially associated with the membrane fractions. Immunoprecipitation assay with cowpea leaf extracts by anti-VuCIPK1 antibody showed that endogenous VuCIPK1 is autophosphorylated at the threonine residues in response to salt stress. These observations suggest that VuCIPK1 is an osmotic stress-activated kinase regulated by its phosphorylation status.
YUASA T, et al (2007) Environmental stresses activate a tomato SNF1-related protein
17 kinase 2 homolog, SlSnRK2C, Plant Biotechnol 24: 401-408 YUASA T et al (2005) Activation of SIPK in response to ultraviolet-C (UV-C) irradiation. Plant Biotechnol 22:7-12
18 15
Position- and substratum-sensing in rhizoid differentiation of Spirogyra
Hisato Ikegaya, Naoko Inoue, and Teruo Shimmen Graduate School of Life Science, University of Hyogo, Hyogo, Japan E-mail: [email protected]
Filaments of Spirogyra are composed of tandem cylindrical cells. Some species of Spirogyra living in running water differentiate the terminal cell to be a rhizoid for anchoring. This differentiation can be easily induced by severing the algal filaments in the laboratory. Before starting the rhizoid differentiation, Spirogyra cells elongate via diffuse growth. When algal filament was severed, the terminal cell starts tip growth at its distal end. Rhizoid is formed only by the terminal cell, suggesting that the cell recognizes its own position before starting the differentiation. Distal end of the terminal cell becomes convex due to its high turgor pressure. When the cell turgor pressure was decreased by adding sorbitol to the external medium, rhizoid differentiation did not start. Upon sorbitol removal, the differentiation started. It is suggested that stretching of the plasma membrane of the distal end is responsible for the position-sensing by the terminal cell. Involvement of stretch-activated channel was suggested. Substratum plays an important role in the rhizoid differentiation. When algal filament did not attach to the substratum, the rhizoid differentiation did not start. Thus, mechanical stimulation of the substratum is necessary for starting the differentiation. In addition, we found that the properties of the substratum is responsible for morphology of rhizoids. On hydrophilic substratum, rod-shaped rhizoids were formed. On the other hand, rosette-shaped rhizoids were formed on the hydrophobic substratum. Involvement of protein phosphorylation in the signal processing was suggested.
19 16
The double water film electrode characterized the electrical properties of the gap-junction in Chara as a function of time
Koreaki, Ogata Brain Science and Engineering, Kyusyu-institute of Technology, Kitakyusyu, Japan E-mail: [email protected]
By scanning the double water film electrode unit (Ogata, 2000) along the length of Chara plant, the resistance (Rm) and capacitance (Cm) at 30Hz across the internode/node interface were studied as a function of time. Rm and Cm were 30*10-3 ohms m2 and 1.5*10-1 Fs m-2 at 20 C, respectively. The series resistance (Rs) of 8*10-3ohms m2 could also be resolved simultaneously. Rm and Cm were strongly depending on the temperature and on a mechanical stimulus. The temperature depencency of Rm generally showed a significant hysteresis, but not for Rs. These observations will be discussed in relation to the dynamic properties of plsmodesma(ta), gap-junction(s) might be responsible for one of the cell-cell comunications.
20 17
Anion channel activity is necessary to induce ethylene synthesis and Programmed Cell Death in response to oxalic acid
Rafik Errakhi,1 Patrice Meimoun,1 Arnaud Lehner,1 Guillaume Vidal,1 Joël Briand,1 Françoise Corbineau,2 Jean Pierre Rona,1 Tomonori Kawano,3 François Bouteau 1 1Biology, Université Paris Diderot, Paris, France; 2Biology, UPMC, Paris, France; 3Graduate School of Environment, University of Kitakyushu, Kitakyushu, Japan E-mail: [email protected]
Oxalic acid is thought to be a key factor of the early pathogenicity stage in a wide range of necrotrophic fungi. Studies were conducted to determine whether oxalate could induce programmed cell death in Arabidopsis thaliana suspension cells and to detail the transduction of the signalling pathway induced by oxalate. A. thaliana cells were treated with millimolar concentrations of oxalate. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements. Involvement of anion channel and ethylene in the signal transduction leading to programmed cell death were determined by using specific inhibitor. Oxalic acid induced a programmed cell death displaying cell shrinkage and fragmentation of DNA into internucleosomal fragments with requirement for active gene expression and de novo protein synthesis, characteristic hallmarks of programmed cell death. Other responses generally associated with plant cell death, such as anion effluxes leading to plasma membrane depolarization, mitochondrial depolarization and ethylene synthesis, were also observed following addition of oxalate. Regarding our results, we propose a model in which oxalic acid activates an early anionic efflux which is a necessary prerequisite for the synthesis of ethylene and for the programmed cell death observed in A. thaliana cells.
21 18
Effects of thermoreceptor agonists on the membrane potential in plants
Kamila Kupisz, Halina Dziubinska, and Kazimierz Trebacz Department of Biophysics, Maria Curie-Sklodowska University, Lublin, Poland E-mail: [email protected]
Effects of menthol and some other "cooling" compounds on the membrane potential in the liverwort Conocephalum conicum were examined. Conocephalum conicum belongs to philogenetically oldest terrestrial plants. It is an excitable plant - generates action potentials, APs, in response to different stimuli including depolarizing current, illumination, and cooling. The temperature increase evokes hyperpolarization, whereas cooling produces depolarization, which, when strong enough, leads to generation of AP. Two enantiomers of menthol: (-)-menthol and (+)-menthol were tested. In animal cold receptors - cation channels belonging to TRP family, (-)-menthol is almost four-times more effective than (+)-menthol in mimicking a response to cold - transient depolarization and a vanishing series of APs. In Conocephalum, in contrast to TRP receptors, (-)-menthol evokes concentration-dependent hyperpolarization of the membrane potential. At 0.01 mM concentration the response to (-)-menthol changed to depolarization and APs were occasionally registered. (+)-Menthol caused generation of APs when applied at concentration as high as 10 mM. More diluted (+)-menthol solutions produced hyperpolarization of the membrane potential. Capsaicin, which activates heat receptors in animals, but also is an agonist of Trpm8 cold receptor, evoked hyperpolarization in Conocephalum cells. Although homologs of TRP encoding genes have not been found in Arabidopsis thaliana, it occurs that their agonists are effective in Conocephalum.
22 19
Ligand-gated Signal Transmission in Sensory Plant
Cells
Rainer Hedrich University of Würzburg, Molecular Plant Physiology and Biophysics, Biocenter, Würzburg University, 97082Würzburg, Germany E-mail: [email protected]
The plant hormone abscisic acid (ABA) is involved in the transmission of environmental changes like drought-, saline-, and cold-periods into stress adaptation processes. Based on the timescale of the individual ABA evoked responses they have been subdivided into fast (membrane transport) and slow (transcription) signalling. In contrast to the latter process the fast ABA response - exemplified by half times of stomatal closure around 5-10 min - seem not involve gene activation. Instead, stomatal closure is accomplished by the release of potassium ions as well as the anions chloride and malate. In search for ABA signalling intermediates the response of ion channels of guard cells in epidermal peels as well as guard cell protoplasts and vacuoles have been challenged with well-characterized modulators effective in signal transduction pathways of animal cells. Isolated, experimentally well controlled guard cell preparations, however, often lack communication with neighbouring cells, turgor or cytosolic components. In addition potential signalling components derived from mutants altered in ABA-induced stomatal closure. Current models, gained from observations on different screens, guard cell preparations, species or not even guard cells, trying to bridge the gap between the still unknown ABA-receptor and stomatal closure, are at least very complex. To online record changes in ion fluxes across the plasma membrane of guard cells in intact plants, we have developed a method, based on multi-barreled microelectrodes introduced into the cytoplasm of these sensory motor cells in combination with spectrocopy. This approach proved suitable when exploring blue- and red light as well
CO2-signalling. Using this online, in planta approach, we have been able to identify signalling
23 elements required for fast ABAinduced stomatal closure. A model on the ABA-based regulation of guard cell ion transport will be presented at the meeting.
24 20
Synergism between reactive oxygen species (ROS), calcium and ABA-induced cell depolarization in
Arabidopsis thaliana suspension cells
Brault M1,2., Trouverie J1., Madiona, K1., Amiar Z1., Vidal G1., Sirichandra C1,2 and J.-P Rona1 1 Electrophysiologie des Membranes, EA 3514, Université Paris VII, 2 place Jussieu, 75005 Paris, France; 2 Present address : Institut des Sciences du Végétal, UPR CNRS 2355, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France and Université Paris-Diderot, 2 place Jussieu, 75251 Paris cedex 05, France
Abscisic acid (ABA) is a plant hormone involved in multiple plant developmental processes. ABA promotes embryo dormancy and seed desiccation tolerance, elicits bud dormancy and is involved in the control of root growth. ABA also mediates some aspects of plant responses to certain environmental stresses such as drought, salinity and cold. In guard cells of several species, plasma membrane (PM) depolarization is one of the earliest responses monitored after ABA perception and it is shown that activation of PM anion channels plays a key role in the depolarization step (see, Verslues and Zhu, 2007; Wasilewska et al, 2008). In A. thaliana suspension cells, PM depolarization was also observed in response to ABA (Jeannette et al., 1999). It results from the activation of PM anion channels and the reduction proton pumping (Brault et al., 2004). In addition, the ABA-signaling pathway is associated with Ca2+ oscillations induced by reactive oxygen species (ROS). The aim of this work was to explore the mechanism behind the synergism between ROS, calcium and ABA-induced cell depolarization. ABA-induced activation of anion currents and proton pumping reduction are both mediated by calcium signaling: in A. thaliana suspension cells, Ca2+ is a second messenger involved in modulating rapid ABA responses promoting the cell depolarization like has been observed in guard cells. We show that upon ABA treatment ROS are rapidly generated and provoke transient calcium spikes.
25 PM Ca2+ channel inhibitors 100 µM La3+; 50 µM Fluspirilène; 50 µM Pimozide) which 2+ reduce ABA-induced frequency modulation of [Ca ]cyt increases were also found to reduce the ABA-induced (i) anion channel activation (ii) proton pumping activity (iii) RD 29a and RAB 18 genes expression (Brault et al., 2004; Zalejski et al., 2006). Similar results were obtained with 5 mM EGTA a chelator of extracellular Ca2+ 2+ (Brault et al., 2004). This suggests that [Ca ]cyt increases we observed in response to ABA mainly resulted from the mobilization of extracellular calcium which generally occurs through calcium influx. This implied that plasma membrane calcium channels were involved. ABA elicits a rapid and transient generation of ROS in suspension cells : reactive oxygen species (ROS) have emerged as second messengers of ABA in the activation of Ca2+ -permeable channels. ROS are generated by the AtRBOH NADPH oxidases in guard cells upon ABA treatment and are found to mimic the ABA-induced stomatal closure. In A. thaliana suspension cells pre-incubated with 10 µM diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, the ROS production elicited by ABA was reduced by 80 %. These results indicate that ABA elicits a ROS production also in A. thaliana suspension cells.
2+ H2O2 treatment induces an increase in [Ca ]cyt and participates to the
ABA-induced plasma membrane depolarization : we used H2O2 because is a more stable
ROS and can diffuse across PM through water channels. We determined the consequence of exogenous application of H2O2 on the cytosolic calcium concentration 2+ 2+ ([Ca ]cyt) in suspension cells. Variations of [Ca ]cyt were evaluated with a cell suspension prepared from leaves of A. thaliana transformed with the apoaequorin gene.
Application of H2O2 on apoaequorin cell suspension triggers immediately a transient 2+ increase in [Ca ]cyt that reaches a maximum 1 min after the introduction of H2O2 and 2+ was dose-dependent from 0.1 mM to 1 mM H2O2. Indeed, after [Ca ]cyt increase promoted by external application of H2O2, we observed anion channel activation and proton pumping reduction as does ABA. In the presence of calcium channel inhibitor 3+ 2+ La (100 µM), the [Ca ]cyt increase provoked by H2O2 (1 mM) was abolished. Similar result was obtained when the extra-cellular calcium was chelated by EGTA (10 mM).
These results support the idea that the calcium mobilized by H2O2 has an extra-cellular origin. In conclusion, we show that ROS are involved in the ABA induce Ca2+ signaling pathways at the plasma membrane. Taken together, the results presented here show that 2+ in A. thaliana cells, [Ca ]cyt oscillation signatures encode specific responses leading to
26 the anion channel activation and proton pumping reduction, including gene expression (RD29a). This indicates that ROS induced Ca2+ increase is one central component of the signaling pathways leading to the plasma membrane depolarization induced by ABA.
References
1- P E Verslues and Jian-Kang Zhu Current Opinion in Plant Biology (2007), 10: 447–452 2- Wasilewska A., , Vlad F., Sirichandra C., Redko Y., Jammes F., Valona C., Frei dit Frey N., and Leung J. (2008). Molecular Plant, vol 2, 198–217. 3- Jeannette, E., Rona, J.P., Bardat, F., Cornel, D., Sotta, B. and Miginiac, E. (1999). Plant J. 18,13-22. 4. Brault M, Amiar Z, Pennarun, AM, Monestiez, M, Zhang, Z, Cornel, D, Dellis, O, Knight, H, Bouteau, F. and Rona, J.-P. (2004) Plant Physiol. 135:231-243. 5. Zalejski C., Paradis S., Maldiney R., Habricot Y., Miginiac M., Rona J.-P., Jeannette E. (2006). Plant Physiol. 141:1555-62
27 21
Mechanosensitive Channel Condidates in Plants
Hidetoshi Iida Department of Biology, Tokyo Gakugei University, Tokyo, Japan E-mail: [email protected]
Mechanosensitive (MS) channels open a conductance pore in response to mechanical stresses, such as touch, gravity, and osmotic shock and thereby convert the stresses into an electrical or chemical signal. Animal and bacterial MS channels have been studied intensively and characterized considerably at the molecular level. The best-understood examples are the bacterial MS channels MscL and MscS. However, the molecular nature of plant MS channels has been unknown until recently, although their physiological roles have long been implicated in thigmotropism and gravitropism. Recently, ten MscS-like proteins have been identified in the genome of Arabidopsis thaliana, and two of them are reported to be present on the plastid envelope and control the size and shape of the plastid. Another candidate of plant MS channels, named Mca1, has been reported by us (Nakagawa et al., PNAS 104:3639-3644, 2007). Mca1 cDNA was isolated from an Arabidopsis cDNA library by functional complementation of a yeast mid1 mutant defective in a putative MS channel component. Mca1 can indeed enhance Ca2+ influx in yeast cells and is localized to the yeast plasma membrane as an integral membrane protein. In Arabidopsis, GFP-tagged Mca1 is also localized to the plasma membrane. Mechanical stress appears to activate Mca1. First, hypotonic shock 2+ increases [Ca ]cyt higher in MCA1-overexpressing MCA1ox) seedlings than in control ones. Second, MCA1ox roots accumulate Ca2+ about 1.7-fold greater than wild-type roots. Third, the expression of CML12 (TCH3), which is known to be induced by touch and Ca2+, is increased in MCA1ox seedlings. Finally, primary roots of mca1-null seedlings fail to penetrate the harder, lower agar medium of two-phase agar medium from the softer, upper agar medium. These results suggest that Mca1 acts as a mechanosensitive Ca2+ channel.
28 22
Membrane topogeneisi of voltage-dependent
K channels
Nobuyuki Uozumi Tohoku University, Japan E-mail: [email protected]
Several kinds of genes encoding K channels and K transporters have been identified in prokaryote and eukaryote. A simple K channel, which is found in almost any kinds of cells consists of membrane-pore-membrane (MPM) motif. HKT/Ktr-type transporters had been classified as a different group from K channels, but the recent study has revealed that they possess four MRM motifs in a single polypeptide. Plants contain two families of potassium (K+) channel. One is the voltage-dependent (Shaker-type) K+ channel family and the other is the TPK (two pore K+) channel family. Shaker-type K+ channels have a voltage sensing domain that controls the open and closed state of ion conducting pore. Membrane-embedded voltage-sensor domains in voltage-dependent K channels contain an impressive number of charged residues. We have studied the membrane topogensis of voltage sensor domains of plant KAT1 and Drosophila neuron Shaker B (Zhang et al. PNAS 104, 8263-8268, 2007). These results indicate that co-operative (‘post-translational’) integration of the voltage-sensor transmembrane segments is a property common to voltage-dependent channels and that a combination of hydrophobic and electrostatic forces involving S2, S3 and S4 controls the membrane insertion of the voltage sensor.
29 23
Ca2+-ROS signaling network regulating stress responses, programmed cell death and development in plants
Kazuyuki Kuchitsu, Takamitsu Kurusu and Hidetaka Kaya Tokyo University of Science, Department of Applied Biological Science, 2641 Yamazaki, Noda 278-8510, Japan E-mail: [email protected]
Ion fluxes including Ca2+ and production of reactive oxygen species (ROS) are induced at the early step of defense signaling triggered by both biotic and abiotic stresses. To reveal the molecular mechanisms and physiological roles of stress-induced Ca2+ mobilization, we established the retrotransposon-insertional knockout lines as well as the overexpressing lines of a putative voltage-gated Ca2+ permeable channel, OsTPC1, in rice. The Ostpc1 overexpressor showed enhanced sensitivity to a proteinaceous elicitor, whereas the elicitor-induced defense responses including activation of a MAP kinase and hypersensitive cell death were strongly suppressed in the knockout cells, which was rescued by expression of Ostpc1 (Kurusu et al. Plant J. 2005). The gene expression profiles as well as changes in cytosolic Ca2+ concentration induced by various stresses are being comparatively analyzed between the Ostpc1 knockout and the wild type lines. Plant respiratory burst oxidase homolog (rboh) proteins, have been implicated in ROS production in stress responses and during development. They have hydrophilic N-terminal regions containing two EF-hand motifs. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD and rbohC/RHD2 were induced by ionomycin, a Ca2+ ionophore. This activation required a conformational change in the EF-hand region, as a result of Ca2+ binding to the EF-hand motifs. AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production. These results suggest that Ca2+ binding and phosphorylation synergistically activate the rboh-mediated
30 ROS-production that governs stress responses and development including root hair growth (Ogasawara et al. JBC 2008; Takeda et al. Science 2008).
31 24
Herbivore-induced early and late responses in
Plant-Insect Interactions
Wilhelm Boland Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, Germany. E-mail: [email protected]
Feeding herbivores elicit defence responses in the damaged plants, typically the emission of a blend of volatile organic compounds (VOCs) that mediates interactions with the parasites or enemies of the herbivore (indirect defenses). Other plants (additionally) respond with the secretion of extrafloral nectar that attracts ants as defenders. In the Lima Bean (Phaseolus lunatus) both indirect defense strategies are utilized simultaneously. These and other defense responses are initiated by the mechanical damage as well as by the oral secretions (OS) of the herbivore. Using the black lipid membrane (BLM) technique, OS was analyzed with regard to their membrane activities. Transmembrane ion fluxes were generated by OS of eight different Lepidopteran larvae, which all displayed comparable ion channel-forming properties in artificial membranes. The herbivore-linked reprogramming of the plant defense was additionally analyzed with microarrays comprising the whole genome of A. thaliana. In total about 5000 genes were either up- or down regulated, even after simple mechanical damage. By Principal Component analysis different treatments of leaves of A. thaliana, such as mechanical damage, feeding by a specialized insect (Diamond Back Moth), and a generalist herbivore (Beet Army Worm), could be clearly distinguished by a typical set of differently affected genes. Interestingly, the salivary secretions of the feeding insects seem to silence locally the gene expression in the damaged leaf, compared to the effect of mechanical wounding, but in distant leaves a significant reprogramming occurs that is not observed after the MecWorm treatment. The complexity of interactions with focus on the very early events will be discussed.
32 25
Heivore-Elicited Events in Legumes’ Terpenoid
Biosynthesis
Gen-ichiro Arimura1,2,3, Wilhelm Boland3 1Global COE Program: Evolution and Biodiversity, Graduate School of Science, Kyoto University, 606-8502 Kyoto, Japan; 2Center for Ecological Research, Kyoto University, 520-2113 Otsu, Japan; 3Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany E-mail: [email protected]
Volatile terpenoids, the major products among the herbivore-induced plant volatiles in legumes, mediate interactions that attract herbivores’ natural enemies and serve as signals to neighboring plants. In this study, we demonstrated cross-talk among the signaling components involving Ca2+, jasmonic acid, and ethylene; together these are responsible for the formation of volatile terpenoids in Medicago truncatula and lima beans (Phaseolus lunatus). We describe that, like the cross-talk among stress-induced phytohormones, herbivore-sti¬mulated Ca2+ transients also influence the blend of terpenoids, whose biosynthesis depends on the jasmonic acid (JA)/ethylene pathway in M. truncatula. Likewise, we investigated the transcriptional mechanisms in Lima bean that underlie herbivory and diurnal responses involved in terpenoid formation. In order to investigate the effect of diurnal versus nocturnal damage on the signaling pathway for legumes’ volatile emissions, we used MecWorm, a robotic device designed to reproduce tissue damage caused by herbivore attack. Lima bean leaves that were damaged by MecWorm during the photophase emitted maximum levels of monoterpenes [β-ocimene] and C6 volatiles [(Z)-3-hexenyl acetate] in the late photophase. Leaves damaged during the dark phase responded differently. JA accumulated locally in direct response to the damage and led to the immediate up-regulation of the β-ocimene synthase gene (PlOS) inde¬pen¬dent of the phase, that is, light or dark. In summary, damage-dependent JA levels directly control the expression level of PlOS, irrespective of light conditions. We discuss a new perspective on possible events (e.g., Ca2+ signaling) leading to terpenoid biosynthesis.
33 26
Ecological Functions of Herbivore-Induced Plant
Volatiles
Junji Takabayashi Center for Ecological Research, Kyoto University, 520-2113, Japan E-mail: [email protected]
In response to feeding by phytophagous arthropods, plants emit volatile chemicals. This is shown to be an active physiological response of the plant and the released chemicals are called herbivore-induced plant volatiles (HIPV). One of the functions of HIPV for the plant is to attract carnivorous natural enemies of herbivores. Depending on which plant and herbivore species interact, blends of HIPV show qualitative and/or quantitative variation. An intriguing question is whether this allows the natural enemies to discriminate between volatiles from plants infested by herbivore species that are either suitable or unsuitable as a food source for the natural enemy. Another question is, whether natural enemies can also recognize HIPV when two or more herbivore species that differ in suitability as a food source simultaneously attack the same plant species. Here, I will show that arthropod parasitoids can tell different HIPV blends apart in single-plant-single-herbivore systems and even in single-plant-multiple- herbivore systems. HIPV further mediate interactions between two plant individuals of the same/different species, and between plants and phytophagous arthropods. The resulting interaction networks mediated by HIPV would have important consequences in ecological community.
34 27
Molecular Mechanisms of the Radical Burst in Plant
Immunity
Hirofumi Yoshioka Laboratory of Defense in Plant-Pathogen Interactions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan E-mail: [email protected]
Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the innate immunity in plants. There are many reports about complementary, synergistic and overlapping functions of NO and ROS in the defense responses. Recent advances provide the molecular mechanisms of NO and oxidative bursts in the defense responses. NOA1 (NO ASSOCIATED1; formerly named NOS1) and membrane-bound NADPH oxidase are believed to participate in the radical burst. Here we describe that two mitogen-activated protein kinase (MAPK) cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NADPH oxidase at the transcriptional level in Nicotiana benthamiana. On the other hand, NOA1-madiated NO burst is regulated by MEK2-SIPK cascade. Furthermore, we introduce the calcium-dependent protein kinase (CDPK) activates NADPH oxidase by the direct phosphorylation of its N-terminal region.
35 28
Disease defense response in rice plants induced by plant defense activators
Kenji Umemura Agricultural & Veterinary Research Labs, Meiji Seika Kaisha Ltd., Yokohama, Japan E-mail: [email protected]
Acquired disease resistance is known to be induced by specific chemical compounds, called plant defense activators. Among known plant defense activators, probenazole (Oryzemate® from Meiji Seika Kaisha, Ltd.) has been used as an agrochemical to control rice blast disease for over thirty years. Figures for recent years indicate it is applied in roughly 30% of all paddy fields in Japan. Our group focused on the defense responses in rice plants induced by probenazole to clarify the defense mechanism of the rice-blast fungus pathosystem. Previously, we isolated a novel rice phytoalexin as a phytocassane from rice plants, and PBZ1—a novel pathogenesis-related (PR) protein. In addition, we purified elicitor molecules from blast fungus. These were found to be cerebrosides, a type of sphingolipid. We have demonstrated that probenazole-induced disease resistance in rice leaves induced by drenching application shares several defense-related responses induced by spray-treatment with cerebroside, including MAP kinase activation, PR protein induction, and signal transduction via G proteins. Among unshared defense responses, treatment with cerebroside induced the production of active oxygen species and phytoalexin accumulation. Treatment with probenazole alone did not induce such phenomena, which may appear only with concurrent blast fungus inoculation. We have recently discovered that probenazole induces the accumulation of conjugated salicylic acid (SA) in rice leaves, whereas no induction of conjugated SA was observed following treatment with a cerebroside elicitor or blast fungus inoculation. Genetic analysis indicates that probenazole requires the accumulation of conjugated SA to exert its full effects, suggesting the existence of an SA-related defense signaling mechanism in rice plants. We will present and discuss the results of our research on rice plant disease response.
36 29
Oxidative and calcium signaling in plants exposed to
UV and photochemical oxidants
Tomonori Kawano,1 Masaru Yukihiro,1 Takuya Hiramatsu,1 Shinpei Etoh,1 Ken Yokawa,1 Rafik Errakhi,2 and Francois Bouteau 2 1Faculty of Environmental Engineering, The University of Kitakyushu, Kitakyushu, Japan; 2LEM, Universite Paris Diderot, Paris, France E-mail: [email protected]
Recently, we are engaged to the monitoring of the seasonal changes in photochemical oxidants and their impacts on the living plants (development of localized cell death on the surface of leaves) in some monitoring sites including an isolated island in western Japan. Reported simulations suggested that the oxidants and their precursors are most likely brought from the highly industrialized areas in China. From both the microscopic celbiological and macroscopic environmental view points, the impacts of photochemical oxidatns (ozone and PAN) and exposure to ultra violet rays (UV-A and C) on induction of cell death in Nicotiana batacum L. and Arabidopsis thaliana (cells and plants) were examined. We observed that both UV and photochemical oxidatnts induce the cell death in plants via stimulating the oproduction of reactive oxygen species (ROS), calcium signaling (rapid and transient influx of Ca2+) and protein kinase casecades. ROS scavengers and calcium chelators completely inhibited the development of photochemical oxidant- and UV-induced cell death. In cases of ozone and UV responses in ozone-sensitive tobacco cell line (Bel-W3) expressing aequorin gene, we observed the nonbiological ROS production and ROS-responsive increase in cytosolic Ca2+ concentration. In case of ozone responses in Arabidopsis cells, drastic changes in anion channel current and membrane potential were shown to be associated with the induced cell death. In addition to such early signaling events, involvement of salicylic acid were also suggested by the use of cell suspensions derived from various mutants and transgenic lines of Arabidopsis, such as npr1, cpr1, cpr5, sid2, NahG, and NPR-overexpression. Our data may contribute for connecting the interests of enviomental researchers, biochemists and plant cell biologists.
37 30
Jasmonic acid and ethylene regulate selenite resistance in Arabidopsis thaliana
Masanori Tamaoki1, John L. Freeman2 and Elizabeth A.H. Pilon-Smits2 1 Environmental Biology Division, National Institute for Environmental Studies, Tsukuba, Japan, and 2 Biology Department, Colorado State University, Fort Collins, USA E-mail: [email protected]
To better understand plant Se toxicity and resistance mechanisms, we compared the physiological and molecular responses of two Arabidopsis accessions, Col-0 and Ws-2, to selenite treatment. Measurement of root length demonstrated a clear difference between selenite-resistant Col-0 and selenite-sensitive Ws-2. Macroarray analysis showed more pronounced selenite-induced increases in mRNA levels of ethylene or jasmonic acid (JA) biosynthesis and -inducible genes in Col-0 than in Ws-2. Indeed, Col-0 exhibited higher levels of ethylene and JA. The selenite-sensitive phenotype of Ws-2 was attenuated by treatment with ethylene precursor or MeJA. Conversely, the selenite resistance of Col-0 was reduced in mutants impaired in ethylene- or JA-biosynthesis or signaling. Furthermore, the generation of reactive oxygen species (ROS) by selenite was higher in Col-0 than in Ws-2. Together these results indicate that JA and ethylene play important roles in Se resistance in Arabidopsis.
38 31
Regulation of transporters responsible for boron transport in response to boron conditions in the environment.
Junpei Takano, Kyoko Miwa, Toru Fujiwara Biotechnology Research Center, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan: SORST, JST E-mail: [email protected]
Boron is an essential micronutrient for plants and is also toxic in high concentrations. Boron homeostasis is important for plant survival. Homeostasis is mainly achieved by regulating transporters responsible in response to B condition in the environment. BOR1 is identified as the first boron transporter required for efficient xylem loading of B (Takano et al., 2002). Arabidopsis and rice have seven and four BOR1 or BOR1-like genes, all likely to encode efflux transporters, with different physiological functions and location within the cell. We also identified NIP5;1, a protein similar to aquaporin, as a transporter required for efficient B uptake (Takano et al., 2006). Expression of BOR1 and NIP5;1 are both upregulated under B deficient conditions but with different mechanisms. BOR1 accumulates in plasma membrane under low B conditions, and degraded through endocytosis under sufficient B supply (Takano et al., 2005). NIP5;1 is transcriptionally upregulated under low B conditions (Takano et al., 2006). With these knowledge in hands, we have successfully generated transgenic plants that are tolerant to low (Miwa et al., 2006) or high boron (Miwa et al., 2007).
Takano et al., Nature 420, 337-340 (2002) Takano et al., Proc. Natl. Acad. Sci. USA 102,12276–12281(2005) Takano et al., Plant Cell 18, 1498-1509 (2006) Miwa et al., Plant J. 46, 1084-1091 (2006) Miwa et al., Science 318, 1417 (2007)
39 32
Mechanisms of salt sensitivity
Mary J Beilby, Sabah Al Khazaaly and Virginia A Shepherd School of Physics, Biophysics, The University of NSW, Kensington 2052, Sydney, Australia E-mail: [email protected]
The current-voltage (I/V) technique was employed to resolve the response of ion transporters to salt stress in salt sensitive charophyte Chara australis. Cells were challenged by 50 mM NaCl/ 0.1 mM Ca2+ medium after pre-treatment in artificial pond water (APW) adjusted to same osmolarity by sorbitol. At this Ca2+/N+ ratio the membrane potential difference (PD) depolarised to -100 mV within minutes. The background current with increased conductance became dominant in the I/V characteristics. The proton pump was inactivated. The PD of -100 mV is close to the excitation threshold and spontaneous repetitive action potentials (APs) were often observed. Each AP depletes the cell of Cl- and K+. The resting PD continued to depolarise. The I/V characteristics became upwardly concave and were modelled by + gradual opening of H channels, with reversal PD, EH, near zero. The presence of the background current kept the resting PD negative of EH, causing cytoplasmic acidification. At this point the cell could still be rescued by replacing the saline medium by APW, which decreased the background conductance, closed the H+ channels and reactivated the pump. However, continued exposure to saline medium depolarised the PD to very low level of -50 to -20 mV, where the outward K+ rectifier channels were activated producing sustained K+ efflux. This was the final step in the cell decline.
40
P01
Magnetic field and root development
Georgina Ponce Romero 1, Nadia Porras González 1, Gregorio Serrano 2, Luis Canedo 3 and Gladys Cassab López 1 1 Biologia Molecular de Plantas, UNAM, Cuernavaca, Mexico 2 CINVESTAV, Zacatenco, México D.F. 2 UAEM, Cuernavaca, Morelos. E-mail: [email protected]
Land plants have developed various mechanisms for regulating their growth orientation according to the environmental signals they face. Magnetic field is an omnipresent accompaniment of Earthly life and hence plants have evolved with it as a background constant. The effects of magnetic field upon living organisms have been thoroughly documented, however, the mechanisms of action are still unclear. Genomic studies have revealed that several genes of the model plant Arabidopsis thaliana are regulated by the application of a high-density (19T) magnetic field (MF). One hundred and twelve genes out of 8000 showed an increase of 2.5 times in their expression compared with untreated plants. The genes induced are also induced by stresses such as heat, cold, drought and obstacles, as well as genes that code for ion transporters (Cl-, - - SO4 and NH4 ). In the present study, we explored the effect of a fixed MF with magnets of 250, 850 and 1000 gauss in 4-day-old Arabidopsis seedlings treated for 8 days. MF affects the development of the primary root and differences were higher depending upon de strength of the MF. Furthermore, an alternating EMF of 8.5 gauss generated by Helmholtz device of 120 Hz applied to 4-day-old seedlings for 8 days and evaluated when seedlings were 12-days old indicated that both the length of primary roots and the number of lateral roots are significantly reduced. Apparently, MF reduced the effect of stresses in living organisms, and thus we also analyzed the effect of both MF and cadmium, a poisonous heavy metal, in root development. Interestingly, the addition of MF increased the cadmium effect on root growth instead of ameliorating it.
41 P02
Characteristics of the artificial electrical activity generated in plant roots under artificial changes in gravity
Elisa Masi 1, Marzena Ciszak 2, Camilla Pandolfi 1, Sergio Mugnai 1, Elisa Azzarello 1, Luciana Renna 1, Giovanni Stefano 1, Boris Voigt 1, Dieter Volkmann 3 and Stefano Mancuso 1 1 Horticulture, Università di Firenze, Firenze, Italy 2 INOA, Istituto Nazionale di Ottica Applicata, Firenze, Italy 3 Zellbiologie der Pflanzen, Rheinische Friedrich-Wilhelms-Universität , Bonn, Germany E-mail: [email protected]
Gravity-changing conditions revealed to generate strong and rapid responses in root of plants and many evidences lead to the hypothesis of an intrinsic capacity of the root apex to generate functional electrical networks. The MEA (multi-electrode array) system is a new tool usually employed in research on animal electrogenic cells that has been recently and for the first time used on plant tissues by our Lab. The MEA approach allows to record extracellular signals from as many as 60 sites simultaneously, thus examining any distributed/synchronized electrical activity of whole cells and tissue, highlighting possibly the link between the electrical activity and the physiological response to external stimuli such as that of temporary gravity-changes conditions during an ESA parabolic flight campaign. Signals from maize roots were recorded continuously during the flight at 20 KHz rate of sampling frequency. No previous data on the use of MEA system in microgravity condition have been ever reported in literature, neither in human/animal nor in plant physiology. For this reason, one of the main goal was to check the capability of the system to perform continuous monitoring of root electrical activity without troubles or crash during the flights. Correlations between spikes and acceleration were analyzed to investigate the role of gravity changes in the onset of naturally occurring electrical spikes. Gravity data
42 from each experimental day were grouped together into classes, respectively 0g, 1g and 1.8 g. For each “g-class”, spike rate, ISI (inter-spike intervals) and grade of synchronization analysis were performed. Results showed a clear and detectable overall root electrical activity during each experiment, with differences in rates, ISI and synchronized events in correlation with gravity-changes. As a conclusion, the trial was successful, with gravity changes proving to affect spike rate generated by maize root tips.
43 P03
Effect of Mitate Conglomerate on root and shoot growth of Raphanus sativus var. radocula with special notice to
Cu in cultivation medium
Mitsuo Kawabata Faculty of Environmental Engineering, Graduate School, University of Kitakyushu, Kitakyushu, Japan E-mail: [email protected]
We have investigated the effect of Mitate Conglomerate, which is practically used as bedrock bath, on the growth and morphology of plants. Growth and mineral nutrition of shoot of Raphanus sativus var. radicula was estimated as the function of root mass and length. When plants were cultured in the Hoagland solution with grounded rock (Mitate Conglomerate or Serpentine, Silica sand; as control) on the hydroponic condition for fifteen days, the amount of Cu in leaves increased significantly when Mitate Conglomerate was added to solution (p<0.05). Hence, Cu is one of the important elements that affect the growth of the plant interacting with Mitate Conglomerate. Main root elongation increased when Mitate Conglomerate or Serpentine was added to the solution containing 0.1ppm of Cu. Main root elongation didn’t differ significantly with increasing Cu concentration in the solution irrespective of rock type. Dry weight of root wasn’t significantly different between rocks or Cu concentrations. Leaf-root ratio of dry weight decreased with increase in Cu concentration in the solution with Mitate Conglomerate. Consequently, mass growth of leaf to unit root mass was inhibited when Mitate Conglomerate was added in the solution under 0.1ppm of Cu concentration.
44 P04
Continuous GABA supply to the root affects nitrate uptake in Brassica napus seedlings
Antonin Leblanc 1, Hugues Renault 2, Philippe Laîné 1, Carole Deleu 2 and Erwan Le Deunff1 1 INRA UMR 950 / Université de Caen Basse-Normandie «Ecophysiologie Végétale et Agronomie, nutritions N, C, S », Esplanade de la Paix, 14032 Caen cedex, France 2 INRA UMR 118 / Agrocampus Rennes / Université de Rennes 1 « Amélioration des Plantes et Biotechnologies Végétales », Campus de Beaulieu, Bât. 14A, 35042 Rennes cedex, France E-mail: [email protected]
In plants, GABA is a major free non protein amino acid metabolized mainly from glutamate and accumulated in response to various biotic and abiotic stresses. The function of GABA in plants as metabolite or signal is still a matter for debate[1]. Emerging literature suggests that GABA may function in plants as a potential modulator of ion transport and consequently mineral acquisition via putative GABA responsive receptors [2]. In this respect, recent data have shown that acute treatment of GABA up regulates transcription and activity of nitrate transporters in Brassica napus L. [3]. In order to quantify and to differentiate the signaling effect of GABA on nitrate uptake and its N nutritional effect via its g amino group, we have used 15N labeled nitrate and GABA. Rape seedlings were submitted to a continuous application of GABA during five days on agarose gel. Our results show that the supply of 0.1 and 1mM GABA to the root did not affect elongation of exploratory root system compared to KN03 treated plants (control). However, 1mM GABA treatment decreased nitrate uptake. Similar results obtained with Glutamate suggested that GABA and glutamate act as important N sources and raised the question of a putative co-regulation of N uptake via organic or mineral sources in long term experiments. [1] Bouché N, Fromm H (2004) GABA in plants: just a métabolite? Trends in Plant Science, 9(3): 110-115. [2] Kinnersley AM, Turano FJ (2000) Crit. Rev. Plant Sci., 19: 479-509 [3] Beuve N, et al. (2004) Plant Cell Env., 27: 1035-1046
45 P05
Over-accumulation of GABA affects development of
Arabidopsis thaliana
Hugues Renault 1, Erwan Le Deunff 2, Abdelhak El Amrani 3 and Carole Deleu 1 1 INRA UMR 118/ Agrocampus Rennes / Université de Rennes 1 « Amélioration des Plantes et Biotechnologies Végétales », Campus de Beaulieu, Bât. 14A, 35042 Rennes cedex, France 2 INRA UMR 950/ Université de Caen Basse-Normandie «Ecophysiologie Végétale et Agronomie, nutritions N, C, S », Esplanade de la Paix, 14032 Caen cedex, France 3 CNRS UMR 6553/ Université de Rennes 1 « EcoBio », Equipe Mécanismes à l’Origine de la Biodiversité, Campus de Beaulieu, Bât. 14A, 35042 Rennes cedex, France E-mail: [email protected]
Gamma-aminobutyric acid (GABA) is a non-protein amino acid found in all kingdoms, from bacteria to animals, known to accumulate in plants in response to a wide range of environmental stimuli [1]. Nevertheless, its roles are still unclear whereas it has been associated to several physiological processes. These role range from regulation of cytosolic pH and C/N balance, protection against oxidative stress, deterrence of insects, osmoregulation and anaplerotic role. In Mammals, GABA plays a major role in the central nervous system as a neurotransmitter and is involved in the establishment of brain networks [2]. The signalling role of GABA in plants has also been previously demonstrated, notably in developmental processes and metabolic regulation. Indeed, GABA is a key component in the growth and orientation of the pollinic tube [3], it is also involved in the down-regulation of the expression of several 14-3-3 genes [4]. Moreover, a negative correlation has been found between the GABA content of the phloem sap and the expression of nitrate transporters in Brassica napus [5]. To study the impact of GABA on Arabidopsis thaliana development, we used pop2 mutants impaired in GABA catabolism as systems that over-accumulate the molecule when subjected to exogenous GABA treatment. Over-accumulation of GABA led to abnormal phenotype. Main physiological traits affected will be presented.
46
[1] Kinnersley AM, Turano FJ (2000) Crit. Rev. Plant Sci., 19: 479-509 [2] Represa A, Ben Ari Y (2005) Trends Neurosci., 28 : 278-283 [3] Palanivelu R, et al. (2003) Cell, 114: 47–59 [4] Lancien M, Roberts MR (2006) Plant Cell Environ., 29: 1430–1436 [5] Beuve N, et al. (2004) Plant Cell Env., 27: 1035-1046
47 P06
Reduced glutathione (GSH) regulates nitrate uptake in winter oilseed rape (Brassica napus L. cv Capitol)
Nicolas Dumez, Echarcki Zerif, Erwan Le Deunff and Philippe Lainé INRA UMR 950 / Université de Caen Basse-Normandie Ecophysiologie végétale, Agronomie et nutritions N, C, S, Esplanade de la Paix, 14032 CAEN cedex, France E-mail: [email protected]
Many reports have shown that sulfate assimilation is well coordinated with the assimilation of nitrate and carbon. It is well known that the capacity to reduce nitrate is decreased in plants starved for sulfate. In this study, glutathione (GSH) has been proposed to be the signal as interconnecting N and S metabolism and more precisely, - 2- the signal peptide regulating NO3 and SO4 uptake in winter oilseed rape (Brassica napus L.). Experiments have been undertaken to study the effect of an exogenous supply of GSH on HATS activity and the root growth in plants deprived or supplied with sulfate. Our results revealed a closed relationship (R2=0.97) between the total N 2- and the total S amounts in plants supplied with SO4 (N/S = 4.3). HATS activity is slightly decreased (approximately by 10 %) by a deprivation of sulfate during the first four days of deprivation. A supply of GSH (1 mM) decreases the HATS activity by 24 and 35% in plants supplied and deprived of sulfate, respectively. A supply of different concentrations of GSH has also been tested on the exploratory root system during seven days of treatment in agarose gel. Low concentrations of GSH (≤ 100 µM) increases the growth of lateral roots whereas a high concentration (1mM) limits the root growth. - From all these data, it can be hypothesized that glutathione could regulated NO3 uptake by exerting negative feedback on HATS activity or by controlling the exploratory root system in function of sulfate availability in soil.
48 P07
A roles of salicylic acid in UV-C induced cell death signaling
Takuya Hiramatsu 1, Takashi Kadono 2, Takashi Yuasa 2 and Tomonori Kawano 1 1 Graduate school of Environmental Engineering, The University of Kitakyushu, Fukuoka, Japan 2 Kyushu University, agricultural department, Fukuka, Japan E-mail: [email protected]
UV-C damages the plants by targeting the DNA, photosynthetic apparatus, a wide variety of physiological processes and generation of reactive oxygen spices (ROS). Our previous study in tobacco cell cultures (Nicotiana tabacum L.; cell line, Bel-B and Bel-W3) were suggested that involvements of ROS and Ca signaling in UV-C-induced cell death. To be analyzed UV-C induced gene expression of Bel-W3 by RT-PCR showed that salicylic acid related gene expression induction. There, to elucidate a role of salicylic acid in UV-C-Induced cell death, the experiments were performed using Arabidopsis thaliana (Ecotype, Columbia) which are present many mutant and transgenic cells(sid2,nah G,npr1,NPR OVER,cpr1,cpr5).Date suggested that UV-C induced cell death is inducted by activation of salicylic acid signaling but SID2 and NPR1 is not related. But NPR OVER was suppressed UV-C induced cell death. There, we preformed analyzing of UV-C induced gene expression by RT-PCR in NPR OVER and nah G.
49 P08
Simple and sensitive bioassays for monitoring of night time ozone in the air using model plant seedlings
Shinpei Etoh, Ken Yokawa, Tomoko Kagenishi, Takuya Hiramatsu and Tomonori Kawano Graduate school of Environmental Engineering, The University of Kitakyushu, Fukuoka, Japan E-mail: [email protected]
We have developed a simple and sensitive bioassay model for monitoring of low concentration ozone in the air nusing model plant seedlings of Bel-B (ozone-tolerant, as the reference) and Bel-W3 (ozone-sensitive, as the probe) tobacco varieties. This system is shown to be responsive to low ozone exposure level (0.08 - 0.1 ppm*h). This assay system was applied for monitoring of night time ozone in the air generated through photochemical reactions during daytime in Japan and Korea.
50 P09
Expression Analysis of Trehalose Biosynthesis Related
Genes in Tomato
Takashi Kadono, Takashi Yuasa and Mari Iwaya-Inoue Department of Plant Resources, Faculty of Agriculture, Kyushu University E-mail: [email protected]
Trehalose, a nonreducing disaccharide in which two glucose molecules are connected in an alpha-1,1-glycosidic linkage, is considered to be an important osmoprotectant that has unique abilities that protect biomolecules from environmental stresses in many organisms, such as bacteria, fungi, lichens, algae and invertebrates. In the plant, since endogenous trehalose levels are very low, the role of trehalose biosynthesis genes, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), were not fully understood. The recent investigation has implicated these genes in important modulators of plant development, e.g. embryogenesis in Arabidopsis and formation of inflorescence architecture in maize. In this study, expression pattern of trehalose biosynthesis related genes in tomato development was investigated by RT-PCR with a set of primers designed from EST data base, MiBASE (http://www.kazusa.or.jp/jsol/microtom/index.html). The 11 trehalose biosynthesis related genes were found by homology analysis for Arabidopsis. As result of expression analysis by RT-PCR, AtTPSs and AtTPPB homolog showed ubiquitous expression. However, AtTPPA homolog expressed during germination, early seedling and fruit formation. In addition, the expression of AtTPPA homolog in the tomato suspension cultured cells was not observed, while almost TPS homologs and AtTPPB homolog were detected. These results indicated that AtTPPA homolog gene is involved in specifically early stage of tomato development and fruit formation.
51 P10
Plant mitochondrial porin regulates defense response against bacterial pathogen and Bax-induced cell death
Chika Tateda, Tomonobu Kusano and Yoshihiro Takahashi Graduate School of Life Sciences, Tohoku University, Sendai, Japan E-mail: [email protected]
Mitochondrial porin, also called voltage-dependent anion channel (VDAC), is a major integral protein of the outer membrane. It is well documented that the protein plays an important role in apoptosis, a kind of programmed cell death, in mammalian system. However, little is known about the role of the plant counterpart during the process of plant-specific cell death such as pathogen-induced hypersensitive response (HR). To address this issue, we isolated three VDAC full-length cDNAs (NtVDAC1-3) from Nicotiana tabacum L. The deduced products, NtVDACs, share 78-85% identity and retain the conserved eukaryotic mitochondrial porin signature (MPS) distal to their C-terminal regions. Mitochondrial localization of three NtVDACs in plant cells was confirmed via a green fluorescent protein fusion method. We further evidenced that the MPS motif is partially responsible for this organelle targeting, because NtVDAC1ΔMPS, eliminating the MPS motif from the intact protein, localized not only to mitochondria, but also to other cellular spaces. After analysing these basic characteristics of the plant VDACs we addressed the main issue concerning pathogenesis relation. The N. benthamiana orthologues of NtVDACs were up-regulated by challenge with the non-host pathogen Pseudomonas cichorii, but not after challenge with the virulent pathogen P. syringae pv. tabaci. Both the pharmaceutical inhibition of VDAC and silencing of NbVDACs genes compromised the non-host resistance against P. cichorii. Involvement of NbVDACs in the mouse Bax-induced cell death was also suggested with a similar approach.
52 P11
Oxidative stress and distortion of calcium signaling by ions of group 13 elements in tobacco cells
Cun Lin1, Izumi C. Mori2 and Tomonori Kawano1
1 Faculty of Environmental Enginnering, The University of Kitakyushu, Kitakyushu, Japan; 2 Research Institute for Bioresources, Okayama University, Kurashiki, Japan E-mail: [email protected]
The group 13 metal elements including Aluminum (Al), Gallium (Ga) and Indium (In) were used for semiconductor industry. Recently, toxicity of the group 13 metal to animals and plants has been reported. On the other hand, Al is main factor which inhibit the plant growth in the acid soil. Previously study shown that Al was induced ROS such superoxide anion - (O2 ) generation and involve in calcium signaling response to various environmental stresses. Furthermore, many trace metal elements shown - inhibition effects on Al induced O2 generation in plant cells (not publish date). In this study, we analysis the group 13 metal elements induced cell death, - O2 generation and change of cytosolic calcium concentration using tobacco cells (Nicotiana tabacum L.) expressing a Ca2+-monitoring luminescent protein - aequorin. Result in that all elements used here shown cell death and O2 generation induction. Cell death induced by the group 13 metals was inhibited by ROS scavenger such as systeine and dimethylthiourea (DMTU) in any case. Whole Al 2+ and Ga were induced [Ca ]c increase and EGTA shown inhibit effects against Al and 2+ Ga induced cell death. In dose not induce [Ca ]c increase and EGTA did not show inhibit effect against In induced cell death. We also investigated the effects of some - trace metal elements that inhibited the Al induced O2 generation against Al induced cell death.
53 P12
Computational simulation of the plant cell responses to microbial physiologically active substances: principles and ongoing approaches
Ken Yokawa 1, Tomonori Kawano 1 and François Bouteau 2 1 Graduate school of Environmental Engineering, The University of Kitakyushu, Fukuoka, Japan 2 Electrophysiologie des Membranes, EA 3514, Université Paris VII, 2 place Jussieu, 75005 Paris, France E-mail: [email protected]
NEURON, a well recognized free software, has been developed specifically for rapid simulation reflecting the status of the nerve cells using the equations used in the electrophysiological area. This approach enables the simulation of the cellular events by building a model based on the known or expected activities of ion channels, pumps, carriers and other biochemical components (or events) involved in the cell signaling. By using NEURON, various factors such as characteristics of ion channels and transporters, influx and efflux of ions, production and release of neurotransmitters and other biochemical events involved in cell signaling can be utilized as the parameters. Here, we would like to propose a use of NEURON simulation system applicable to the simulation of the time-dependent changes in cellular signal transduction status in plant cells challenged by various stimuli such as microbial components after defining the parameters based on the actual electrophysiological and biochemical data obtained through experiments (such as voltage-clumping). Here, we would like to describe a likely approach for constructing a reliable artificial plant cell in silico capable of simulating some of the cellular responses.
54 P13
Specific capture of phosphoprotein by immobilized
Zirconium ion affinity chromatography
Hidenobu Mizuki, Tomonori Kawano and Kazuya Uezu Graduate school of Environmental Engineering, The University of Kitakyushu, Fukuoka, Japan E-mail: [email protected]
The development of new efficient methods for highly specific enrichment of phosphorylated proteins and peptides is one of most active research fields in phosphorproteome analysis. Enrichment of phosphorylated proteins and peptides from complex Fig.1 Immobilized Zr(IV) Affinity chromatography peptides mixtures by immobilized prepared by surface template polymerization metal affinity chromatography (IMAC) is a poplular way to perform phosphoproteome analysis. IMAC is originally based on the affinity of the phosphate group with metal ions (usually Fe 3+ or Ga 3+) immobilized on a choromatographic support. However the specificity of those IMAC adsorbents is still not high enough 1). To characterize phosphoproteins more efficiently, it was necessary to develop novel affinity suface with higher selectivity for phosphoproteins. In this study, we focused that interaction between Zirconium phosphonate (Zr-Phos complex) and phosphate groups is strong 2). Taking advatage of strong interaction, we have developed a novel IMAC adsorbent immobilized Zr(IV) on the polymer surface (Zr-Phos IMAC) by surface template polymerization with W/O/W emulsion 3) (Fig. 1). Phosphoric acid oleyl ester (DOLPA), sorbitan monooleate
55 (Span80), divinyl-benzene (DVB), polystyrene, and toluene were employed as the functional monomer, emulsion stabilizer, matrix-forming monomer, porogen, and diluent, respectively. Fig.2 Calculated molecular model for So far, we investigated conformation of the F-Zr-Phosphate complex by using Zr-Phos complex on the polymer surface by MOPAC PM5 method using fluoride ion 4). Fig. 2 shows calculated molecular model for the F-Zr-Phosphate complex. Molar ratio of F-Zr-Phosphate complex is 3：1：3. So we expect that binding site with phosphorylated proteins and peptides exist enough on the polymer surface. Because of the multicoordination effect, the interaction between Zr-Phos complex and phosphopeptide is much stronger. Model phosphoproteins were employed to investigated the performance of a novel Zr-Phos IMAC. The separation performance of the phosphoproteins (Phosphorylase a , phosphorylase b, β-casein, bovine serum albumin (BSA)) was evaluated by using electrophoretic. Phosphoproteins (Phosphorylase a, β-casein) sepalated from complex proteins mixtures at the following condition: Buffer solution (5 mL) is 0.1 M acetic acid/ NaOH, pH 3.1. The concentration of model phosphoproteins is 10 mg/L. We also discussed about phosphopeptide enrichment and MALDI-TOF MS analysis.
References 1. S. Feng et. al., Molecular & Cellular Proteomics, 2007, 6.9, 1656-1665 2. G. Nonglaton et. al., J. Am. Chem. Soc, 2004, 126, 1497-1502. 3. K. Uezu et. al., Macromolecules, 1997, 30, 3888-3891. 4. H. Mizuki et. al., Proceedings of the Japan-Taiwan Joint Symposium on Environmental Science and Technology: Environmental chemistry, bioscience and managements, pp 129-131, 2007
56 P14
Quantum analysis of interaction between fungal polysaccharide and single polynucleotide
Tadashi Okobira 1, Kentaro Miyoshi 1, Kazuya Uezu 1, Kazuo Sakurai 1 and Seiji Shinkai 2 1 Chemical Processes and Environments, Kitakyushu University, Kitakyushu, Japan 2 Chemistry and Biochemistry, Kyushu University, Fukuoka, Japan E-mail: [email protected]
β-1,3-D-glucans (polysaccharide) have been isolated from fungi as right-handed 61 triple helices, in which the constituting three glucan chains are underpinned with one another by intermolecular hydrogen bonds. Among these polysaccharides, Curdlan is very simple structurally since it only contains linear 1,3-linked repeat units with no 1,6-linked side chain glucosyl units. Recently, we found that when the single chain of these is renatured together with a homo nucleic-acid [ex. poly (cytidylic acid): poly(C), poly (adenylic acid): poly(A), and poly (deoxyadenylic acid): poly(dA)], the polysaccharide and polynucleotide chains form a macromolecular complex, instead of forming the polysaccharide triple helix. We examined the structure of the poly(C)/polysaccharide (poly(C)/Curdlan and poly(C)/Schizophyllan) complex by the semi-empirical molecular-orbital package (MOPAC).
57 P15
Molecular Dynamics Studies of Side Chain Effect on the
Microbial Polysaccharides Triple Helix in Aqueous
Solution
Kazuya Uezu 1, Tadashi Okobira 1, Kentaro Miyoshi 1, , Kazuo Sakurai 1 and Seiji Shinkai 2 1 Chemical Processes and Environments, Kitakyushu University, Kitakyushu, Japan 2 Chemistry and Biochemistry, Kyushu University, Fukuoka, Japan E-mail: [email protected]
β-1,3-D-glucans have been isolated from fungi as right-handed 61 triple helices. They are categorized by the side chains bound to the main triple helix through β-(1→ 6)-D-glycosyl linkage. Indeed, since a glucose-based side-chain is water-soluble, the presence and frequency of glucose-based side-chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side-chains, self-assembles to form an water-insoluble triple helical structure while Schizophyllan, which has a 1,6-D-glucose side chain of every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side-chains and water molecules. Our recent research reported on the formation of stoichiometric complexes of Schizophyllan with polynucleotides experimentally and computationally. We simulated three kinds of β-1,3-D-glucan: Curdlan; a hypothetical glucan with a side-chain at every sixth glucose unit; and Schizophyllan. And we investigated how water molecules interact to glucan side chains and affect the glucan structure in aqueous solution at room temperature.
58 P16
A Dynamic Measurement Technique Characterizes the
Turgor pressure Change in Characean Internodal Cells: the Hydraulic Conductivity of the Plasma Membrane is
Isotropic and Independent of the External Hydrostatic
Pressure
Koreaki Ogata Brain Science and Engineering, Kyusyu-institute of Technology, Kitakyusyu, Japan E-mail: [email protected]
Assuming that the magnitude of the peak-to-peak difference in strain △Sn caused by an external constant sinusoidal stress is proportionate to the turgidity of the material, I applied this method, a dynamic measurement technique, to characterize the relative turgor pressure P of cylindrical Characean internodal cells as a function of time. In contrast to the ordinary transient methods, the present method “vibro-method”, is not only simpler than any of those introduced by other authors, but also enables the measurement of P without being invasive to the living cell, with the temporal resolution of 100msec. The cell length l and diameter d could be measured simultaneously in the present apparatus. The resolution estimated is 1μm or less. A rectilinear correlation was found between l and △Sn within at least 5 sec immediately after a step change in external hydrostatic pressure πe: the Young’s modulus along the length of the cell can be assumed to be constant in a short term study. On the other hand, d was found to be practically unchanged during this period, suggesting that the absolute volume of water flow across the membrane can be estimated by changes in l, △l. Therefore, the velocity of water flow across the membrane V can be explained thus: V = d△Sn / dt.
Consequently, because the change in intracellular hydrostatic pressure πi is negligibly small, the hydraulic conductivity of the plasma membrane L p can then be calculated as:
L p= V / (πi -πe )= V /△π. Results suggest there is no evidence to prove the plasma membrane itself is a
59 rectifier of water volume flow, but that the apparent rectification is due to the mechanical properties of the cell wall and/or to the change in water motive force across the membrane. Furthermore, there is no evidence to prove a change in L p in response to
πe up to 1.5MPa within at least 5 sec immediately after the onset.
60 P17
The relationship between vegetational succession and water environmental change in a warm-temperate, volcanic peat mire in south-western Japan
Yoshiumi Shinohara 1, Keiji Matune 2, Ayumi Nakazono 1 and Tutomu Iyobe 1 1 Faculty of Environmental Engineering, Graduate School, University of Kitakyushu, Kitakyushu, Japan 2 National Federation of Agricultural Cooperative Associations (Zen-Noh Group), Tokyo, Japan E-mail: [email protected]
The relationship between vegetational succession and water environmental change was investigated in Tadewara mire, in a warm-temperate, volcanic peat mire in south-western Japan. Water chemical environment are monitored every month from August 2006 to July 2007 and changes in species abundance at the correspondence sites were investigated. Vegetational group classified by TWINSPAN did not correspondence sites grouped by water chemistry using cluster analysis. Then we tested the correlation between changes in species abundance and the corresponding environmental change within the investigation period.Abundance of Moliniopsis japonica (Hack.) Hayata show significant positive correlation Sphagnum fimbriatum Wils. in Hook. , Hydrangea paniculata Sieb. et Zucc. , and Phragmites australis (Cavanilles) trinius ex steudel. Abundance of M. japonica show significant negative correlation S. palustre. Abundance of S. fimbriatum show significant positive correlation M. japonica, H. paniculata, and P. australis. Abundance of S. fimbriatum show significant negative correlation S. palustre. Abundance of H. paniculata decreased at sites with low Mn concentration. Abundance of S. fimbriatum decreased at sites with low Na+ concentration. Change in abundance S. palustre increased at sites with low Mg2+ and low Ca2+ concentration. H. paniculata 2+ 2- decreased at sites with low pH, Mg , SO4 , TN, Fe and low Mn concentration.
61 P18
Hydrological Control of River Systems
Enen Ryu 1, Chiharu Ikeda 2, and Tutomu Iyobe 1 1 Faculty of Environmental Engineering, Graduate School, University of Kitakyushu, Kitakyushu, Japan 2 SEIKAN CO., LTD., Fujieda, Japan E-mail: [email protected]
Many researches on hydrological control by vegetation have been reported. Over grown vegetation, however, had negative effect on river management because the vegetation is removed by high water level during the flooding, and consequently blocked the river channel. In this research, we evaluated the response of Pharagmites japonica Steudel community in the Iwatake River to flow rate and water table at various flooding condition, we measured the vegetation density and flow rate distribution in a P. japonica community, and then calculate the threshold of community resistance to increasing flow rate. In situ density of P. japonica was 76‐106 m-2, and column diameter was 0.4‐0.6 cm in the Iwatake River and the estimated threshold of vegetation resistance to flow rate and water table was 0.45‐0.6 ms-1 and 80－100 cm respectively.
62 P19
Effect of volcanic activity on mire vegetation in
Tadewara mire, southwest Japan
Ayumi Nakazono and Tsutomu Iyobe Faculty of Environmental Engineering, Graduate School, University of Kitakyushu, Kitakyushu, Japan E-mail: [email protected]
Peat stratigraphy and macrofossil composition of three – 210 cm, 270 cm, 420 cm peat cores obtained from Tadewara mire (Ooita) were investigated to clarify the relationship between volcanic activity and vegetational change with special reference to chemical deposition from volcano. Tow distinct horizons consist of volcanic glass were observed at 160 and 252 cm depth, however most layers contained volcanic glass implying frequent impact of volcanic activity on Tadewara mire. Distinct peak of sulfur content in soil core was found from depth at 110 cm by elemental analysis of peat soil. Content of carbon, nitrogen, and hydrogen decreased corresponding to the increase of sulfur. Dominant species of macrofossil community started to change from Sphagnum SPP. to Phragmites australis just corresponding to the increase of sulfur at 110 cm. By 14C dating, increase in sulfur content started at 970±40 y.B.P and it just corresponded to the peak of volcanic activity of mount Kurotake near by Tadewara mire. Thus we obtained the evidence that mire vegetation changed from ombrotrophic to minerotrophic community by sulfur deposition due to the volcanic activity.
63 P20
Inhibition of copper-induced calcium influx by prion- drived peptide in suspension-cultured tobacco cells
Tomoko Kagenishi, Ken Yokawa and Tomonori Kawano Graduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu 808-0135, Japan E-mail: [email protected]
Recently, we have been engaged to the designing of artificial peptides for protection of the plant cells from various metal stresses. Copper is known to induce an 2+ increase in cytosolic fee calcium concentration ([Ca ]c) in cultured tobacco cells (Inoue et al. 2005). Since our recent reports suggested that the human prion protein-derived copper-binding peptide functions as the chelators and the catalysts of generating the reactive oxygen species such as superoxide anion radical (Kawano, 2007), we expected the effects of such peptides as modulators of copper toxicity. Here, we examined the effects of copper-binding peptides derived from prion on the action of copper in n tobacco BY-2 suspension culture expressing aequorin gene. Addition of copper resulted 2+ in rapid and transient increase in [Ca ]c. When the cells were pre-treated with a prion-derived copper-binding peptide fragment (corresponding to the neurotxic region), 2+ the copper-induced [Ca ]c was effectively lowered. While the copper-sequestering action of prion neurotoxic peptideis likely attributed to the chelating activity of the neurotoxic peptide, other prion-derived copper-binding peptides (including those corresponding to octa-repeat region and helical region) were shown to be less active in sequestering of the copper action. Further examinations are required for designing the peptidic or biochemical agents for protection of plant cells from metal toxicity.
Inoue, H., T. Kudo, H. Kamada, M. Kimura, I. Yamaguchi and H. Hamamoto (2005) Copper elicits an increase in cytosolic free calcium in cultured tobacco cells. Plant Phyol Biochem. 43(12): 1089-1094. Kawano, T. (2007) Prion-derived copper-binding peptide fragments catalyze the generation of superoxide anion in the presence of aromatic monoamines. Int. J. Biol. Sci. 3(1): 59-65.
64 P21
Possible use of green paramecia in development of photo-controlled micro-particle transport system
Chiaki Karaki, Furukawa Shunsuke and Tomonori Kawano Graduated Shool of Environmental Enginnering, The University of Kitakyusyu, Kitakyusyu, Japan E-mail: [email protected]
Paramecium bursaria is known to harbor endosymbiotic algae in its cytoplasm, and responds to light stimuli to show photoaccumulation or photoavoidance as the consequences of phototactic cellular migration in either positive or negative manner. Early studies indicated that P. bursaria has rhodopsin-like protein as a photoreceptor in plasma membrane. On the other hand, calcium ion (Ca2+) is known as one of the important regulatory elements for ciliary movements. However, the mechanism for photosensory signal transduction of P. bursaria is not fully understood to date. In this study, the migration by P. bursaria irradiated with ultraviolet lights (UV-A and UV-C) in fine capillary tube was demonstrated. Both UV-A and -C induced the negative phototaxis while UV-C is lethal and UV-A is not harmless. The UV-C-dependent phototaxis was performed in the presence of various pharmacological inhibitors. The data suggested that Ca2+ signaling (sensitive to flunarizine, a T-type Ca2+/Na+ channel inhibitor but insensitive to tetrodotoxin, a Na+ channel inhibitor) is required for the light-dependent cell migration. However, despite of expectation, the signaling mechanism is likely free from phosphorylation events (thus insensitive to KT5720, staurosporine and K252a) and G-protein-mediated sensing (thus insensitive to cholera toxin and pertussis toxin).
65 P22
Development of micro-particle transport system using galvanotactically migrating green paramecium
Shunsuke Furukawa, Chiaki Karaki and Tomonori Kawano Graduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu, Japan E-mail: [email protected]
It is well known that protozoan species including Paramecium species towards the cellular movements to anodic electrode when exposed to the electric field in the medium. This phenomenon is known as the galvanotaxis. We have developed a system for controlled transportation of the microparticles packed in the cells of a ciliate (Paramecium bursaria, pre-conditioned for maximizing the particle-loading capacity) migrating inside the capillary tubes (φ, 2 mm) by galvanotaxis. We succeed in controlled cell migration by galvanotaxis in the complexed routes made of fine capillaries. Since the demonstration was successful, we could conclude that P. bursaria has a potential to be used as one of the micro-biorobotic devices.
66 P23
Response to the presence of glucose depending on
NDHD in Synechocystis sp. PCC6803
Yuta Jin, Yuto Tamura and Hiroshi Ohkawa Faculty of Agriculture and Life Science, Hirosaki University, Japan
Cyanobacteria have several growth characteristics and are able to grow under photoautotrophic, photomixotrophic and heterotrophic conditions. However, little is known about the mechanisms how cyanobacteria respond to the presence of glucose in their environment. Moreover, while glucose sensitive (GS) and glucose tolerant (GT) strains of Synechocystis sp. PCC 6803 are available, the mechanisms distinguishing their response to glucose are poorly understood. Synechocystis sp. PCC6803 genome contains multiple copies of ndhD and ndhF genes. Analysis of these double mutants in a pair of homologous have been reported that there were functionally distinct multiple of NAD(P)H dehydrogenase complexes
(NDH-1) in GT strains. One is classified into CO2 uptake NDH-1, which include
NdhD3 related to low CO2 induced CO2 uptake system and NdhD4 related to constitutive CO2 uptake system. There were the distinct role between NdhD3 and NdhD4. On the other hand, the other is classified into cyclic electron transport and respiration NDH-1, which include NdhD1 or/and NdhD2. However, mechanisms of the response to glucose in these mutant cells have not been poorly clarified. To clarify response to the presence of glucose depending on NDH-1 in Synechocystis sp. PCC6803, we have constructed each single disruption mutant in GS strains. Both ndhD1 and ndhD2 mutant shows higher sensitive to glucose than GS strains, but ndhD3 and ndhD4 mutant did not show significant differences. Analysises of these mutants are in progress.
67 Satelite Session 1
Abscisic acid-induced anion currents activation mediated by cyclic ADP-ribose / ryanodine receptor
(RyR) in Arabidopsis thaliana suspensions cells
G. Vidal1, K. Madiona1, P.Meimoun1, A Lehner, J.Briand1, F, Kawano T12., Bouteau1, JP Rona1. 1 Electrophysiologie des Membranes, EA 3514, Université Paris VII, 2 place Jussieu, 75005 Paris, France. 2Graduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu, Japan.
2+ 2+ The cytosolic Ca activity [Ca ]cyt controls essential and multiple animal and plant cellular processes, including the plant growth regulator abscisic acid (ABA) 2+ signal transduction. Increase of [Ca ]cyt, is the major trigger for ABA-induced anion currents activation in Arabidopsis thaliana suspension cells. (Brault et al., 2004; Zalejski et al., 2006).
We previously shown that ABA-induce H2O2 production triggers an increase in 2+ [Ca ]cyt and participates to the fast depolarization of the plasmalemma, due to anion 2+ channel activation. H2O2 generation was shown to trigger the second of the two [Ca ]cyt increases observed in response to ABA, probably by promoting extracellular Ca2+ entry 2+ since addition of EGTA impaired this H2O2 generated [Ca ]cyt increase. In addition, L, N and T-type Ca2+ channel inhibitors, fluspirilene (50 µM), pimozide (50 µM) and EGTA (5mM) considerably altered ABA induced RD29a expression. These first results support the idea that the calcium mobilized by H2O2 can at least has an extra-cellular origin (Ghelis et al., 2000).
2+ [Ca ]cyt is also critically affected by the release from intracellular stores (vacuole, reticulum) which are controlled by two major channel/receptor complexes, the inositol trisphosphate receptor (IP3R) and the cADP-ribose/ryanodine receptor (RyR). Here, we report that ABA-Signaling mechanism implies the regulation of vacuolar Ca2+ release.
68 2+ [Ca ]cyt oscillation signatures may be controlled through the balance of processes of Ca2+ influx and release into the cytosol against those of Ca2+ sequestration and elimination. The major approach to the study of the RyR’s role has been to use the inhibitory effects of drugs effective on IP3-mediated Ca2+ increase. Previous studies have indicated that the IP3-mediated Ca2+ signal requires subsequent RyR activity. In A. thaliana suspension cells, we used two tonoplast Ca2+ -receptor/channel inhibitors, (i) 8-bromo-cADP-ribose an antagonist of endogenous cADP-ribose, which induces Ca2+ release from the stores through both ryanodine channel-receptor (RyR) and via mechanisms independent of RyR channels, and (ii) Dantrolene an agent which interacts with the ryanodine receptor (RyR) to modulate the channel function inhibiting one part of the vacuolar Ca2+ efflux. Both inhibitors, blocked the release of calcium from the vacuole. 8-bromo-cADP-ribose (100 µM) decrease the ABA-induced activation of anion channels (65%). Dantrolene (100µM) decrease the ABA-induced activation of anion channels (52%) and reduce the expression of ABA-responsive gene (reduction of 20% for RD29a). The finding that 8-bromo-cADP-ribose and Dantrolene can at least partially block 2+ [Ca ]cyt, increase induced anion efflux current in response to ABA, demonstrates that vacuolar Ca2+ is also clearly involved in the ABA-signaling pathways. In conclusion, we show that tonoplast cADPR/ryanodine receptor (RyR) is probably involved in the ABA induce Ca2+ signaling pathways. In addition, our results suggest that ABA activates indirectly several Ca2+ channels at the tonoplast and provide strong evidence that vacuolar Ca2+ release is an important component in the ABA signal transduction pathway.
References 1. Brault M, Amiar Z, Pennarun, AM, Monestiez, M, Zhang, Z, Cornel, D, Dellis, O, Knight, H, Bouteau, F. and Rona, J.-P. (2004) Plant Physiol. 135:231-243. 2. Zalejski C., Paradis S., Maldiney R., Habricot Y., Miginiac M., Rona J.-P., Jeannette E. (2006). Plant Physiol. 141:1555-62 3. Ghelis, T., Dellis, O., Jeannette, E., Bardat, F., Cornel, D., Miginiac, E., Rona, J.-P., and Sotta, B. (2000). FEBS Lett. 474, 43-47.
69 Satelite Session 2
Thaxtomin A-induced defense responses in
Arabidopsis thaliana cells require an early Ca2+ influx
Errakhi R1, Dauphin A1, Meimoun P1, Lehner A1, Reboutier D1, Vasta P2, Briand J1, Rona JP1, Wendehenne D2, Beaulieu C3, Bouteau F1. 1 LEM (EA 3514), Université Paris 7, 2, place Jussieu 75251 Paris cedex 05 France. 2 UMR INRA 1088/CNRS 5184/Université de Bourgogne, Plante-Microbe-Environnement, Dijon, France 3 GRBA, Département de Biologie, Université de Sherbrooke, Québec, Canada JIK 2RI
The pathogenicity of various Streptomyces scabies isolates, involved in the potato scab disease, was correlated with the production of thaxtomin A. Since calcium is known as an essential second messenger associated with pathogen-induced plant responses and cell death, we investigate on Arabidopsis thaliana suspension cells, a convenient model to study plant-microbe interaction, whether thaxtomin A could induce a Ca2+ influx rela]ted to cell death and to other putative plant responses. A. thaliana cells were treated with micro-molar concentrations of thaxtomin A. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements, with the apoaequorine Ca2+ reporter protein and by external pH measurement. Involvement of anion and calcium channels in the signal transduction leading to programmed cell death was determined by using specific inhibitors. Our data suggest that this toxin induces a rapid Ca2+ influx and cell death in A. thaliana cell suspension. Moreover, our data provide strong evidence that the Ca2+ influx induced by thaxtomin A is necessary to achieve this cell death and is a prerequisite to early thaxtomin A-induced responses: anion current increase, alkalization of the external medium and the expression of PAL a key enzyme of the phenylpropanoid pathway.
70 Satelite Session 03
Similarity between plant redox enzymes and copper-bound prion protein
Tomonori Kawano, Ken Yokawa, Tomoko Kagenishi Graduate School of Environmental Engineering, The University of Kitakyushu, Fukuoka, Japan E-mail: [email protected]
Recently, our group has shown that human prion-derived copper-binding motifs (including well characterized octarepeat region and helical region) may be good model for studying the neurotransmitter-dependent oxidative burst in which superoxide is generated while aromatic monoamines such as phenylethylamine and tyramine are oxidized in the presence of trace amount of hydrogen peroxide. The type of reaction revealed was shown to be very much similar to the reactions catalyzed by plant enzymes (peroxidases). This presentation focusses on the mechanisms how prion protein and plant enzymes catalyze the generation of reactive oxygen species. Lastly the consequence of reactions in vivo both in plant and animal systema will be discussed.
71 Satelite Session 04
Simulation of the signal transduction in artificial plant cells using NEURON: Inspired from the artificial retinal model
Ken Yokawa, Tomonori Kawano Graduate School of Environmental Engineering, The University of Kitakyushu, Fukuoka, Japan E-mail: [email protected]
Human retinal tissue has several nerve cell layers and realizes complicated signal processing by communicating within layers. Artificial retina, a bioengineering implement for re-construction of vision by stimulating degenerated-retinal nerve cells directly with electrode, has been studying in many places. However, limitation of experiment of human body prevents the progression of research and the development of these devices. One of solution is that a environment of dry-experimentation for exploring a optimized parameters such as electrical intensity, electrode position and cell responses has been conducted. Number of computational simulators for constructing a mathematical system to monitor the conditions of living cell or organisms has been developed. At the field of neurophysiology, a well recognized free software, NEURON has been highly exploited for calculating the parameters of the nerve cells based on the differential equations. Various factors such as characteristics of ion channels and transporters, influx and efflux of some kind of ions, production and release of neurotransmitters and other biochemical events involved in cell signaling can be utilized as the parameters. NEURON has definitely been used for construction the artificial retinal model. Here, we would like to propose a use of NEURON system for the simulation of cellular signal transduction in plant cells after defining the parameters based on the actual electrophysiological and biochemical data obtained through experiments.
72
LIST OF PARTICIPANTS
73 ARIMURA, Gen-ichiro E-mail: Global COE Program: Evolution and [email protected] Biodiversity, Graduate School of Science, Kyoto University CASSAB, Gladys I. E-mail: Biologia Molecular de Plantas [email protected] Instituto de Biotecnologia Universidad Nacional Autonoma de Mexico AZZARELLO Elisa E-mail: [email protected] Department of Horticulture, University of Florence E-mail: [email protected] ETOH, Shimpei Graduate School of Environmental Engineering BALUSKA, Frantisek The University of Kitakyushu IZMB E-mail: [email protected] University of Bonn E-mail: [email protected] FUKAO, Yoichiro The Graduate School of Biological BEILBY, Mary J. Sciences Biophysics, School of Physics, Nara Institute of Science and Technology The University of NSW E-mail: [email protected] E-mail: [email protected] FUJIWARA, Toru Biotechnology Research Center, The BOLAND, Wilhelm University of Tokyo Max Planck Institute for Chemical E-mail: [email protected] Ecology, E-mail: [email protected] FURUKAWA, Shunsuke Graduate School of Environmental BOUTEAU, François Engineering LEM, Univ. Paris 7 The University of Kitakyushu
74 E-mail: [email protected] HIRSCH, Ann M. Dept. of Molecular, Cell & Developmental Biology GEISLER, Markus UCLA University of Zurich E-mail: [email protected] E-mail: [email protected]
HOSON, Takayuki HARADA, Akiko Biology Department of Biology Osaka City University Osaka Medical College E-mail: [email protected] E-mail: [email protected]
IIDA, Hidetoshi HARAGUCHI, Akira Department of Biology Faculty of Environmental Engineering, Tokyo gakugei University University of Kitakyushu E-mail: [email protected] E-mail: [email protected]
INABA, Takehito HEDRICH, Rainer The 21st Century COE Program, Molecular Plant Physiology and Cryobiofrontier Research Center Biophysics, Biocenter, Iwate University Würzburg University, E-mail: [email protected] E-mail: [email protected] IWATA-INOUE, Mari Kyushu Univ. HIRAMATSU, Takuya E-mail: [email protected] Graduate School of Environmental Engineering The University of Kitakyushu KADONO, Takashi E-mail: Faculty of Agriculture [email protected] Kyushu University E-mail: [email protected]
75 nutritions N,C&S KAGENISHI, Tomoko Universite de Caen Graduate School of Environmental E-mail: [email protected] Engineering The University of Kitakyushu E-mail: [email protected] LIN, Cun The University of Kitakyushu E-mail: [email protected] KARAKI, Chiaki Graduate School of Environmental Engineering MANCUSO, Stefano The University of Kitakyusyu University of Florence E-mail: [email protected] E-mail: [email protected]
KAWABATA, Mitsuo MASI, Elisa Faculty of Environmental Engineering Università di Firenze The University of Kitakyushu E-mail: [email protected] E-mail: [email protected]
MIZUKI, Hidenobu KAWANO, Tomonori The University of Kitakyushu Faculty of Environmental Engineering E-mail: [email protected] The University of Kitakyushu E-mail: [email protected] NAKAZONO, Ayumi Graduate School of Environmental KUCHITSU, Kazuyuki Engineering Applied Biological Science The University of Kitakyusyu Tokyo University of Science E-mail: [email protected] E-mail: [email protected]
OGATA, Koreaki LE DEUNFF, Erwan Y-A. Brain Science and Engineering, UMR INRA 950 Laboratoire Kyusyu-institute of Technology, Ecophysiologie vegetale et agronomie, E-mail:
RYU, Enen OHKAWA, Hiroshi Faculty of Environmental Engineering Faculty of Agriculture and Life Science, The University of Kitakyushu Hirosaki University E-mail: [email protected] E-mail: [email protected]
SCHLICHT, Markus OKOBIRA, Tadashi Department of Plant Cell Biology Chemical Processes and Environments University of Bonn The University of Kitakyushu E-mail: [email protected] E-mail: [email protected]
SHIMAZAKI, Ken-ichiro PONCE-ROMERO, Georgina Department of Biology, Kyushu University Biologia Molecular de Plantas E-mail: [email protected] Instituto de Biotecnologia
E-mail: [email protected]
SHIMMEN, Teruo
Graduate School of Life Science, RENNA, Luciana University of Hyogo University of Florence E-mail: [email protected] E-mail: [email protected]
SHINOHARA, Yoshiumi RONA, Jean-Pierre Faculty of Environmental Engineering Electrophysiologie des Membranes, The University of Kitakyushu Université Paris VII, E-mail: [email protected] E-mail: [email protected]
SUZUKI, Akihiro
Saga University ROSSI, Marika E-mail: [email protected] University of Florence
E-mail: [email protected]
77
TAKABAYASHI, Junji Center for Ecological Research, UEZU, Kazuya Kyoto University Chemical Processes and Environments E-mail: [email protected] The University of Kitakyushu E-mail: [email protected]
TAKAHASHI, Hideyuki Graduate School of Life Sciences UMEMURA, Kenji Tohoku University Agricultural & Veterinary Research Labs E-mail: [email protected] Meiji Seika Kaisha, Ltd. E-mail: [email protected]
TAKAHASHI, Yoshihiro Graduate School of Life Sciences UOZUMI, Nobuyuki Tohoku University Tohoku University, Japan E-mail: [email protected] E-mail: [email protected]
TAMAOKI, Masanori VAN VOLKENBURGH, National Institute for Environmental Elizabeth Studies Biology E-mail: [email protected] University of Washington E-mail: [email protected]
TOYOKURA, Koichi Department of Science YOKAWA, Ken Kyoto university Graduate school of Environmental E-mail: Engineering [email protected] The University of Kitakyushu E-mail: [email protected]
TREBACZ, Kazimierz Department of Biophysics YOSHIOKA, Hirofumi Maria Curie-Sklodowska University Laboratory of Defense in Plant-Pathogen E-mail: [email protected] Interactions, Graduate School of
78 Bioagricultural Sciences, Nagoya University E-mail: [email protected]
YUASA, Takashi Faculty of Agriculture Kyushu University E-mail: [email protected]
79
80 List of PNB2008 Organizing Committee
Members
Symposium Co-chairs Takashi YUASA (Kyushu Univ.), [email protected]
Tomonori KAWANO (Univ. Kitakyushu),
Members (alphabetical order) François BOUTEAU (Univ. Paris 7; Univ. Kitakyushu), [email protected]
Toru FUJIWARA (Univ. Tokyo), [email protected]
Akira HARAGUCHI (Univ. Kitakyushu), [email protected]
Takehito INABA (Iwate Univ.), [email protected]
Mari IWATA-INOUE (Kyushu Univ.), [email protected]
Maki KATSUHARA (Okayama Univ.), [email protected]
Izumi C. MORI (Okayama Univ.), [email protected]
Hiroshi OHKAWA (Hirosaki Univ.), [email protected]
Teruo SHIMMEN (Univ. Hyogo), [email protected]
Akihiro SUZUKI (Saga Univ.), [email protected]
81
Koji TAKAHASHI (Nagoya Univ.), [email protected]
Masanori TAMAOKI (National Institute for Environmental Studies), [email protected]
Kazuya UEZU (Univ. Kitakyushu)(In charge of sponsored sessions), [email protected]
Hirofumi YOSHIOKA (Nagoya Univ.), [email protected]
K. YOKAWA (Univ. Kitakyushu) (Technical staff in charge of web site), [email protected]
82
PNB2008 The 4th International Symposium on Plant Neurobiology Fukuoka, Japan (June 6-9 2008)
Proceedings
5th Symposium on Plant Neurobiology
25 – 29 May 2009 Florence – Italy Programme
Monday 25th May
11:30 – 14:00 Registration
14:30 – 15:00 Stefano Mancuso, Opening and welcome addresses Liz Van Volkenburgh
15:00 - 15:40 Frantisek Baluska Animal-like behavior in plants: avoidance and escape tropisms of roots
15:40 – 16:20 Peter Barlow The influence of the lunar-solar tidal acceleration on trees gives a glimpse of how the plant- neurobiological system came into being
16:20 – 17:00 Markus Geisler Regulation of the auxin export complex
18:00 - 20:00 Welcome drink
Tuesday 26th May
9:30 – 10:10 Aart Van Bel Forisomes: molecular mammoths unveil calcium- associated sieve-element biology
10:10 - 10:40 Clement Thomas Plant LIMs: simple proteins, intricate functions
10:40-11:10 Coffee break
11:10 - 11:50 Miguel Botella The role of plant synaptotagmins in plasma membrane integrity and cell survival
11:50 - 12:20 Narendra Tuteja Cloning and identification of a novel function of pea lectin receptor-like kinase in salinity stress tolerance
12:20 - 12:50 Luciana Renna AGD5, an ARF-GAP, interacts with ARF1 GTPase at the Trans-Golgi Network
12:50 - 14:30 Lunch
14:30 - 15:10 Tomonori Kawano Development of model peptides as the research tools for studying the dynamic signaling events in living plant cells
15:10 - 15:40 Axel Mithofer Plant-insect interactions: General aspects and dissection of mechanical and chemical challenges
15:40 - 16:10 Haiyun Ren AtFH8, an actin filament nucleator and bundler, has a relationship with root development in arabidopsis
16:10 - 16:40 Coffee break
2 16:40 - 17:20 Manuela Flow of nutrients and information in mycorrhizal Giovannetti networks
17:20 - 17:50 Ton Timmers Cellular mechanisms and signal transduction associated with endosymbiotic root infection
Wednesday 27th May
9:30 -10:10 Sergey Shabala GORK and NSCC channels as components of salinity tolerance mechanism in plants
10:10 - 10:40 In Sun Yoon Sucrose nonfermenting1 (SNF1)-related protein kinase 2 (SnRK2) family function in hyper-osmotic stress signaling of rice
10:40-11:10 Coffee break
11:10 - 11:30 Beom-gi Kim Na+ measurements using a fluorescent dye in plant
11:30 - 12:00 Veronique The 7B-1 mutation in tomato confers a blue light- Bergougnoux specific lower sensitivity to coronatine
12:00 – 12:30 Maria-Carmen Changes in cell wall polymers and pectin methyl Risueno esterase expression are developmentally regulated during pollen maturation and embryogenesis
12:30 – 12:50 Jinxing Lin Actin turnover is required for myosin-dependent mitochondrial movements in Arabidopsis root hairs
12:50 - 14:30 Lunch
14:30 - 16:00 Poster session
Thursday 28th May
9:30 – 10:10 Rainer Hedrich Sensory transduction in guard cell
10:10 - 10:40 Mathias Rudi System potentials, a novel electrical long distance Zimmerman apoplastic signal in plants induced by wounding
10:40-11:10 Coffee break
11:10 - 11:50 Patrick H. Masson WDL proteins control root growth behavior and anisotropic cell expansion in Arabidopsis
11:50 - 12:20 Paul Jarvis Genetic analysis of the protein import machinery of Arabidopsis plastids
12:20 - 12:50 Vladimira Both electrical and chemical signals may act in a Hlavackova triggering of rapid systemic responses of tobacco or tomato plants to local burning
3 12:50 - 14:30 Lunch
14:30 - 15:10 Bettina Hause Jasmonates functions in symbiotic interactions and plant response to wounding
15:10 - 15:40 Cristian Mazars Nuclear calcium signaling in plant cells
15:40 - 16:10 Doreen Apocarotenoids - signaling compounds of Schachtschabel Zygomycetes and plants?
16:10-16:40 Coffee break
16:40 - 17:10 Lorella Navazio Sensing plant symbiotic signals by nitrogen-fixing bacteria
17:10 - 17:40 Yoko Nakamura Chemical Biology of Leaf-movement of Albizzia saman
20:00 Gala dinner
Friday 29th May
9:30 -10:10 Mary Beilby Membrane potential fluctuations in Chara australis: a characteristic signature of high external sodium.
10:10 - 10:40 Francois Bouteau Anion channel activation is an early event in ozone- induced cell death in Arabidopsis cell suspension
10:40-11:10 Coffee break
11:10 - 11:50 Fernando Arabidopsis root movements and symmetry Migliaccio
11:50 - 12:20 Mark Staves Increasing the density of the external medium inhibits and reverses root gravitropism
12:20 – 12:50 Joseph Neumann Panpsychism - past and present
12:50 - 13:00 Closing of the meeting
13:00 – 14:30 Lunch
4 ABSTRACTS
Oral Presentation
Animal-like behavior in plants: avoidance and escape tropisms of roots 10 Baluska, F
The influence of the lunar-solar tidal acceleration on trees gives a glimpse of how 11 the plant-neurobiological system came into being Barlow, PW, Klingele, E, Mikulecky, M
Regulation of the auxin export complex 14 Geisler, M.M., Bailly, A., Sovero, V., Wang, B., Mancuso, S.
Forisomes: molecular mammoths unveil calcium-associated sieve-element biology 15 van Bel, A.J.E., Hafke, J.B., Furch, A.C.U., Will, T., Fricker, M.D., Knoblauch, M.
Plant LIMs: simple proteins, intricate functions 16 Thomas, C, Hoffmann, C, Dieterle, M, Moes, D, Moreau, F, Papuga, J, Tholl, S, Steinmetz, A
The role of plant synaptotagmins in plasma membrane integrity and cell survival 17 Botella, MA, Voigt, B, Jasik, J, Rosado, A, Menzel, D, Salinas, J, Mancuso, S, Valpuesta, V, Baluska, F, Schapire, AL
Cloning and identification of a novel function of pea lectin receptor-like kinase in 18 salinity stress tolerance Tuteja, N, Joshi, A, Vaid, N, Dang, H-Q
AGD5, an ARF-GAP, interacts with ARF1 GTPase at the Trans-Golgi Network 19 Renna L., Stefano G., Baluska F., Mancuso S.
Development of model peptides as the research tools for studying the dynamic 20 signaling events in living plant cells Kawano, T , Yokawa, K , Kagenishi, T
Plant-insect interactions: General aspects and dissection of mechanical and 21 chemical challenges Mithofer A
AtFH8, an actin filament nucleator and bundler, has a relationship with root 22 development in arabidopsis Xue X, Guo C, Lu Q, Zhang C, Ren H
Flow of nutrients and information in mycorrhizal networks 23 Giovanetti M
Cellular mechanisms and signal transduction associated with endosymbiotic root 24 infection Timmers, T, Sieberer, B, Chabaud, M, Genre, A, Fournier, J, Barker, D
GORK and NSCC channels as components of salinity tolerance mechanism in 25 plants Shabala S., Cuin T.A.
5 Sucrose nonfermenting1 (SNF1)-related protein kinase 2 (SnRK2) family function 26 in hyper-osmotic stress signaling of rice Yoon, I.S., Choi, S.R., Nam, M.H., Lee, I.S., Kim, D.Y., Lee, J.S., Byun, M.O.
Na+ measurements using a fluorescent dye in plant 27 Kim B.G.
The 7B-1 mutation in tomato confers a blue light-specific lower sensitivity to 28 coronatine Bergougnoux, V, Hlaváčková, V, Plotzová, R, Novák, O, Fellner, M
Changes in cell wall polymers and pectin methyl esterase expression are 29 developmentally regulated during pollen maturation and embryogenesis Risueno, MC, Barany, I, Prem, D, Rodríguez-Serrano, M, Testillano, PS
Actin turnover is required for myosin-dependent mitochondrial movements in 30 Arabidopsis root hairs Zheng M., Beck M, Müller J., Chen T., Wang X., Baluška F., Logan D.C., Šamaj J., Lin J
Sensory transduction in guard cell 31 Hedrich R.
System potentials, a novel electrical long distance apoplastic signal in plants 32 induced by wounding Zimmermann , M.R., Maischak, H., Mithöfer, A., Boland, W., Felle, H.H.
WDL proteins control root growth behavior and anisotropic cell expansion in 33 Arabidopsis Masson, PH, Jia, G, Perrin, R, Wang, Y
Genetic analysis of the protein import machinery of Arabidopsis plastids 34 Jarvis P.
Both electrical and chemical signals may act in a triggering of rapid systemic 35 responses of tobacco or tomato plants to local burning Hlaváčková, V.
Jasmonates functions in symbiotic interactions and plant response to wounding 36 Hause, B.
Nuclear calcium signaling in plant cells 37 Mazars, C., Xiong, TC., Brière, C., Coursol, S., Mithöfer, A.
Apocarotenoids - signaling compounds of Zygomycetes and plants? 38 Schachtschabel D., Schlicht M., Baluska F., Boland W.
Sensing plant symbiotic signals by nitrogen-fixing bacteria 39 Navazio, L, Moscatiello, R, Squartini, A, Mariani, P
Chemical Biology of Leaf-movement of Albizzia saman 40 Nakamura, Y., Hamamoto, S., Inomata, S., Uozumi, N., Ueda, M.
Membrane potential fluctuations in Chara australis: a characteristic signature of 41 high external sodium Beilby, MJ, Al Khazaaly, S, Walker, NA, Shepherd, VA
6 Anion channel activation is an early event in ozone-induced cell death in 42 Arabidopsis cell suspension Kadono, T, Errakhi, R , Hiramatsu, T, Meimoun, P, Tran, D, Briand, J, Kawano, T, Bouteau, F
Arabidopsis root movements and symmetry 43 Migliaccio, F, Fortunati, A, Tassone, P
Increasing the density of the external medium inhibits and reverses root 44 gravitropism Staves, M P, Heldt, L
Panpsychism - past and present 45 Neumann, J
Epigenetic memory in plant responses to environmental stimuli 46 Shen, W.-H., Berr, A., Gao, J., Meyer, D., Ménard, R., Molitor, A.
Poster
Sieve-element Ca2+ channels link remote stimuli and sieve-tube occlusion in Vicia 47 faba Furch, A.C.U., van Bel, A.J.E., Fricker, M.D., Felle, H.H., Fuchs, M., Hafke, J.B
Short-time effects of coumarin along the maize primary root axes 48 Lupini, A., Sorgonà, A., Araniti, F., Abenavoli, M.R.
Monoterpene-mediated modulations of Arabidopsis thaliana phenotype: effects on 49 stomata, actin-cytoskeleton and on the expression of selected genes Baluška F., Šamajová O., Schlicht M., Braun S., Ulbrich A., Jansen M., Ka Hahn, Kriegs• B., Schulz M.
Effects of acetylcholine on blue-light response of dark-grown Arabidopsis seedlings: 50 nutrition, light quality, and the effect of mutations in the pigment cryptochrome Bisson, MA, Diamond, A
Lectins as determinants of cell recognition in cyanolichens through Peltigera canina 51 Díaz, EM , Sacristán, M , Legaz, ME, Alarcón, B, Vicente, C
Scald-susceptible cultivars of sugarcane promote signalling to induce the synthesis 52 of a virulence factor in Xanthomonas albilineans Blanch, B, Vicente, C, Alarcón, B, Díaz, EM, Quintana, J
Cytology of compatible and incompatible hyphal interactions in arbuscular 53 mycorrhizal fungi Sbrana, C, Giovannetti, M
Lectins as determinants of cells recognition in cyanolichens through Peltigera 54 canina Díaz, EM, Sacristán, M, Legaz, ME, Alarcón, B, Vicente, C
Towards functional characterization of plant class II formins: first lessons from 55 outliers. Cvrčková, F, Oulehlová, D, Grunt, M, Zárský, V
7 Involvement of auxin-binding proteins and auxin in response of maize seedling to 56 blue light Čudejková, M, Pěnčík, A, Rolčík, J, Fellner, M
Light alters plant elongation responses to auxin 57 Fellner, M, Čudejková, M, Juricić-Knezev, D, Plotzová, R, Pěnčík, A, Rolčík, J, Bořucká, J, Vaclová, T, Řehulka, J, Zalabák, D, Bergougnoux, V.
Shotgun proteomics of protein complexes using mass spectrometry. 58 Fukao, Y
Reaction of a light-induced chloroplast movement to local and systemic stimuli and 59 relation to photoinhibition. Naus, J., Hlavackova, V., Rolencova, M.
Blebbistatin inhibits chemotactic response of smut teliospores towards high and 60 middle molecular mass glycoproteins present in sugarcane juices Quintana, J, De Armas, R, Vicente, C, Legaz, ME, Santiago, R, Sacristán, M
Regulation of aquaporin by protein phosphorylation in fruit and flower 61 Shiratake, K, Nakagawa, Y, Sakakibara, I, Miyashita, K, Yamaki, S
Ozone-inducible glycine-rich peptides as plant prions? 62 Yokawa, K, Kagenishi, T, Kawano, T
Role of auxin, auxin-binding proteins and light in the development of maize 63 seedlings Juricić-Knezev, D, Čudejková, M, Pěnčík, A, Rolčík, J, Bergougnoux, V, Fellner, M
Role of acetylcholine in plant cell elongation 64 Fornaciari, S, Anceschi, E, Arru, L
The model of cell recognition in phycolichens through a fungal lectin that binds to 65 an algal ligand could be expanded to cyanolichens. Sacristán, M, Vivas, MP, Díaz, EM, Santiago, R
Scald-susceptible cultivars of sugarcane promote signalling to induce the synthesis 66 of a virulence factor in Xanthomonas albilineans. Blanch, M, Vicente, C, Alarcón, B, Díaz, EM, Quintana, J
System potentials, a novel electrical long distance apoplastic signal in plants 67 induced by wounding Zimmermann, M.R., Maischak, H., Mithöfer, A., Boland, W., Felle, H.H.
Microgravity alters the expression of actin and tubulin genes, of some ROS 68 scavenging factors, and of the auxin transport genes aux1 and eir1 in arabidopsis Tassone, P., Fortunati, F., Meloni, M.A., Pippia, P., Migliaccio, F
Serotonin and melatonin influence somatic embryogenesis in Coffea canephora 69 P.ex.Fr. Ramakrishna, A., Giridhar, P., Ravishankar, G.A
Network connectance and autonomy analyses of the photosynthetic apparatus in 70 tropical tree species from different successional groups under contrasting irradiance conditions. Souza MG; Ribeiro RV, De Oliveira RF, Machado EC
8 β-1,3 glucanase activity as a response to xanthomonas signalling in permeabilized 71 leaf discs from healthy and scald-diseased sugarcane plants Santiago, R, Alarcón, B, Blanch, M, Quintana, J, Legaz, ME
Action of neurotoxic peptide in plant cells 72 Kagenishi, T, Yokawa, K, Kawano, T
The 7B-1 mutation in tomato confers a blue light-specific lower sensitivity to 73 coronatine Bergougnoux, V, Hlaváčková, V, Plotzová, R, Novák, O, Fellner, M
Investigation of BL-mediated de-etiolation in the spontaneous 7B-1 mutant in 74 tomato Bergougnoux, V, Plotzova, R, Fellner, M
Changes in abscisic acid distribution in heat-stressed pepper seedlings 75 Cui, J, Liu, TX, Zhang, ZS
List of Participants 76
9 Animal-Like Behavior in Plants: Avoidance and Escape Tropisms of Roots
Baluska, F 1
1IZMB, University of Bonn, Bonn, 53115 E-mail: [email protected]
In the last two-three decades, plants have been demasked as very sensitive organisms, monitoring and integrating immense amount of abiotic and biotic parametres from their environment. Plants are unique as their development and morphogenesis are plastic throughout their life. Plants retrieve properties of their environment via sensory perceptions which are critical for their survival. Especially light and gravity are essential in this respect. Plants seem to actively experience environment and can both store and retrieve memories in order to drive active life-style evident especially in growing roots. There are several critical situations requiring ‘centralized’ decisions in growing roots, such as, for instance, search for water and avoidance of dangerous soil patches. Although these root decisions are based on information retrieved preferentially at the root cap, they imply some central ‘processor’ which would reliably control the root tropisms. Importantly, any wrong decision at root apices would have detrimental consequences for the whole plant. Recently, dramatic salt-induced modification of root growth direction has been reported which represent new salt-avoidance tropism of root apices. This salt-avoidance behavior of growing root apices might represent an active adaptive mechanism for roots approaching saline areas. Similarly as in the root gravi- and photo-tropisms, polar auxin transport and PIN2 auxin exporter play the central role in this new type of root tropism. It was also shown that root apices recognize in advance dangerous substrate (soil) patches having high aluminium content and avoid them using similar active avoidance root tropism. Interestingly in this respect, root apices exposed to light have been reported to increase their growth rate in association with more active role of PIN2 in driving auxin transport. These responses are mediated by blue-light receptor PHOT1 which is localized to PIN2-enriched synaptic domains in root cortex cells. As roots are evolutionarilly optimised for exploitation of the dark environment in soil, this blue light induced root escape tropism emerge as serious factor affecting in vivo studies using Arabidopsis roots in confocal microscopy. All authors using living roots in confocal microscopy should be aware of this phenomenon and keep this in mind when intrerpreting their data obtained in vivo. Our recent study revealed that neuronal molecules like synaptotagmins are relevant for coping of plant cells with salt stress and these proteins are relevant also for adaptation to cold stress. All these reports document that plant roots have a fine and sophisticated sensory and communicative systems that enable them to dynamically and efficiently cope with rapidly changing environment via animal-like active behavior.
References Baluška F et al. (2004) Biologia 59:9-17. Baluška F et al. (2005) Trends Plant Sci 10:106-11. Baluška F et al. (2009) Acta Nova Leopold, In press Hodge A (2009) Plant Cell Environm, In press Laxmi A et al. (2008) PLoS ONE 3:e1510 Li X, Zhang WS (2008) Plant Signal Behav 3:351-3. Schapire AL et al. (2008) Plant Cell 20:3374-88. Schlicht M et al. (2008) Plant J 55:709-17. Wan Y et al. (2008) Mol Plant 1:103-17.
10 The influence of the lunar-solar tidal acceleration on trees gives a glimpse of how the plant-neurobiological system came into being
Barlow, PW 1, Klingele, E 2, Mikulecky, M 3
1School of Biological Sciences, University of Bristol, Bristol, BS8 1UG 2Institute of Geodesy and Photogrammetry , ETH Hoenggerberg, Zurich, CH 8093 3Department of Biometrics and Statistics, Neuroendocrinology Letters, Bratislava, SK E-mail: [email protected]
”The Universe in which we find ourselves and from which we cannot be separated is a place of Law and Order. It is not an accident, nor chaos. It is organized and maintained by an Electro-dynamic Field capable of determining the position and movement of all charged particles.” H. S. Burr
Two separate and independent systems in plants appear to be affected by the lunar-solar tidal acceleration, δg. The first is the bean leaf (of Phaseolus spp., Canavalia ensiformis) whose ‘sleep’, or nastic, movements inspired the idea of an endogenous physiological ‘clock’. The dilatation cycles (diurnal variations of trunk diameter, δDi) of tree trunks and branches comprise the second system; these cycles have also provoked controversy as to whether or not they are governed by δg (Zürcher et al. 1998). The time courses of the leaf movements of bean plants grown in darkness were recorded in the 1990s by the late Dr Gunter Klein. He proposed that the initiation of rapid upward or downward movements of leaves occurred at times when δg was changing from low to high, or vice versa (Klein 2007). In order to place Klein’s proposal on a firmer footing, we re-examined his original and unpublished data and used the statistical methods of cosinor analysis and cross-‘correlation’ in conjunction with estimates of δg, derived from a geophysical-mathematical program, as they applied to the geographical location and times when leaf movements were recorded (Barlow et al., 2008). Other data were extracted from published literature (notably results obtained by Anthonia Kleinhoonte in the 1930s) in which individual leaf movements (i.e., not averaged results) were recorded at specified times, dates and location (Barlow 2007). In all these cases, temporal leaf turning points coincided with temporal turning points of δg. Published time courses of δDi for trunks of Picea abies (Zürcher et al. 1998) were examined, also with the aid of statistical methodologies (Barlow et al. 2009a, b). The varying values of of δDi of trees placed in darkness were positively correlated with the pattern of δg (the paper of Zürcher et al. 1998 contains an erroneous conclusion in this respect). Analogous data for δDi from trunks of other species of trees showed similar correlations with δg. Illumination seemed to regulate the dilatation cycle, apparently over-riding the effect of lunar-solar gravity. Other physiological features that seem to be affected by lunar gravity are the onset and termination of sapflow, transpiration, and the related rapid rises and falls of electrical potential. All data sources are given in Barlow et al. (2009b). The conclusion drawn from the observations on the two mentioned biological systems is that when plants are screened from ‘strong’, solar-driven environmental influences, ‘weak’, lunar gravitational forces can be perceived and responses initiated. In the particular cases discussed, the lunar tidal accelerations probably regulate hydrostatic pressures within the plant. As commented by Harold Saxton Burr (1945), Professor of Anatomy at Yale University and one of the pioneers of plant bioelectricity, “... since growth in trees is in part a matter of hydration, it may eventually turn out that the effect of the lunar cycle on the growth of the tree is as direct as on the tide level”. How do these conclusions impinge upon ‘Plant Neurobiology’? Burr’s comments above may give us a clue. First, according to the data sets which we analysed, sapflow follows changes in the electrical potential of the young secondary xylem. Second, the stem dilatation cycle seems to be brought about by the rhythmic concertina-like changes to the radial structure of the secondary phloem (Zweifel et al. 2000). These changes could affect, also in a rhythmical manner, the internal pressures within the secondary phloem. As Fensom et al. (1994) have proposed, pressure waves in the phloem might regulate the movements of H+ and K+ in and out of the sieve elements and companion cells. Pressure waves initiated in the phloem may also be transmitted to the secondary xylem via the rays and thereby
11 initiate electrical signalling. And for certain simple algae, Bisson et al. (2006) have reviewed the variations in the electrical potentials of their protoplasts which can be induced by turgor changes (i.e. variations in pressure exerted upon the cytoplast membrane). The variation δDi in trees, driven by δg, may therefore provide a constant and ever-present internal physiological condition by means of which electrical potentials are supported and caused to exhibit rhythms. Cycles of δDi may also be linked with sapflow via internal water movements in the manner proposed by De Pauw et al. (2008) and, indeed, this process may even be re-inforced by the action of ‘muscular’ groups of cells in the bark (Holdheide 1951), such as the phloem fibre sclereids (in Populus, e.g.). Explorations have also begun of the correlations between certain geomagnetic indices (Thule index and Disturbance storm index) and δg, and their link with sapflow. It could be that the lunar tidal acceleration δg is important for ‘focussing’ geomagnetic fluxes upon the Earth’s surface and these fluxes can be perceived by living systems. The effect of the aurora borealis on plant growth is suggestive in this respect [see Lodge (1908) reporting on the work of Karl Selim Lemström]. Perhaps it is not fortuitous that the relationships between sapflow, stem dilatation and electrical potential have been discovered in trees! And it may even be safe to speculate that an autonomic plant ‘neural’ system for the transmission of action potentials developed in the giant progymnosperms which appeared in the mid-Devonian era, 374 million years BP. Before this time, vascular cambium, if present at all, was unifacial and produced secondary xylem only; and this xylem may or may not have possessed electrical variation potentials. However, the progymnosperms (e.g., Triloboxylon hallii) developed a bifacial cambium and were therefore a new cell type – the secondary phloem – could be produced (Scheckler and Banks 1971). Besides enabling more sugars from photosynthesis to be transported between the extremities of the plant, we can speculate that secondary phloem became the prototypic neural channel of plants due to the electrical properties inherent to cells of this type. The development of a ‘neural’ channel might also have been an evolutionary adaptation to the increasing dryness of the primitive soil and provided the means of communicating stress signals which, in the form of action potentials passing from the roots to more remote regions of the plant, became more critical than would have been the case in earlier epochs when the equally large, tree-like Lycopsids flourished in wetter and more steamy environments. As mentioned, the tidal force due to the Moon has been ever-present, even during those earlier epochs when sunlight to the planet was blotted out by clouds of volcanic ash, or when persistent clouds deposited the ages of ice and snow upon the Earth. Life on Earth – ranging from the most ancient archaic forms to the forms of today – evolved always and everywhere in the company of lunar-solar tidal acceleration, δg. Besides the ‘gift’ of a neural system from the Moon to plant life, another gift from the Moon is the ability of organisms to keep time – viz. the clock-like bean leaf movements – and maybe also the ability to anticipate events within the flow of time. Perception of the cosmic lunar δg rhythm has become a fundamental feature of living systems, and has perhaps also become internally assimilated into their physiology. We propose that to the five Aristotelian ‘senses’ should be added the ‘sensing of the passage of time’.
Barlow PW. 2007. Foreword. In: G. Klein – ‘Farewell to the internal clock’. New York, Springer. Pp.vii-xx. Barlow PW, Klingelé E, Klein G, Mikulecký M, Sr. 2008. Leaf movements of bean plants and lunar gravity. Plant Signaling and Behavior 3: 1083-1090. Barlow PW, Střetík J, Mikulecký M, Sr. 2009a. Mutual interplay of gravitational, electrical and geomagnetic potentials influences the sap flow in trees? Abstracts: Conference “Man in his terrestrial and cosmic environment”, Úpice, Czech Republic, May 19-21, 2009. Barlow PW, Klingelé E, Mikulecký M, Sr, Střetík. 2009b. Tree stem diameter and sap flow fluctuates with the lunar tides and maybe with geomagnetic activity. (To be submitted) Bisson MA, Beilby MJ, Shepherd VA. 2006. Electrophysiology of turgor regulation in marine siphonous green algae. Journal of Membrane Biology 211:1-14. Burr HS. 1945. Diurnal potential in the maple tree. Yale Journal of Biology and Medicine. 17:727- 735. De Pauw DJW, Steppe K, De Baets B. 2008. Identifiability analysis and improvement of a tree water flow and storage model. Mathematical Biosciences. 211:314-332.
12 Fensom DS, Thompson RG, Caldwell CD. 1994. Tandem moving pressure wave mechanism for phloem translocation. Russian Journal of Plant Physiology 41:118-130. Holdheide W. 1951. Anatomie mitteleuropäischer Gehölzrinden. In H Freund’s Handbuch der Mikroskopie in der Technik, vol 5/1 Frankfurt, Umschau. pp. 193-367. Klein G. 2007. Farewell to the internal clock. New York, Springer. Lodge O. 1908. Electricity in agriculture. Nature 78:331-332. Scheckler SE, Banks HP. 1971. Anatomy and relationships of some Devonian progymnosperms from New York. American Journal of Botany 58:737-751. Zürcher E, Cantiani M-G, Sorbetti-Guerri F, Michel D. 1998. Tree stem diameters fluctuate with tide. Nature 392:665-666. Zweifel R, Item H, Häsler R. 2000. Stem radius changes and their relation to stored water in stems of young Norway spruce trees. Trees 15:50-57.
13 Regulation of the auxin export complex
Geisler, M.M. 1, Bailly, A. 1, Sovero, V. 1, Wang, B. 1, Mancuso, S. 2
1Molecular Plant Physiology, University of Zurich, Zurich, 8008 2Department of Horticulture, University of Firenze, Firenze, 50019 E-mail: [email protected]
Cellular efflux provided by the independent and interactive action of ABCB/PGP- and PIN catalysts is the rate-limiting step of polar auxin transport. Here, we summarize recent progress of ABCB interaction with immunophilin-like FKBP42, TWISTED DWARF1 (TWD1). Using yeast and in planta BRET (bioluminescent resonance energy transfer) assays, we show that ABCB1-TWD1 interaction is disrupted by synthetic and native auxin transport inhibitors, like NPA and quercetin, leading to inactivation of ABCB1. NPA binds to ABCBs but surprisingly also to the N- terminus of TWD1. As a consequence, auxin fluxes and gravitropism of twd1 roots are NPA insensitive while gain-of-function alleles perform faster bending kinetics. Our data demonstrate that the TWD1 and ABCB1 are key components of the NPA-binding protein complex. Moreover, we suggest a protein-protein interaction feedback loop building the basis for the establishment and control of plastic asymmetric auxin fluxes.
14 Forisomes: molecular mammoths unveil calcium-associated sieve-element biology van Bel, A.J.E. 1, Hafke, J.B. 1, Furch, A.C.U. 1, Will, T. 1, Fricker, M.D. 2, Knoblauch, M. 3 1Plant Cell Biology Research Group, Institute of General Botany, Justus-Liebig-University, Giessen, 35390 2Department of Plant Sciences, University of Oxford, Oxford, OX1 3 RB 3School of Biological Sciences, Washington State University, Pullman, 99164-4236 E-mail: [email protected]
Cutting paradermal windows in leaf veins of Vicia faba allowed in vivo observation of events in intact sieve elements (SEs). In SEs, we found contractile protein bodies (forisomes) of considerable size (10- 100 microns in length), which disperse in response to Ca2+ supply and re-contract after Ca2+ withdrawal. Forisome genes are exclusively expressed in the SE/CC precursors of legumes. Forisomes may possess nanobiological potential as shown by their behaviour in artificial silicate-based sieve tubes. Forisomes have further been used as in vitro indicators for Ca2+-binding capacity of aqueous saliva. Their calcium-binding proteins appear to prevent sieve-tube occlusion in response to wounding (inflicted by insertion of the stylet) and, hence, help to maintain food supply. Presence of forisomes enabled to identify and select SE protoplasts for physiological tests. In these protoplasts, touch and osmotic treatments induced forisome dispersion, most likely as result of changes in calcium concentration. Forisomes also demonstrated Ca2+-induced sieve-tube occlusion in response to remote stimuli and the apparent involvement of Ca2+-channels in long-distance signalling. Passage of an electric potential wave (EPW) often coincided with dispersion of forisomes. The sensitivity of forisome dispersion was dependent on its intracellular location which conforms to an uneven distribution of Ca2+-channels over SEs. Ca2+ channels were found to be located both on the PM and EM cisternae. Both channels may cooperate in amplifying the amounts of calcium released into the SE mictoplasm. The Ca2+ threshold for forisome dispersion may only be reached in the ER interstices, where the forked forisome ends are inserted. That nature and strength of the stimuli were related to forisome behaviour led to a model in which the release of Ca2+-ions during EPW propagation results in various local reactions in SEs and adjacent cells. References Furch ACU, Hafke JB, Schulz A, van Bel AJE (2007) Calcium-mediated remote control of reversible sieve-tube occlusion in Vicia faba. J. Exp. Bot. 58: 2827-2838. Furch ACU, Hafke JB, van Bel AJE (2008) Plant- and stimulus-specific variations in remote- controlled sieve-tube occlusion. Plant Sign. Behav. 3, 858-861. Hafke JB, Furch ACU, Reitz M, van Bel AJE (2007) Isolation of functional sieve-element protoplasts. Plant Physiol. 165: 95-103. Knoblauch M, van Bel AJE (1998) Sieve tubes in action. Plant Cell 10: 35-50. Knoblauch M, Peters WS, Ehlers K, van Bel AJE (2001) Reversible calcium-regulated stopcocks in legume sieve tubes. Plant Cell 13: 1221-1230. Knoblauch M, Noll GA, Müller T, Prüfer D, Schneider-Hüther I, Scharner D, van Bel AJE, Peters WS (2003, 2005) ATP-independent contractile proteins from plants. Nature Materials 2: 600-603; 4:353. Noll GA, Fontanellaz ME, Rüping B, Ashoub A, van Bel AJE, Fische R, Knoblauch M, Prüfer D (2007) Spatial and temporal regulation of the forisome gene for1 in the phloem during plant development. Plant Mol. Biol. 65: 285-294. Pelissier HC, Peters WS, Collier R, van Bel AJE, Knoblauch M (2008) GFP tagging of sieve element occlusion (SEO) proteins results in green fluorescent forisomes. Plant Cell Physiol 49: 1699-1710. Will T., Tjallingii WF, Thönnessen A, van Bel AJE (2007) Molecular sabotage of plant defense by aphid saliva. PNAS 104: 10536-10541.
15 Plant LIMs: simple proteins, intricate functions
Thomas, C 1, Hoffmann, C 1, Dieterle, M 1, Moes, D 1, Moreau, F 1, Papuga, J 1, Tholl, S 1, Steinmetz, A 1
1Laboratory of Plant Molecular Biology, CRP-Santé, Luxembourg, L-1526 E-mail: [email protected]
The LIM domain defines a tandem zinc finger motif that primarily functions as a protein-protein binding interface in eukaryotic cells. In animals, LIM domain-containing (LIM) proteins are abundant and display a wide range of cellular functions as regulators of gene expression, cytoarchitecture, cell adhesion, cell motility and signal transduction. Although LIM proteins are less numerous and diverse in plants, a comprehensive picture of their biological roles only starts to emerge. This presentation highlights central information regarding the biology of plant LIM proteins and discusses the most recent advances in the field. Plant LIM proteins are relatively short (~200 AA) proteins consisting of two LIM domains, an inter- LIM spacer and a C-terminal domain. In the cytoplasm they bind to, stabilize and bundle actin filaments into thick actin cables. Based on its expression pattern and specific responsiveness to regulatory factors such as calcium and pH, one LIM protein subset is expected to play a central role in actin cytoskeleton organization of growing pollen tubes. Another subset of LIM proteins is assumed to respond to mechanical cues by massively accumulating along actin filaments thereby reinforcing their strength via bundle formation. In a transient protoplast system, one LIM protein was found to enhance the activation of a target promoter. A direct interaction between the LIM protein and cis-elements of this target promoter is supported by in vitro data. In conclusion, plant LIM proteins appear as multifunctional proteins that connect the cytoskeleton to the nucleus. We speculate that they serve as biosensors to modulate the actin cytoskeleton organization/dynamics and gene expression in response to abiotic signals.
16 The role of plant synaptotagmins in plasma membrane integrity and cell survival
Botella, MA 1, Voigt, B 3, Jasik, J 3, Rosado, A 1, Menzel, D 3, Salinas, J 4, Mancuso, S 2, Valpuesta, V 1, Baluska, F 3, Schapire, AL 1
1Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, Málaga, 29071 2Department of Horticulture, University of Florence, Sesto Fiorentino, 50019 3Institute of Cellular & Molecular Botany, University of Bonn, Bonn, 53115 4Departamento de Biología de Plantas, CIB-CSIC, Madrid, 28040 E-mail: [email protected]
Calcium dependent vesicular trafficking (CDVT) is involved in many essential physiological processes in animals. Synaptotagmins, proteins containing a transmembrane domain and two C2 domains in tandem, have been identified as key players in CDVT. Plasma membrane of animal cells can rapidly reseal disrupted sites through a tightly regulated CDVT process that is dependent on Synaptotagmin VII. In fact, this process is essential for survival and defective plasma membrane repair produces muscular dystrophies, a diverse group of myogenic disorders characterized by progressive loss of muscle strength and integrity. Despite the importance of plasma membrane repair in animals, this process has not been reported in plants. In our screening for salt hypersensitive Arabidopsis mutants we identified that mutations in the SYT1 gene, that shows homology and conserved domains similar to animal synaptotagmins, produce hypersensitivity to different abiotic stresses by decreasing the integrity of the plasma membrane. This result implicates CDVT as an essential uncharacterized process in plant abiotic stress tolerance. We made an exhaustive analysis of the mutant and a biochemical characterization of the SYT1 protein reporting its Ca2+ and phospholipid binding characteristics. The SYT1 protein is localized predominantly to the plasma membrane, an aspect that is likely to be critical for its function. We also found that the homologous SYT3 gene has partially redundant function with SYT1 as the doble mutant shows decreased plasma membrane integrity than single mutants. Our data indicate that Ca2+ dependent plasma membrane repair mediated by SYT1 and SYT3 is essential for plasma membrane integrity and cell survival.
17 Cloning and identification of a novel function of pea lectin receptor-like kinase in salinity stress tolerance
Tuteja, N 1, Joshi, A 1, Vaid, N 1, Dang, H-Q 1
1Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, Delhi 110067 E-mail: [email protected]
The plant lectin receptor-like kinases are involved in various signaling pathways but their role in salinity stress tolerance has not heretofore been described. Salinity stress is one of the major factors which negatively affects plant growth/productivity and threatens food security worldwide. Based on functional gene-mining assay, we have isolated 34 salinity tolerant genes out of one million Escherichia coli (SOLR) transformants containing pea cDNAs grown in 0.8 M NaCl. Sequence analysis of one of these revealed homology to lectin receptor-like kinase (LecRLK), which contains N-myristilation site and N-glycosylation sites thus corroborating that the protein is a glycoconjugate. Structurally the plant RLKs are similar to animal receptor kinases because both consist of: 1) N- terminal, extracellular, ligand-binding domain, 2) a hydrophobic transmembrane domain and 3) a C- terminal intracellular, kinase catalytic domain (Ser/Thr). The homology based computational modeling of the kinase domain suggested the high degree of conservation with the protein already known to be stress responsive in plants. The NaCl tolerance of LecRLK in bacteria was further confirmed by using another strain of E. coli (DH5α) transformants. In planta, the expression of LecRLK cDNA was also upregulated in response to NaCl. Howerver, there was no significant effect of K+ and Li+ ions on the expression level of the gene in planta as well in E coli, suggesting the Na+ ion specific response. Transcript of the PsLRK gene accumulate mainly in roots and shoots. The purified 47 kDa recombinant pea LecRLK protein has been and shown to contain autophosphorylation activity and also phosphorylate the MBP substrate. This suggests that the cellular response to high salinity stress is conserved across prokaryotes and plant kingdom. Overall, this study urges to develop novel concepts about the role of plant LecRLK in high salinity stress tolerance and this study shall provide a highly significant new contribution for our better understanding of stress tolerance in plants. It remains to be tested whether the expression of these genes will confer durable resistance to high salinity tolerance in crops, but the successful identification of the salinity stress induced gene reveals a clear new pathway for the direction for further experimentation.
18 AGD5, an ARF-GAP, interacts with ARF1GTPase at the trans Golgi network
Renna L.1, Stefano G.1, Baluska F.2, Mancuso S.1
1Department of Horticulture, University of Florence, Viale delle Idée 30, Sesto Fiorentino, 50019 FI, Italy 2Institut für Zelluläre und Molekuläre Botanik, Universität Bonn, Kirschallee 1, D-53115 Bonn, Germany. E-mail: [email protected]
Eukaryotic cells are characterized by a complex of endomembranes. The endomembranes system identity and interconnection is preserved by an active intracellular trafficking mediated by vesicles, which shuttle cargo molecules such as proteins, polysaccharides, and lipids between these organelles. During the intracellular trafficking, vesicle fusion and budding is driven by the assembly and disassembly of the coat proteins from the membrane. In turn, assembly and disassembly of coat proteins are regulated by other proteins called ADP-Ribosylation Factors (ARFs) [1]. As in animal and yeast cells, ARFs have different effectors and regulator proteins that can control the trafficking pathway, like the ARF-GAP proteins. In general, GAP proteins play a crucial role in regulating the disassembly and dissociation of vesicle coats. In the Arabidopsis thaliana genome there are 15 proteins with an ARF-GAP domain, which are classified as ARF-GAP Domain proteins (named AGD1-15) [2]. Here we characterized an Arabidopsis ARF-GAP (AGD5) that contains the AGD domain at the amino terminus. This protein is structurally related to the yeast ARF-GAPs (Age2p, Gcs1p and Glo3p) who perform their function at the trans-Golgi network (TGN) [3,4].This study provide evidence that AGD5 represent a new interactor for the small GTPase ARF1 protein at the TGN organelle suggesting a role in vesicle transport along the endocytic pathway. Furthermore, analysis of transgenic Arabidopsis plants for this protein, showed various defects in root growth, as well as root hairs and pollen tubes development.
References:
[1] Matheson, L.A., Hanton, S.L., Rossi, M., Latijnhouwers, M., Stefano, G., Renna, L. and Brandizzi, F. (2007) Multiple roles of ADP-ribosylation factor 1 in plant cells include spatially regulated recruitment of coatomer and elements of the Golgi matrix. Plant Physiol, 143, 1615-1627.
[2] Vernoud, V., Horton, A.C., Yang, Z. and Nielsen, E. (2003) Analysis of the small GTPase gene superfamily of Arabidopsis. Plant Physiol, 131, 1191-1208.
[3] Cukierman, E., Huber, I., Rotman, M. and Cassel, D. (1995) The ARF1 GTPase-activating protein: zinc finger motif and Golgi complex localization. Science, 270, 1999-2002.
[4] Jurgens, G. and Geldner, N. (2002) Protein secretion in plants: from the trans-Golgi network to the outer space. Traffic, 3, 605-613.
19 Development of model peptides as the research tools for studying the dynamic signaling events in living plant cells
Kawano, T 1, Yokawa, K 1, Kagenishi, T 1
1The University of Kitakyushu, Laboratory of Chemical Biology and Bioengineering, , 808-0135 E-mail: [email protected]
Recently, we have been engaged to the development of oligopeptide based artificial model signaling peptides behaving as artificial enzymes and/or ion channels. This presentation focuses on the development of artificial redox-active enzyme mimics capable of catalyzing the redox reactions such as decomposition of hydrogen peroxide, oxidation of amines and phenolics and generation of superoxide anion, by analogy to the redox nature of human prion protein and plant peroxidases. By designing artificial peptides encompassing the prion-like metal-binding domain, a series of peroxidase mimics with thermo-stable (heat-tolerant and freezing/thawing-tolerant) nature was materialized. Among such metal-binding and redox active peptides synthesized and tested, some acted as pro- oxidants and some behaved as anti-oxidants. Pro-oxidative peroxidase mimics were capable of catalyzing the phenol-dependent robust superoxide generation (Kawano et al., 2007; Yokawa et al 2009a, b) and the aniti-oxidative peptides were capable of plant cell protection from the metal-induced cell death possibly by removal of both the toxic metals and resultant reactive oxygen species (Kagenishi et al., 2009). Furthermore, by inserting certain motifs into the artificial peroxidase sequences, the mode of reaction could be converted from the phenol-requiring peroxidative mode to the catalase-like phenol-independent mode by decomposing hydrogen peroxidase and evolving molecular oxygen.In addition to the redox-active nature of above artificial enzyme mimics, we found that these peptides can actively interact with other proteins or peptides. Such intermolecular interactions include oxidation of amino acid residues (enzyme mimics as catalysts) and modification by phosphorylation (enzyme mimics as substrates), suggesting that controls of signal transduction pathway by properly designing the peptide sequences. For an instance, by designing the oligo-peptides composed of artificial peroxidase moiety and MAP kinase phosphorylation domain, we could develop a novel articial enzyme capable of altering the catalytic activity by phosphorylation of tyrosine and threonine. Lastly, application of these artificial peptides for the studies of plant signaling mechanism will be discussed.
20 Plant-insect interactions: General aspects and dissection of mechanical and chemical challenges
Mithöfer, A 1
1Max Planck Institute for Chemical Ecology, Bioorganic chemistry, Jena, 07745 E-mail: [email protected]
Interactions between plants and insects are central to almost all ecosystems. Although numerous types of interaction have evolved, there is considerable overlap in recognition, signal transduction and gene expression events that orchestrate the plants’ reactions. The activation of any specific responses requires efficient recognition of the interacting organism, conversion of the perceived signal into downstream signalling cascades, and eventually, the onset of appropriate reactions. Responses to insect herbivore damage can be triggered by simple wounding or insect-derived elicitors such as certain enzymes, fatty acid-derived conjugates, other low molecular weight aliphatic compounds, and peptides generated from degradation of ingested plant material. Early induced signalling processes in host plants are characterised by membrane depolarization, intracellular [Ca2+] transients and ROS production, followed by the activation of protein kinases and downstream phytohormone networks that coordinate particular pathways leading to defences. These defences include physical factors (barriers) as well as the deployment of toxic or harmful phytochemicals that are constitutive or induced. Such defensive phytochemicals belong to different classes of secondary metabolites, such as phenylpropanoids, alkaloids, terpenoids, or are fatty acid derivatives. In addition, diverse indirect and direct strategies that plants have evolved to defend themselves against phytophagous insects will be addressed. For example, emissions of induced volatile organic compounds influence local and long range interactions by repelling herbivores and attracting parasites and parasitoids from a distance, thus employing a third trophic level. I will present data on the early phase of plant-insect interactions: from recognition and subsequent signalling processes to the activation of defence related genes and appropriate defence responses towards an attacking enemy. In detail, we studied the individual contributions of wounding and chemistry, which is introduced by any feeding insect. Therefore, we tried to dissect mechanical and chemical treatments during herbivory in order to understand their particular impact on the induction of the whole set of plant responses.
21 AtFH8, an Actin Filament Nucleator and Bundler, Has a Relationship with Root Development in Arabidopsis
Xue X1, Guo C1, Lu Q2, Zhang C2, Ren H1
1Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Science, Beijing Normal University, Beijing 100875, China
2The Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education and the State Key Laboratory of Bio-membrane and Membrane Bio-engineering, College of Life Science, Peking University, Beijing 100871, China
Formins have been paid much attention for their potent nucleating activity. In addition to nucleate actin filament assembly, some formins can bundle actin filaments. In this study, we characterized the bundling activity of AtFH8 (Arabidopsis thaliana Formin Homologue 8) in vitro. Biochemical analysis showed that AtFH8(FH1FH2) could form dimers and bundle preformed actin filaments. However, during the polymerization processes, it not only bundled actin filaments but also induced stellar structures consisting lots of actin bundles. To investigate the localization and function of AtFH8 in vivo, full-length cDNA and truncated forms of AtFH8 were expressed as GFP fusion protein in Arabidopsis. It was found that AtFH8 localized to nuclear envelope in interphase and to the forming cell plate during cytokinesis, which was significantly dependent on its N-terminal transmembrane domain. The immunolocalization of AtFH8 confirmed the nuclear envelope and cell plate localization. Overexpression of AtFH8 promoted mitosis of root tip cells and increased the primary root growth rate in the young transgenic seedlings and vise verse in its N-terminal transgenic lines. The lateral root initation of T-DNA insertion mutant seedlings was partially inhibited by a F-actin-depolymerizing drug, latrunculin B, treatments for 8 days, and the wild-type AtFH8 transgene complements the lateral root phenotype, indicating that AtFH8 linked stabling of actin filament structures may associate with the initiation of lateral roots. Our results suggest that AtFH8 is a potent actin bundle organizer that contributes to primary root growth and lateral root initiation in Arabidopsis young seedlings.
22 Flow of nutrients and information in mycorrhizal networks
Giovannetti, M 1
1Department of Crop Plant Biology, University of Pisa, Pisa, 56124 E-mail: [email protected]
Arbuscular mycorrhizal fungi (AMF) are soil microorganisms which live in symbiosis with the roots of most land plants and are fundamental for plant nutrition and ecosystem biodiversity and productivity. Their extraradical mycelium is the key structure for soil nutrient uptake and transfer to the host plants: it can spread from mycorrhizal roots into the soil and simultaneously colonise many different plants, representing a means for nutrient exchange and resource allocation in plant communities. Recent studies revealed that hyphal fusions (anastomoses) between compatible hyphae represent the main mechanism allowing the formation of large interconnected mycorrhizal networks, whose structure directly influence the nutrition and growth of host plants (1, 2). Anastomoses allow also mycorrhizal networks originating from plants belonging to different species, genera and families to become interconnected, thus giving raise to indefinitely large mycelial webs (3, 4). The continuous bidirectional protoplasmic flow established between self-compatible hyphae, easily detected by time- lapse video microscopy and vital staining, shows that cytoplasm, cellular organelles and nuclei can migrate in AMF mycelium by means of anastomosis. Since genetic exchange has been recently shown between genetically different individuals, and specific genetic markers were transmitted to the progeny (5), a considerable promiscuity can be assumed to occur in the mycorrhizal network, where important nutritional, genetic and information flows are active.
Refereces 1) Giovannetti M., Fortuna P., Citernesi A. S., Morini S., Nuti M. P. (2001). The occurrence of anastomosis formation and nuclear exchange in intact arbuscular mycorrhizal networks. New Phytologist, 151, 717–724.
2) Avio L., Pellegrino E., Bonari E., Giovannetti M.(2006). Functional diversity of arbuscular mycorrhizal fungal isolates in relation to extraradical mycelial networks. New Phytologist, 172, 347– 357.
3) Giovannetti M., Sbrana C., Avio L., Strani P. (2004). Patterns of below-ground plant interconnections established by means of arbuscular mycorrhizal networks. New Phytologist, 164, 175–181.
4) Giovannetti M., Avio L., Fortuna P., Pellegrino E., Sbrana C., Strani P. (2006). Self Recognition and Non-Self Incompatibility in Mycorrhizal Networks. Plant Signaling and Behavior, 1, 1-5.
5) Croll D., Giovannetti M., Koch A., Sbrana C., Ehinger M., Lammers P. J., Sanders I.R. (2009). Nonself vegetative fusion and genetic exchange in the arbuscular mycorrhizal fungus Glomus intraradices. New Phytologist, 181, 924-937.
23 Cellular mechanisms and signal transduction associated with endosymbiotic root infection
Timmers, T 1, Sieberer, B 1, Chabaud, M 1, Genre, A 2, Fournier, J 1, Barker, D 1
1Lab. Plant Microorganism Interactions, CNRS/INRA, Castanet-Tolosan, 31326 2Dept. of Plant Biology, University of Turin and IPP-CNR, Turin, 10125 E-mail: [email protected]
The mutual beneficial relationship between plants and nitrogen-fixing bacteria known as rhizobia is very important both from the agricultural and ecological viewpoint. Rhizobia possess the enzyme complex nitrogenase which enables them to convert atmospheric nitrogen into ammonia, which can then be utilised by the plant host. In exchange, the plant provides the bacteria with a carbon source and a privileged ecological niche in a newly formed plant organ, the root nodule. The rhizobial symbiosis is limited to leguminous plants for which the interaction between Medicago truncatula and Sinorhizobium meliloti is a model system for nodulation. Rhizobia enter the M. truncatula root hair following entrapment within the curled tip and gain access into the root internal tissues via a specialised intracellular plant-derived apoplastic compartment called the infection thread (IT). The path of the IT is determined by the transient formation of a nuclear-driven cytoplasmic assembly which precedes the construction of the IT. The formation of this assembly requires a major intracellular reorganisation involving the plant cytoskeleton and endomembrane system. By using an in vivo confocal microscopy approach we have recently discovered a number of novel and important characteristics of the rhizobial infection process (Fournier et al. 2008, Plant Physiol. 148: 1985-95). These include the relationship between the position of the migrating plant cell nucleus and the progression of the IT, as well as the mechanisms of rhizobial colonization of the thread. One important observation is the frequent presence of a bacterial-free space behind the tip of the extending IT, which shows that direct physical contact between the two organisms is not required for IT growth. Time-lapse studies indicate that a precise coordination exists between IT extension and progressive bacterial invasion. Together with genetic and molecular evidence, this strongly suggests that IT initiation and progression requires continued signalling between the two symbionts. A likely candidate for this signalling is a rhizobial secreted molecule, the Nod factor, a lipochitooligosaccharide, which has been shown to play a prominent role in the host-bacteria recognition and early events of nodulation. When applied externally to plant roots, purified Nod factors elicit a number of plant responses including changes in the cytoskeleton and cytoplasmic Ca2+ levels. By using live-cell imaging and fluorescent microtubule markers we have shown that Nod factors elicit a reorganisation of the microtubular array and modifies microtubule dynamics in root hairs (Sieberer et al. 2005, MPMI 18: 1195-1204; Timmers et al. 2007, Eur J Cell Biol. 86: 69-83). NF-induced cytoplasmic Ca2+ spiking has been reported to arise in the perinuclear region of root hairs (Oldroyd and Downie 2006, Curr. Opin. Plant Biol. 9: 351-357). In order to evaluate NF-elicited Ca2+ responses in the nuclear compartment we have developed a nuclear-targeted cameleon sensor. Our experiments reveal that Ca2+ spiking also occurs within the root hair nucleus and that this response is cell-autonomous. Nuclear Ca2+ spiking is highly variable in terms of frequency and spike duration. Future work will focus on the role of, and the spatio-temporal relationship between the plant cytoskeleton and Ca2+ spiking during rhizobial infection in different root tissues and the potential role of NFs within this process.
24 GORK and NSCC channels as components of salinity tolerance mechanism in plants
Shabala, S 1, Cuin, TA 1
1School of Agricultural Science, University of Tasmania, Hobart, Tasmania 7001 E-mail: [email protected]
Salinity stress tolerance is mediated by multiple physiological and biochemical mechanisms, implying the orchestrated regulation of numerous membrane transporters. In this talk, I summarise our recent findings to show that both outward-rectifying depolarisation-activated K+ (GORK) and non-selective cation (NSCC) channels play a key role in plant salinity responses. We show that salt tolerance correlates with K+ retention, and GORK channels are primarily responsible for mediating salt-induced K+ loss [1]. Interestingly, GORK channels density was similar in barley cultivars contrasting in their salt tolerance, highlighting the importance of channel regulation, not their physical number [2]. Voltage gating was one of the key features of this regulation. We show that tolerant plants had superior control of membrane potential by intrinsically higher H+-pump activity, thus reducing K+ loss through GORK, as well as improving the thermodynamics for K+ uptake. We also show that GORK channels are the downstream target for all substances known to ameliorate detrimental effects of salinity on plants (e.g. exogenous application of calcium or compatible solutes). Furthermore, a link between intracellular potassium homeostasis and Reactive Oxygen Species (ROS) signalling (a well known component of salinity stress) is established and discussed. We show that application of ROS 2+ (either H2O2 or OH -generating Cu /ascorbate mixture) to plant tissues results in a massive, dose- dependent efflux of K+. Pharmacological experiments [2, 5] and patch-clamp data [3] indicate outward-rectifying K+ (KOR) and NSCC as downstream targets of such signalling. We further show that expression of animal CED-9 anti-apoptotic gene significantly increases plant oxidative stress tolerance by regulating KOR and NSCC activity [4], thus providing the first link between ‘ion flux signatures’ and mechanisms involved in regulation of PCD in plants. Finally, we show that physiologically relevant concentrations of various compatible solutes significantly reduce ROS impact on major ion transporters [5]. Importantly, we demonstrate a significant reduction in K+ efflux (associated with increased stress tolerance) using osmolytes with no reported free radical scavenging activity. This indicates that compatible solutes must play other (regulatory) roles in addition to free radical scavenging in mitigating the damaging effects of oxidative stress.
References [1] Shabala and Cuin (2008) Physiol Plantarum 133: 651-669
[2] Chen et al (2007) Plant Physiol 145: 1714-1725
[3] Demidchik et al (2003) J Cell Sci 116: 81-88
[4] Shabala et al (2007) Planta 227: 189-197
[5] Cuin and Shabala (2007) Plant Cell Environ 30: 875-885
25 Sucrose nonfermenting1 (SNF1)-related protein kinase 2 (SnRK2) family function in hyper-osmotic stress signaling of rice
Yoon, I. S. 1, Choi, S. R. 1, Nam, M. H. 2, Lee, I. S. 1, Kim, D. Y. 1, Lee, J. S. 1, Byun, M. O. 1
1Department of Agricultural Bio-resources, National Academy of Agricultural Sciences, Suwon, Kwonsun-Gu, Seodun-Dong 441-707 2Environmental and Metabolomics Research Team, Korea Basic Science Institute, Seoul, Sungbuk-Gu, Anam- Dong 136-713 E-mail: [email protected]
Plant SnRK2 family has been implicated as important regulators of abiotic stress and ABA signaling. Although rice genome contains 10 SnRK2 members, their biological function is largely unknown yet. In the present study, we have characterized biochemical properties and physiological functions of rice SnRK2 subclass 1a proteins. GST-fused recombinant proteins showed autokinase activity, strong substrate preference for rice bZIP transcription factors and cofactor requirement for Mn2+ over Mg2+. Autophosphorylation of catalytic domain was important for enzyme activation and C terminus domain was involved in activity regulation. Noticeable differences in optimum pH, autokinase activity, substrate specificity and inhibitor sensitivity were found among different SnRK2 proteins. We made transgenic rice over-expressing each SnRK2 proteins under the control of 35S promoter and hyper- osmotic stress responses were analyzed. Two SnRK2 transgenic rice showed similar phenotype, i.e. salt-sensitive, but drought-tolerant phenotype. Salt-responses of rice roots were further analyzed. In gel kinase assay revealed that salt and ABA responsive protein kinase are highly activated in the roots of 35S-OSRK1 transgenic rice. Comparison of proteome changes during early salt response of roots showed that enzymes associated with glycolysis, pentose phosphate pathway, nitrogen assimilation and branched amino acid catabolism were rapidly induced by salt treatment. Furthermore, it was noted that those salt-induced enzymes were constitutively up-regulated in roots of 35S-OSRK1 transgenic rice under non-stressed condition. Our data suggests that OSRK1 is upstream regulator of salt stress signaling and possibly be involved in stress-induced carbon and nitrogen metabolic changes. This work was supported by the grants from National Academy of Agricultural Sciences, RDA.
26 Na+ measurements using a fluorescent dye in plant
Park , Mh 1, Lee, HS 1, Lee, JS 1, Byun, MO 1, Kim, BG 1
1National Academy of Agricultural Science, RDA, Suwon, 441-707 E-mail: [email protected]
Fluorescent indicators for Na+ are valuable for nondestructive monitoring of spatial and temporal distribution of Na+ in plants. We tested whether CoroNa Green fluorescent dye, a newly developed sodium indicator, is suitable for measuring the relative concentration of Na+ in planta. To determine the ideal conditions for measuring Na+ content in planta using CoroNa Green dye, NaCl pre-treated Arabidopsis thaliana seedlings were incubated with different concentrations of CoroNa Green, and fluorescence in each organ was visualized using a fluorescein isothiocyanate (FITC) filter. When 50 M of dye was used, we found that fluorescence was distributed more uniformly and intensely in root tip than in other organs. Under these conditions, we showed that fluorescence gradually increased in root tip upon binding of Na+ to CoroNa Green for concentrations up to 100 mM NaCl. Consequently, confocal fluorescence microscopy revealed that when Arabidopsis seedlings were incubated with the same concentration of NaCl, the sos1 mutant exhibited much stronger fluorescence than that of wild type. This study marks the first report of the properties of CoroNa Green used to measure Na+ in intact plants, and demonstrates the usefulness of this technique for investigating the mechanism of Na+ homeostasis in plants.
27 The 7B-1 mutation in tomato confers a blue light-specific lower sensitivity to coronatine
Bergougnoux, V 1, Hlaváčková, V 2, Plotzová, R 1, Novák, O 3, Fellner, M 1
1Department of Cell Biology and Genetics, Palacky University in Olomouc, Olomouc, 78371 2Department of Experimental Physics, Palacky University in Olomouc, Olomouc, 77146 3Laboratory of Growth Regulators, Institute of Experimental Botany, Olomouc, 78371 E-mail: [email protected]
Male sterility of crop species, spontaneous or induced, is a criterion of importance for breeders. In almost all crop species male-sterile mutants have been reported but their use in breeding programs has been limited as some of them were sensitive to abiotic stresses, such as drought or cold temperature. In tomato (Solanum lycopersicum L.), one of the most important crops worldwide, the spontaneous mutant 7B-1, isolated for its photoperiod-dependent male-sterility, has been described as resistant to various abiotic stresses specifically under blue light. Since this finding improved potential of 7B-1’s use in breeding programs, its susceptibility to coronatine (COR)-induced stress, the phytotoxine produced by several Pseudomonas syringae strains, was assessed in this study. The 7B-1 mutant was found to be less sensitive than the corresponding wild-type (WT) to COR treatment in a blue light dependent manner. Treatment of WT and 7B-1 plants with COR induced a strong accumulation of salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) in hypocotyls. Interestingly, accumulation of ABA and SA in the 7B-1 mutant was distinctly greater than in WT, especially in blue light. Based on the cross-talk between SA- and JA-signaling pathways, expression analysis of NPR1 and COI1 genes, respectively involved in these pathways, was investigated in COR-stressed plants. The blue light-specific lower sensitivity of 7B-1 plants to COR was found to be associated with blue light-specific over-expression of the NPR1 gene. This data suggests that the SA-dependent NPR1- dependent pathway could be involved in the lower sensitivity of the 7B-1 mutant to COR. The role of anthocyanins and ABA accumulation during the response to COR is also discussed.
The work was supported by grant from the Ministery of Education of the Czech Republic (grant no. MSM6198959215).
Key-words: blue light-specific response, COI1, coronatine, growth, NPR1, SA-signaling pathway, 7B- 1 mutant, tomato (Solanum lycopersicum L.).
28 Changes in cell wall polymers and pectin methyl esterase expression are developmentally regulated during pollen maturation and embryogenesis
Risueno, MC 1, Barany, I 1, Prem, D 1, Rodríguez-Serrano, M 1, Testillano, PS 1
1Center of Biological Research, CIB, National Research Council, CSIC, Madrid, 28040 E-mail: [email protected]
Plant cell walls are involved in many mechanisms of growth and development but the specific roles of the different plant cell wall polymers are still unclear. They are the repository of chemical signals and also contain information for the direction of cell fate. Changes in the composition and mechanical properties of the cell wall seem to be crucial for coordinated growth and development during morphogenesis. Pectins are complex polysaccharides of the cell walls and are formed by different structural domains (HG, RGI and RGII) which can be modified. The methyl-esterification degree of pectins has a crucial role in various growth and development processes in plants. Pectin methylesterases (PME, EC. 3.1.1.11) are the enzymes involved in the de-methyl-esterification of plant cell wall pectins in muro. Modifications in pectin residues, oligosaccharides and other wall components have been reported as potential signals regulating growth and development. In the present work in situ analysis of the presence and distribution of various cell wall components has been performed to monitor cell wall changes during two different developmental programmes followed by the pollen: the gametophytic development, a differentiation process, and stress-induced reprogramming to embryogenesis, in Capsicum annuum L and Brassica napus L. Specific antibodies recognizing the major hemicellulose, xyloglucan (XG), the rhamnogalacturonan II (RGII) domain of pectins, and high and low-methyl-esterified pectins were used for dot-blot and immunolocalization assays at light and electron microscopy levels at defined developmental stages. Also, the expression profile of the PME was analysed during the pollen developmental processes of B. napus by semiquantitative RT-PCR. Results showed differences in the presence and abundance of these molecular complexes, as well as changes in the esterification level of pectins at specific developmental stages of gametophytic pollen differentiation and pollen embryogenesis. An increase in the expression of PME was found accompanying embryogenesis progression and differentiation events. These changes were related to cell wall growth and maturation during proliferation and differentiation processes followed by both pollen developmental programmes, suggesting that cell wall complexes could contain information on the cell fate and the direction of the cell development.
References BARANY I, FADÓN B, RISUEÑO MC, TESTILLANO PS (2009) Cell wall polymers and pectin esterification levels as markers of proliferation and differentiation events during pollen development and embryogenesis. Submitted.
BARANY I., GONZÁLEZ-MELENDI P., CORONADO M.J., RAMÍREZ C., TESTILLANO P.S., RISUEÑO M.C. (2006) Plant cell wall components rearrange during cell reprogramming to embryogenesis. In: ‘Electron Microscopy 2006’, Iijima S. (ed.) Microscopy Society of Japan. Sapporo, Japan. pp. 245.
Work supported by grants of the Spanish Ministry of Science and Innovation, MICINN, BFU2008- 00203 and AGL2008-04255. D.P. and M.R.S. are recipients of postdoctoral contracts at the CIB-CSIC funded by the MICINN programmes ‘Postdoctoral stays of foreign doctors in Spain’ (SB2006-0074) and ‘Juan de la Cierva’ respectively.
29 Actin turnover is required for myosin-dependent mitochondrial movements in Arabidopsis root hairs
Zheng M.1, Beck M2, Müller J.2, Chen T.1, Wang X.1, Baluška F.2, Logan D.C.3, Šamaj J.2, Lin J.1
1Key Laboratory of Photosynthesis and Molecular Environmental Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 2Institute of Cellular and Molecular Botany, Rheinische Friedrich-Wilhelms-University Bonn, Department of Plant Cell Biology, Kirschallee 1, D-53115 Bonn, Germany 3Department of Biology, University of Saskatchewan, Saskatoon, S7N 5E2, Saskatchewan, Canada (D.C.L.)
E-mail: [email protected] Background: Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.
Methodology/Principal Findings: We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP- FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.
Conclusions/Significance: Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.
Key words: mitochondrial movements, actin turnover, FABD2-GFP, evanescent wave microscopy, velocity, myosin
30 Sensory transduction in guard cell
Hedrich Rainer 1
1Molecular Plant Physiology and Biophysics, Biocenter, University of Würzburg, Würzburg, D-97082 E-mail: [email protected]
Stomata of higher plants close in response to darkness, drought and CO2. The plant hormone abscisic acid (ABA) is involved in the transmission of environmental changes. This process is induced by the activation of guard cell anion channels. To online record changes in ion fluxes across the plasma membrane of guard cells in intact plants, we have developed a method, based on multi-barrelled microelectrodes introduced into the cytoplasm of guard cells in combination with calcium imaging. Using this online, in planta approach, we have been able to identify signalling elements required for fast ABAinduced stomatal closure. Recently mutants were shown to lack a gene encoding a putative guard cell anion transporter named SLAC1. SLAC1 function and stomatal closure-related signaling components leading to anion channel activation, however, remained still unknown. Using protein- protein interaction assays we identified a protein kinase and -phosphatase within the ABA transduction pathway as regulators of SLAC1. Our studies demonstrate that SLAC1 represents the slow inactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation- dephosphorylation. A model on the ABA-based regulation of guard cell ion transport will be presented at the meeting.
31 System potentials, a novel electrical long distance apoplastic signal in plants induced by wounding
Zimmermann , M.R. 1, Maischak, H. 2, Mithöfer, A. 2, Boland, W. 2, Felle, H.H. 1
1Institute of General Botany, Justus-Liebig-University, Giessen, 35390 2Chemical Ecology, Max-Planck-Institute, Jena, 07745 E-mail: [email protected]
We present a recently discovered electrical long-distance signal which, unlike action potentials or variation potentials, is transmitted systemically through hyperpolarization. These signals, called ‘system potentials’ (SPs), are induced by cut-wounding and simultaneously added agents like inorganic salts or glutamic acid. Although these agents produce a depolarization at the stimulation site, the plants respond with a transient hyperpolarization which is transmitted at 5 to 10 cm min-1 to the target (leaf). It is suggested that this hyperpolarization is due to a stimulation of the plasma membrane H+ATPase (H+-pump), a notion which is supported by the actions of fusicoccin and vanadate. Ion movements, measured within the leaf apoplast of the target leaf using ion-selective ion electrodes (K+, Ca2+, Cl-), follow the SP-voltage changes. So far, SPs have been demonstrated in all thereupon tested plants : Hordeum vulgare, Vicia faba, Nicotiana tabacum, Phaseolus vulgaris, Zea mays. This indicates that SPs may be a universal long- distance signal which is used by plants in general to respond to injuries.
32 WDL proteins control root growth behavior and anisotropic cell expansion in Arabidopsis
Masson, PH 1, Jia, G 1, Perrin, R 1, Wang, Y 1
1Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706 E-mail: [email protected]
In plants, anisotropic cell expansion is a tightly regulated process that contributes to morphogenesis. In addition to modulating overall growth rates, this process orchestrates directional growth responses to both endogenous and external cues, allowing plant organs to grow toward environments that are better suited for their primary functions. To better understand how mechanical information and other surface-derived stimuli modulate root growth behavior and anisotropic cell expansion, we screened a population of Arabidopsis thaliana activation-tagging mutants for root behavioral defects on tilted hard-agar surfaces. Under such conditions, wild-type roots are exposed to a combination of gravity, touch and other surface-derived stimuli. Consequently, they tend to skew their growth toward the right of the gravity vector (when viewed through the medium), and they wave on the surface, a phenomenon that is accompanied by reversible helical growth. This complex growth behavior is believed to facilitate obstacle avoidance by growing roots. wvd2-1 was identified in this screen, showing opposite root skewing and dampened or no waves on tilted hard-agar surfaces. This mutant also displayed altered anisotropic cell expansion in all organs of the plant, and enhanced thigmomorphogenesis suggested a role in the control of mechano-transduction or response. WVD2 encodes a microtubule (MT)-associated protein that promotes the bundling of MT in vitro, and affects the organization and dynamic instability of cortical MTs in expanding cells of the root. It shares a 95 amino-acid motif with 7 other Arabidopsis proteins, called WDL1-7, and initial phenotypic analyses of over-expression and knockout mutants suggest distinct, though overlapping roles for the WDL proteins in the regulation of MT-dependent morphological processes. Interestingly, brassinosteroids, which appear to modulate root waving on hard-agar surfaces and anisotropic cell expansion, may regulate WVD2 and WDL proteins activity, as suggested by in vitro studies of WVD2/WDL – BRI1 kinase-domain (KD) interactions and of BRI1-KD-mediated phosphorylation of WVD2 and at least some of the WDL proteins. We hypothesize that WVD2 and related WDL proteins contribute to the control of organs growth behavior by regulating the organization and/or dynamic properties of cortical MTs in expanding cells, thereby modulating the patterns of anisotropic cell expansion and complex growth responses to the environment.
33 Genetic analysis of the protein import machinery of Arabidopsis plastids
Jarvis, P 1
1Department of Biology, University of Leicester, Leicester, Leics LE1 7RH E-mail: [email protected]
While plastids retain a fully-functional genetic system, the plastid genome encodes less than 10% of the proteins required to build a fully-functional organelle. The majority of plastidic proteins are encoded in the nucleus and translated on free cytosolic ribosomes. They are synthesized in precursor form, each one bearing an amino-terminal targeting signal, or transit peptide. The transit peptide directs the protein through a post-translational targeting pathway, and is cleaved upon arrival inside the plastid. This targeting or import process is mediated by the coordinate action of two proteinaceous import machines, one in each of the envelope membranes. The import machinery of the outer envelope membrane is called the TOC complex, and that in the inner membrane is called the TIC complex. Over the last decade, several components of the TOC and TIC complexes have been identified using biochemical approaches and isolated pea chloroplasts. Interestingly, many of these components (particularly receptor components of the TOC complex) have been found to have multiple homologues in Arabidopsis. We have used genetic approaches to dissect the functional significance of these different TOC protein isoforms. Our results suggest that the different isoforms operate in different import pathways with distinct precursor recognition specificities; i.e., different import pathways exist for different precursor protein classes. The existence of such substrate-specific import pathways might play a role in the differentiation of different plastid types, and act to prevent deleterious competition effects between abundant and non-abundant precursors.
34 Both electrical and chemical signals may act in a triggering of rapid systemic responses of tobacco or tomato plants to local burning
Hlaváčková, V. 1
1Laboratory of Biophysics, Department of Experimental Physics, Faculty of Science, Palacký University, Olomouc, Czech Republic CZ-771 46 E-mail: [email protected]
Higher plants respond to adverse environmental factors by various defence processes, e.g. stress protein accumulation, changes in stomata aperture, changes in rates of respiration or photosynthesis. An important characteristic of the defence processes is their occurrence also in undamaged plant tissues that are distant from the damaged ones. This feature suggests an existence of a mobile signal that is transmitted from the damaged part of the plant to the distant undamaged tissues where it evokes systemic stress responses. We studied mainly short-term (up to one hour) systemic reactions of tobacco (Nicotiana tabacum cv. Samsun) or tomato (Lycopersicon esculentum Mill. cv. Moneymaker) to local wounding (burning) of an upper leaf. In both plant species, significant changes were detected in gas exchange parameters of undamaged leaf growing under the burned one. In tobacco, a decrease of the stomatal conductance and the rates of transpiration and CO2 assimilation has already been observed 5-7 minutes after local stimulation. In tomato, an initial (within 5 min) increase of the stomatal conductance and the rate of transpiration was reversed by their marked decrease up to 25 min after local burning when steady-state values were reached. Values of the rate of CO2 assimilation only slowly decreased to the steady-state levels. We detected no considerably rapid systemic changes in chlorophyll a fluorescence parameters in tobacco. Similarly, no significant changes were detected in systemic chloroplast movement in the non-treated tobacco leaf after local burning of the distant leaf. Electrical and chemical signals that could be involved in a triggering of the above mentioned systemic responses were investigated. A sharp decrease followed by an increase (seconds to minutes) of surface electrical potential (SEP) was detected in several leaves growing below the burned one in tobacco plants. In tomato, an increase of SEP was detected in distant untreated leaves within seconds after burning of the upper leaf. Our low-noise multi-channel device for the monitoring of SEP propagation throughout plants will be presented. SEP changes in tobacco were followed by rapid systemic increases in endogenous concentration of abscisic acid (ABA, 8-15 min) and jasmonic acid (JA, 15-60 min) after local burning. In tomato mutants - sitiens (ABA-deficient plants), SEP changes propagated more rapidly down the leaf trace of the wounded leaf and SEP amplitude was dependent on the position of the measured leaf with respect to the leaf trace of the wounded leaf. An amplitude of the SEP wave in sitiens mutants was approximately twice lower (30 mV) than that in wild-type (60 mV). Our results indicate a possible participation of both, electrical (SEP) and chemical (ABA, JA) signals in rapid long-distance (systemic) responses of plants to local stress. ABA seems to be involved in the electrical signal generation and its long-distance propagation after local wounding of tomato. A scheme of possible signaling pathways including generation of the chemical and electrical signals, their interactions and participation in triggering of the responses will be discussed.
The project was supported by grant from the Ministry of Education of the Czech Republic, No. MSM 6198959215.
35 Jasmonates functions in symbiotic interactions and plant response to wounding
Hause, B. 1
1Leibniz Institute of Plant Biochemistry, Department of Secondary Metabolism, Halle, D-06018 E-mail: [email protected]
Jasmonic acid (JA) and its derivatives, commonly termed jasmonates, are hormonal regulators involved in plant responses to abiotic and biotic stresses as well as in plant development. Jasmonates are lipid-derived signals, they are synthesized by the octadecanoid pathway via the allene oxide synthase branch of the so-called lipoxygenase pathway. The role of jasmonates is well established as part of a complex signal transduction pathway activated upon wounding of leaves by insects and interaction of plants with microorganisms. Among these interactions, also the mutualistic interactions between plants and arbuscular mycorrhizal (AM) fungi or nitrogen-fixing rhizobacteria are believed to be regulated from the plant side among other signals by action of JA. To get deeper insights, functional analyses by transgenic approaches were performed to investigate the role of JA during the interaction between Medicago truncatula and Glomus intraradices or Shinorhizobium meliloti. The capacity of M. truncatula roots to synthesize JA was changed by transformation with Agrobacterium rhizogenes leading to chimeric plants. This was achieved by modulation of the transcript level of the MtAOC1 gene encoding the allene oxide cyclase (AOC), a crucial enzyme involved in JA biosynthesis. Transgenic roots exhibiting partial suppression of MtAOC1 and lower JA levels showed a significant delay in the process of colonization with G. intraradices. Analyses of global transcript profiles by microarrays in non-mycorrhizal and mycorrhizal roots with modulated MtAOC1 expression, revealed a large number of regulated genes, among them are those encoding enzymes involved in phenylpropanoid metabolism. Analyses of isoflavonoids showed that mycorrhizal roots with enhanced and decreased MtAOC1 expression exhibited decreased and increased levels, respectively. In contrast to mycorrhization, a role of JA in the interaction of M. truncatula with S. meliloti leading to the formation of nodules could not be demonstrated. Here, overexpression and partial suppression of MtAOC1 did not lead to an altered nodule phenotype: Neither the morphology of nodules nor the number of nodules were different in these plants in comparison to the empty vector control. As wounding of plants leads to endogenous rise of JA accompanied by the expression of a distinct set of genes, we addressed the question, how roots and shoots of M. truncatula respond to wounding. Surprisingly, M. truncatula plants responded very sensitively to mechanostimulation caused by harvest. Mechanostimulation led to a rapid increase in JA levels followed by increased transcript accumulation of JA-responsive genes. Even repeated mechanostimulation performed by touching obviously changed the phenotype of plants. Moreover, repeated wounding of leaves affects mycorrhization of M. truncatula with G. intraradices pointing to a systemic effect of JA raised in shoots. In conclusion, our results indicate that jasmonates act as positive regulator of mycorrhization, but do not have a role in nodule formation of M. truncatula.
36 Nuclear calcium signalling in plant cells
Mazars, C. 1, Xiong, TC. 2, Brière, C. 1, Coursol, S. 3, Mithöfer, A. 4
1UMR 5546,Surfaces Cellulaires et Signalisation chez les Végétaux, CNRS/Université de Toulouse , Castanet- Tolosan, 31326 2BPMP-IBIP, UMR 0386 INRA/UMR 5004 CNRS, INRA/CNRS/Montpellier SupAgro/Université Montpellier , Montpellier, 34060 3UMR 320 INRA/8120 CNRS, INRA/CNRS/Université Paris XI/INA-PG de Génétique Végétale, Ferme du Moulon, Gif-sur-Yvette, 91190 4Institut Für Chemische Ökologie Bioorganische Chemie, Max Planck-Institut , Jena, 07745 E-mail: [email protected]
In plants, Ca2+ is a ubiquitous intracellular second messenger, known to couple a diverse array of signals and responses such as the plant responses to biotic and abiotic stress [1,2]. Using tobacco BY-2 cell suspension cultures harbouring the bioluminescent Ca2+-responsive protein aequorin in the nucleoplasm, we show here that isolated plant nuclei generate Ca2+ signals on their own in response to mechanical and thermal stimuli, independently of a cytosolic environment (3).By combining mathematical modeling and pharmacological approaches, we predict that the buffering capacity of the nucleoplasm and the activities of putative calcium transporters/exchangers located on the inner nuclear membrane might be highly coordinated (4). We have also addressed the possible connections between sphingolipid metabolites and the modulation of [Ca2+]nuc in tobacco BY-2 cells and in isolated nuclei. Experimental evidences suggest that an active metabolism of sphingolipid takes place in the nucleus. Thus, exogenously supplied LCBs, elicited increases in [Ca2+]nuc in intact cells and isolated nuclei in a dose-dependent and structure-related manner. In contrast, sphingosine-1-phosphate and C2-ceramide were completely inactive. Importantly, we found that pre-treatment of isolated nuclei with transient receptor potential (TRP) channel inhibitors significantly inhibited the effect of DMS on Δ[Ca2+]nuc (5). We also show that jasmonate derivatives or their synthetic mimics differentially induced calcium transients in the cytosol and the nucleus. We show further that some of them were active on [Ca2+]nuc only giving a physiological relevance to the calcium signalling autonomy of the nucleus(6). Collectively, our results reinforce the theory suggesting that the nucleus is autonomous in terms of calcium signaling and may impact on response specificity by controlling downstream nuclear Ca2+-dependent events. Current work investigates the role of calcium compartmentation in sphingolipid-induced cell death-processes.
References
1. Hetherington AM, Brownlee C. (2004) Annu. Rev. Plant Physiol. Plant Mol. Biol. 55, 401-427.)
2. Sanders D.,Pelloux J.,Brownlee C.,Harper J.F. (2002) Plant Cell S401-417
3. Xiong TC, Jauneau A, Ranjeva R, Mazars C. (2004) Plant J. 40, 12-21.
4. Briere C, Xiong TC, Mazars C, Ranjeva R. (2006) Cell Calcium 39, 293-303.
5. Xiong T.C., Coursol S., Grat S., Ranjeva R., Mazars C. (2008). Cell Calcium 43,29-37
6. Walter A.,Mazars C, Maitrejean M.,Hopke J.,Ranjeva R. Boland W. Mithöefer A. (2007) Angew. Chem. Int. Ed. 46 , 4783-4785
37 Apocarotenoids - signaling compounds of Zygomycetes and plants?
Schachtschabel D.1, Schlicht M. 2, Baluska F.2, Boland W. 1
1Max Planck Institute for Chemical Ecology, Bioorganic chemistry, Jena, 07745 2IZMB, University of Bonn, Kirschallee 1, 53115 Bonn E-mail:[email protected]
Zygomycetes are common heterotrophic microorganisms which naturally occur on terrestrial habitats. For sexual reproduction and parasitic interaction, the zygomycete fungi interact via an elaborate series of carotene derived compounds, namely trisporic acids and their biosynthetic precursors. However, details of their metabolism and the biological significance of the various intermediates remained unclear.[1] Therefore we generated a trisporoid library including deuterium labeled intermediates by a combination of synthesis and biotransformation using cultures of Blakeslea trispora.[2] These references enabled us to study the biosynthesis and the biological function of individual trisporoids in more detail.[2-4] The results prompted us to postulate a new sequence of molecular interaction between both mating partners, which includes two different metabolic pathways.[5] Moreover, apocarotenoids are strongly discussed as new branching hormones in plants and fungi.[6] Therefore we decided to determine an influence on plant cells and mycorrhizal interaction. Amazingly the β-C18-ketone (D’orenon), an early trisporoid precursor, strongly inhibited root hair development of A. thaliana as results of disruption of the auxin signaling network.[7]
[1] R. P. Sutter et al., Fung. Gen. Biol. 1996, 20, 268-279.
[2] D. Schachtschabel, W. Boland, J. Org. Chem. 2007, 72, 1366-1372.
[3] M. Richter et al., Environ. Microbiol. in prep.
[4] D. Schachtschabel et al. Phytochem. 2005, 66, 1358-1365.
[5] D. Schachtschabel et al., ChemBioChem 2008, 9, 3004-3012.
[6] D. Schachtschabel, W. Boland ChemBioChem Highlight 2008, 10, 221-223.
[7] M. Schlicht et al. Plant J. 2008, 55, 709-717.
38 Sensing plant symbiotic signals by nitrogen-fixing bacteria
Navazio, L 1, Moscatiello, R 1, Squartini, A 2, Mariani, P 1
1Dipartimento di Biologia, Università di Padova, Padova, 35131 2Dipartimento di Biotecnologie Agrarie, Università di Padova, Legnaro (Padova), 35020 E-mail: [email protected]
The rhizosphere represents a highly dynamic forefront for interactions between plant roots and soil microbes. In this space, encompassing few millimeters, complex biological and ecological processes occur, such as the exchange of chemical signals among organisms. Under nitrogen-limited conditions, the Gram-negative soil bacteria rhizobia are able to establish a symbiotic interaction with leguminous plants. This host plant-specific association results in the development of root nodules in which rhizobia differentiate into nitrogen-fixing bacteroids. A successful symbiosis is the result of an elaborate developmental program, regulated by the exchange of molecular signals between the two partners. During growth in the rhizosphere of the host plant, rhizobia sense compounds such as flavonoids secreted by the host root and respond upon induction of the nodulation genes by synthesizing the Nod factors. These diffusible rhizobial signal molecules of lipochito-oligosaccharide nature are perceived by plant roots and trigger a Ca2+-dependent signaling pathway leading to specific physiological responses, which culminate in the nodule organogenesis. In comparison with the large knowledge of the signalling pathway active in plants, limited information is available on the rhizobial perception and transduction of symbiosis-inherent signals generated by the plant host. By using aequorin-expressing rhizobia (Mesorhizobium loti and Rhizobium leguminosarum bv. viciae) we demonstrated that host plant root exudates, and signal molecules therein contained, such as flavonoids (nod gene inducers), are sensed by nitrogen-fixing bacteria through transient intracellular Ca2+ elevations. The significant inhibition of nod gene expression obtained when the Ca2+ response is blocked indicates that an upstream Ca2+ signal is required for nod gene activation. Ca2+ changes were not triggered by flavonoids unable to induce nod gene expression (anti-inducers). A rhizobium strain cured of the symbiotic plasmid, impaired in flavonoid-induced nod gene expression, retained its ability to respond to flavonoids with an unchanged Ca2+ response. The possible placement of the Ca2+ signal within the NodD-flavonoid gene expression paradigm will be discussed. These data indicate a newly described early event in the molecular dialogue between plants and rhizobia and are consistent with a crucial role played by Ca2+ in the symbiotic signalling.
39 Chemical biology of leaf-movement of Albizzia saman
Nakamura, Y.1, Hamamoto, S. 2, Inomata, S.1, Uozumi, N. 2, Ueda, M. 1
1Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, 9808578 2Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, 9808579 E-mail: [email protected]
Albizzia plants close their leaves in the evening, as if to sleep, and open them in the morning according to the circadian rhythm. Potassium β-D-glucopyranosyl-12-hydroxyjasmonate (1) was isolated as leaf- closing factor (LCF) of Albizzia saman. We developed molecular probes consisting of modified LCF 1 in order to identify its mode of action. We have already demonstrated that a specific binding protein is involved in the motor cell of A. saman1). We synthesized natural-type photoaffinity probe and biologically inactive enantiomer-type probe. We utilized them for photoaffinity labeling of the receptor for LCF 1. By using protoplasts of motor cell, we found membrane protein of 38 kDa which strictly recognizes the stereochemistry of 1, and it is highly likely that the protein is the specific receptor for LCF2). Recently, we observed that LCF shrank motor cell protoplasts prepared from A. saman. And comparing the results of several bioassay using glucosyl jasmonate-type LCF and jasmonic acid, it is also interesting that the mode of action of LCF is completely different with that of jasmonate3).
References 1) Y. Nakamura, R. Miyatake, A. Matsubara, H. Kiyota, M. Ueda, Tetrahedron, 62, 8805 (2006).
2) Y. Nakamura, R. Miyatake, M. Ueda, Angew. Chem. Int. Ed., 47, 7289 (2008).
3) Y. Nakamura, R. Miyatake, S. Inomata, M. Ueda, Biosci. Biotechnol. Biochem., 72, 2867 (2008).
40 Membrane potential fluctuations in Chara australis: a characteristic signature of high external sodium
Beilby, MJ 1, Al Khazaaly, S 1, Walker, NA 1, Shepherd, VA 1
1The University of NSW, School of Physics, Sydney, NSW 2052 E-mail: [email protected]
We have studied fluctuations in membrane potential difference (PD) in Chara australis at frequencies between 1 and 500 mHz, by classical noise analysis and by inspection of the PD time-course. The former shows (1) a quasi-Lorentzian (1/f2) rise of noise power as frequency falls, and (2) a marked increase in noise power when the cell is exposed to high salinity (Chara australis is a salt-sensitive species). Inspection shows that as well as initiating depolarization, exposure to high Na+ usually initiates a continuous but random series of small depolarizations with median amplitude of 2 mV, frequency of 0.2 Hz, depolarization rate of 2 mV/s and repolarization of 0.9 mV/s (7 cells). The noise 2+ + - 2- is abolished by substituting Mg for Na , but remains unchanged by exchanging Cl for SO4 . We hypothesize that the noise is caused by transient openings of H+ or OH- (H+/OH-) channels in the early exposure to sodium. With time in saline medium the membrane PD depolarizes due to inactivation of the proton pump. The noise diminishes, as progressively greater numbers of the H+/OH- channels remain open, modifying the current-voltage characteristics of the membrane and shifting the membrane PD towards zero. The involvement of H+/OH- channels in salt sensitivity has not been considered before. Similarities between charophyte cells and roots of land plants are discussed.
41 Anion channel activation is an early event in ozone-induced cell death in Arabidopsis cell suspension
Kadono, T 1, Errakhi, R 1, Hiramatsu, T 2, Meimoun, P 1, Tran, D 1, Briand, J 1, Kawano, T 2, Bouteau, F 1
1EA3514, Université Paris Diderot, Paris, 75005 2Graduate School of Environmental Engineering, University of Kitakyushu, Kitakyushu, 808-0135 E-mail: [email protected]
Ozone (O3) produced by a complex series of photochemical reactions from primary precursor emissions of nitrogen oxides and volatile organic compounds, is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight. By exposing cells to the pulse of ozonized air, O3 induced acute cell death was observed in suspension cells of Arabidopsis thaliana depending on the exposure time. In this study we demonstrated that the activation of plasma membrane anion channel is an early key component of the O3-induced cell death in A. thaliana. Previous data obtained on tobacco cells suggested the action of Ca2+ as a secondary messenger initiating the oxidative cell death. As in tobacco cells, impairment of Ca2+ influx with BAPTA or La3+ allowed to decrease the extent of O3-induced cell death. The increase in anion channel activity also seemed to be dependent on an increase in cytosolic Ca2+ since BAPTA or La3+ allowed to decrease the O3-induced depolarization and anion channel activity increase. Reactive oxygen species scavengers also allowed to decrease O3-induced cell death, but failed to inhibit anion channel increase suggesting that the involvement of ROS in O3-induced cell death doesn’t concern the early events.
42 Arabidopsis root movements and symmetry
Migliaccio, F 1, Fortunati, A 1, Tassone, P 1
1Institute of Agro-Environmental and Forest Biology, Consiglio Nazionale delle Ricerche, Monterotondo (Roma), 00016 E-mail: [email protected]
When Arabidopsis plants are grown on an agar dish inclined at an angle, the primary roots do not grow straight down but by making a characteristic wavy pattern, which direction, in the wild-type, slants to a side that was defined as the right-hand. This because the half-waves made to the right-hand are deeper than those made to the left-hand. In addition, the waves normally show a twsting that is alternatively left-handed when the wave is made to the right-hand, and right-handed when the wave is made to the left hand. Furthermore, when the roots are grown on a plate set horizontally they make large or strict clockwise coils, that show also a strong torsion to the left-hand on themselves. The reason of all these movements are still not totally clear, and there is more than a single hypothesis about, but generally they are supposed to be the consequence of three forces acting together on the root, i.e. positive gravitropism, circumnutation and a form of thigmotropism. Gravity in fact controls the direction down the plate, the waving seems to be the consequence of typical circumnutation, with the complication of an alternative switching from the right-handed to the left-handed symmetry, that seems to be induced by negative thigmotropism. As concerns the coils they seems to be circumnutation circles flattened on an agar dish, that possibly show a strong torsion due to the necessity of discharging the tension produced by the flattening of the space right-handed helix. Following a different hypothesis the coils, however, could be also a consequence of thigmotropism. These are the results of several researches made both on the wild-type and on mutants, in 1g , in simulated microgravity, and in one case also in space. The various possibilities are discussed and confronted with data coming from different plants.
43 Increasing the density of the external medium inhibits and reverses root gravitropism
Staves, M P 1, Heldt, L 2
1Biology Department, Grand Valley State University, Allendale, Michigan 49401 2Department of Cell and Molecular Biology, Grand Valley State University, Allendale, MI 49401 E-mail: [email protected]
As an alternative to the statolith model, we have presented a model for plant gravity sensing in which the entire protoplast functions as the gravity sensor. This gravitational pressure model was developed as a result of experiments with the large, statolith-free, intermodal cells of Chara. These cells exhibit a polarity of cytoplasmic streaming in response to gravity such that, under normal conditions when the density of the external medium is less than that of the protoplast, streaming takes place ca. 10% faster in the downward direction than in the upward direction. However, when the density of the external medium is equal to that of the protoplast the polarity of cytoplasmic streaming is abolished. When the density of the external medium is greater than that of the protoplast (and the protoplast becomes buoyant within the extracellular matrix) the gravity response is reversed. The question remains whether the gravitational pressure model can explain the gravity responses of higher plants containing statocytes. We tested the gravitational pressure model by monitoring gravitropic curvature of statolith-containing roots in media of differing densities. Changing the density of the external medium will affect the static buoyancy of the protoplast but not the sedimentation of intracellular particles. We find that increasing the density of the external medium inhibits, and in some cases reverses the direction of gravitropic curvature of these roots. These data are consistent with the gravitational pressure model for plant gravity sensing and inconsistent with the statolith model.
44
Panpsychism - Past and Present
Neumann, J 1
1Department of Plant Sciences &Philosophy, Tel-Aviv University, Tel-Aviv, 69978 E-mail: [email protected]
Panpsychism is the doctrine that all matter has a psychical aspect. Not only human beings and animals, but also plants and even inanimate objects. It was a favored doctrine in the past, echoed in many prescientific cultures. In the 18-th and 19-th century panpsychism was related to the idealistic philosophy. Among the supporters of panpsychism should be mentioned - Fechner who is known for establishing the science of psychophysics, and who wrote a book about the soul of plants, and Wilhelm Wundt . Other supporters include the important psychologist and philosopher William James, and the philosopher Alfred Whitehead. Most people would say that plants are not conscious, yet a case could be made that the first stirrings of consciousness came with having senses and interacting with the world, and plants can sense the world; they respond to gravity, light temperature and moisture. An argument in favour of panpsychism is related to the theory of evolution. How so enormous a jump from one creature to another should have occurred, as the introduction of a fact entirely different from the physical fact. Panpsychism became much less attractive with the ongoing powerful scientific picture of the physical world and the assumption (based on a very large amount of evidence) about the closure of the physical world. Maintaining otherwise would amount to believe in miracles .As a result of our immense scientific knowledge of the physical world, panpsychism has become an implausible view. However, recently the possibility of panpsychism surfaced in relation to the problem of consciousness. The philosophy of mind in the modern era begins with Rene Descartes in the 17-th century. His famous doctrine was dualism, the idea that the world divides into two different substances : the mental (consciousness) and the physical (being extended in space). Humans are composite beings, consisting both of a mind and a body; animals and plants are mere machines with no mental life. The problem of dualism is how can there be an interaction between two different substances, despite the undeniable fact that that such an interaction does take place. Because of the failure of dualism (in its different forms) there is in contemporary philosophy a turn to physicalism – according to which, the only reality that exists is physical. Mental states are either eliminated, reduces to physical states or identified with them. An attractive theory is Searle\'s biological naturalism, which avoids both dualism and physicalism; consciousness is caused by the behavior of neurons in the brain and at the same time, it is realized in the brain system, which is composed itself of neurons, but there still remains the question how exactly to integrate consciousness with the brain. There are two possibilities: panpsychism and emergentism. It seems imperative to decide whether and how mind emerges; whether it exists only under some specifiable conditions in specific places (the brain) or whether it is a part of the fundamental structure of the world. The advances in science: the theoretical success of physical science which explained how chemical complexity arises from physical principles point to the latter. Thus, all modern physicalistic theories of mind implicitly rest upon a theory of emergence (either epistemological or ontological), but none is satisfactory. Until such an account will be achieved, panpsychism remains an open possibility.
45 Epigenetic memory in plant responses to environmental stimuli
Shen, W.-H. 1, Berr, A. 1, Gao, J. 1, Meyer, D. 1, Ménard, R. 1, Molitor, A. 1
1CNRS, Institute of Plant Molecular Biology (IBMP), Strasbourg, 67084 E-mail: [email protected]
Epigenetics refer to heritable changes (during mitosis and meiosis) of genome function that occur without a change in DNA sequence. The advantage of epigenetic changes is that they are stable and also reversible. DNA methylation and histone modifications are thought to have crucial roles in organism adaptive response to environmental stimuli and in inheritance of stress memories. Our group is interested in characterization of regulatory factors involved in the deposition of histone modifications, such as methylation and ubiquitylation on histone lysine residues. We will show and discuss our recent data to highlight roles of histone modifications in plant growth and development and in plant responses to environmental stimuli, including abiotic and biotic stresses.
Selected recent publications from our group:
XU, L., MENARD, R., BERR, A., FUCHS, J., COGNAT, V., MEYER, D., and SHEN, W.-H. (2009) The E2 ubiquitin-conjugating enzymes, AtUBC1 and AtUBC2, play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana. Plant J. 57, 279- 288. (Epub online 14 october 2008) XU, L. and SHEN, W.-H. (2008) Polycomb silencing of KNOX genes confines shoot stem cell niches in Arabidopsis. Curr. Biol.,18, 1966-1971. XU, L., ZHAO, Z., DONG, A., SOUBIGOU-TACONNAT, L., RENOU, J.-P., STEINMETZ, A. AND SHEN, W.-H. (2008) Di- and tri- but not mono-methylation on histone H3 lysine 36 marks active transcription of genes involved in flowering time regulation and other processes in Arabidopsis thaliana. Mol. Cell. Biol., 28, 1348-1360. LIU, S.M., YU, Y., RUAN, Y., MEYER, D., WOLFF, M., XU, L., WANG, N., STEINMETZ, A. and SHEN, W.-H. (2007) Plant SET- and RING-associated domain proteins in heterochromatinization. Plant J., 52, 914-926. ZHU, Y., DONG, A., MEYER, D., PICHON, O., RENOU, J.P., CAO, K. and SHEN, W.-H. (2006) Arabidopsis NRP1 and NRP2 encode histone chaperones and are required for maintaining post- embryonic root growth. Plant Cell, 18, 2879-2892. ZHAO, Z., YU, Y., MEYER, D., WU, C. and SHEN, W.-H. (2005) Prevention of early flowering by expression of FLOWERING LOCUS C requires methylation of histone H3 K36. Nature Cell Biol., 7, 1256-1260. DONG, A., LIU, Z., ZHU, Y., YU, F., LI, Z., CAO, K. and SHEN, W.-H. (2005) Interacting proteins and differences in nuclear transport reveal specific functions for the NAP1 family proteins in plants. Plant Physiol., 138, 1446-1456.
46 Sieve-element Ca2+ channels link remote stimuli and sieve-tube occlusion in Vicia faba
Furch, A.C.U. 1, van Bel, A.J.E. 1, Fricker, M.D. 2, Felle, H.H. 3, Fuchs, M. 1, Hafke, J.B. 1
1Plant Cell Biology Research Group, Institute of General Botany, Justus-Liebig-University, Giessen, 35390 2Department of Plant Sciences, University of Oxford, Oxford, OX1 3RB 3Institute of General Botany, Justus-Liebig-University, Giessen, 35390 E-mail: [email protected]
This study addresses the role of Ca2+ as a link between propagation of electropotential waves (EPWs) and forisome dispersion in intact legume plants. Here, Ca2+-induced dispersion of forisomes induced by a remote stimulus was used as an optical marker for Ca2+ influx during EPW passage as result of Ca2+-channel activity. In response to a number of remote stimuli of different strength and nature, EPWs, activation of Ca2+ channels, Ca2+ influxes into sieve elements (SEs), and forisome reactions could be correlated semi-quantitatively. The magnitude of initial EPW phase was reduced by La3+ and Gd3+, impermeant Ca2+-channel antagonists, comparable with Ca2+ influx from the cell wall during EPW propagation. The permeant Ca2+-channel blockers, nifedipine and verapamil had a more marked effect on the prolonged plateau phase which rather suggests interference with intracellular Ca2+ channels. All Ca2+-channel blockers inhibited forisome dispersion. Resting levels of cytosolic Ca2+ around ~50 nM were measured using both Ca2+-selective electrodes and Oregon Green BAPTA-1 (OGB-1). Transient increases in cytosolic Ca2+ were observed in intact phloem tissue in response to remote stimuli, but were below the threshold of forisome dispersion (~30 µM Ca2+). The discrepancy between observed Ca2+ changes and Ca2+ threshold necessary for forisome dispersion implies that forisome ends must be located close to Ca2+ channels. We showed a tight association between forisomes and ER stacks at the EM level. We used high-affinity binding of fluorescent nifedipine (Kd 600 nM) to map the density of Ca2+ channels over the SEs. Ca2+ channel density was higher in the vicinity of sieve plates and pore plasmodesma units with the lowest densities in central parts of SEs. Fluorochrome studies revealed that Ca2+ accumulation and Ca2+-channel patches overlayed the unevenly distributed ER stacks. Ca2+ channels that co-localize with the plasma membrane were deployed mainly on the plasma membrane at the companion cells. To reach the functional threshold, Ca2+ ions must remain limited to the SE margins, most likely in the unstirred interstices between ER stacks. Insertion of forked forisome ends between the ER stacks and a higher dispersion rate of ER- attached forisomes upon distant burning seem to demonstrate an optimal localization of Ca2+ stores and channels for occlusion reactions.
47 Short-time effects of coumarin along the maize primary root axes
Lupini, A. 1, Sorgonà, A. 1, Araniti, F. 1, Abenavoli, M.R. 1
1Dipartimento Bio.M.A.A., Università degli Studi Mediterranea di Reggio Calabria, Reggio Calabria, Calabria 89124 E-mail: [email protected]
Coumarin, the simplest allelopathic compound, induces a broad range of effects on plant root morpho- physiology. Recent studies indicate that coumarin selectively inhibites or stimulates growth of individual roots of Zea mays [1] and Arabidopsis thaliana [2]. Nevertheless, coumarin-effects on different zones along the root axes are still missing. Thus, the main objective of the present study was to analyze the responses of three development regions (tip, middle and basal zone) of the primary maize root exposed to localized coumarin concentrations. For this purpose, tip (3 mm), middle (20 mm) and basal regions (50 mm) of primary maize root were exposed to 0, 25, 50 or 100 µM coumarin and the root elongation rate (RER), proton efflux (PE) and plasma-membrane potential response of each root region were evaluated. The root tip treatment (3mm) with 25 and 50 µM coumarin did not modify the primary root RER, which, instead, was significantly increased by 100 µM coumarin (0.0274 mm/min) respect to the control (0.0146 mm/min). Conversely, the middle and basal zone treatment, at all coumarin concentrations, did not influence the primary root RER. The proton efflux was also enhanced when the root tip was exposed to the coumarin, but this effect was not observed on the other root zones. An unchanged depolarization phase of plasma-membrane potential was observed in root tip zones, followed by an increased, coumarin concentration-dependent, hyperpolarization phase. In a different way, only an increased depolarization phase was induced by coumarin treatment in the middle root zones, which did not modify plasma-membrane potential of basal root zones. In conclusion, the results show that tip is the root zone much more sensitive to coumarin. Further, coumarin-induced effect on proton efflux and plasma-membrane potential (hyperpolarization) on root tip could consider, as reported by Zimmermann et al (2009), a third systemic electrical signal in higher plants [3].
References
1. Abenavoli et al. 2004. J. Chem. Ecol. 30: 1871-1883
2. Abenavoli et al. 2008. Allelopathy J. 22 (1):245-252
3. Zimmermann et al. 2009. Plant Physiol. 149: 1593-1600
48 Monoterpene-mediated modulations of Arabidopsis thaliana phenotype: effects on stomata, actin-cytoskeleton and on the expression of selected genes
Baluška F. 1, Šamajová O. 1, Schlicht M. 1, Braun S. 2, Ulbrich23 A., Jansen M. 2, Ka Hahn4, Kriegs• B. 4 and Schulz M. 4
1IZMB (Institut für Zelluläre und Molekulare Botanik) Universität Bonn 53115 Bonn, Germany 2JPPC Jülich Plant Phenotyping Centre, ICG-3-Phytosphäre, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany 3Fachhochschule Osnabrück, Gemüseproduktion und Verarbeitung 49090 Osnabrück, Germany 4IMBIO (Institut für Molekulare Physiologie und Biotechnology der Pflanzen), Universität Bonn 53115 Bonn, Germany
Monoterpenes are reported to have beneficial effects on plants as they protect them from oxidative damage, increase high temperature tolerance and are essential for plant defence strategies against herbivors. On the other hand, monoterpenes can act as allelochemicals by suppressing the growth of neighbouring plants. Those negative effects are developed during long term exposure and fumigation with higher concentrations of monoterpenes but also by prolonged exposure to volatiles of aromatic plants. Thus, the manifestation of positive or negative responses of a target plant exhibits a dose and time dependency. In the presented study negative effects of camphor and menthol on 3 three weeks old Arabidopsis thaliana used as a target plant were investigated. Two fumigation periods of 96 h with 10 mg camphor / L resulted in phenotypic modifications such as reduced growth, alterations in plant body shape and rosette structure, as well as decrease of the quantum yield indicating a lowered photosynthetic performance. Camphor and menthol, as well as volatile bouquet of aromatic plants, induced permanently opened stomata which were unable to close. By studying the actin-cytoskeleton of cotyledones of a stably transformed line of Arabidopsis thaliana transformed with the actin reporter, the 35S::GFP:FABD construct, after fumigation with 10 mg / L camphor and 5 mg / L menthol; a reorganisation and partial disruption of F-actin was observed. The aberrant organization of the actin cytoskeleton is supposed to be the major reason for the unability to accomplish closure of stomata after monoterpene fumigation, resulting finally in desiccation and plant death. The response of Arabidopsis plants on fumigation with camphor (10 mg/L) and menthol (5 mg/L) is also characterized by time dependent alterations in the expression of several genes involved in stomatal movement and stress response, such as RD29B (At5g52300); AREB1, AREB2 (accession No. AB017160; AB017161), CER5 (At1g51500); CER6 (At1g68530); LOX2 (At3g45140; MPK3 (At3g45640); PEPCase (At2g42600). These results demonstrate the deleterious effects of monoterpenes, when the target plants are permanently exposed to higher concentrations as they appear naturally; for instance in the Citrus orchards or in the High Chaparral vegetation of South California
49 Effects of acetylcholine on blue-light response of dark-grown Arabidopsis seedlings: nutrition, light quality, and the effect of mutations in the pigment cryptochrome
Bisson, MA 1, Diamond, A 1
1Dept. Biological Sciences, University at Buffalo, Buffalo, New York 14260 E-mail: [email protected]
Dark-grown (etiolated) Arabidopsis plants grow rapidly in the dark, and this high growth rate is rapidly inhibited in response to blue light. The pigment cryptochrome has been implicated in this pathway. The earliest response to light is a transient depolarization, which has been proposed to be due to activation of a chloride channel in a ligand-gated channel family (Lewis & Spalding, 1998). They report that blocking this channel function prevents the light-dependent growth inhibition. At a previous meeting (Slovakia, 2007) we reported that the neurotransmitter acetylcholine (ACh) prevented the light-dependent depolarization and increased decreased the inhibitory effect of light. We suggested that ACh (or a related endogenous molecule) could act as a ligand that modulates the effect of light on the channel in question. We report here further experiments that eliminate some confounding possibilities. We employ blue light instead of white, to narrow range of pigments that might be responsible. We also minimize the use of green as a safe light, since it has been suggested to be antagonist to blue light (Folta 2004). We investigated the possibility that the nitrogen in choline might be acting as a nutrient in increasing plant growth. Choline alone had less effect than ACh, which suggests that this is not true. We also utilized more nutrient-rich media, to relieve any nitrogen limitation on growth. In all cases, we show that ACh inhibits the growth response to light. We also utilize double mutants in the two cryptochrome genes implicated in the pathway (cry1cry2) to determine whether the effect is due to inhibition of the cryptochrome-initiated pathway or a competing pathway that masks the cryptochrome response. We explore models of the mechanism of ACh action, taking into account new models of broader activities of ACh in animals.
Lewis, B, Spalding E (1998). J Membrane Biol 162: 81-90 Folta, KM (2004). Plant Physiol 135: 1407-1416
50 Lectins as determinants of cell recognition in cyanolichens through Peltigera canina
Díaz, EM 1, Sacristán, M 1, Legaz, ME 1, Alarcón, B 1, Vicente, C 1
1Faculty of Biology. Dpeartment of Plant Biology , Complutense University, Madrid, Madrid 28040 E-mail: [email protected]
Lichen are symbiotic between a fungus and a cyanobacterium (cyanolichens) or green alga (chlorolichens), joined to form a new biological entity different from its individual components. Both bionts appear in nature among a mixture of millions of non-symbiotix microorganisms, and mechanisms of compatible combination are required. Thus, specificity is required for the lichen association. Specificity can be defined in this context as the preferential, but not exclusive, association of a biont with another. Recognition of compatible algal cell is carried out by specific lectins produced and secreted by the potential mycobiont. Some lectins from phycolichens have been characterized as glycosylated arginases which bind to an algal cell wall receptor indentified as an α-1.4- polygalactosylated urease. The binding is improved by Ca+2 and Mn+2 in a similar way to that described for legume lectins. Two forms of glycosylted arginases seem to be involved in cell contact between phyco- and mycobiont, whereas a second, secreted arginase produces recruitment of compatible algal cells near the surface of fungal arginase is internalised, increasing the levels of algal putrescine, which promotes chloroplast disorganization, activation of glucanases and breakdown of the cell wall with loss of the prototoplast. Evolution of symbiotic relationships implies then the synchronization of cell division and lectin receptor production, probably as a consequence of the perception of environmental factors. The structural and functional similarities between lectins from cyano- and phycolichens, it could be expected that the recognition mechanism in phycolichens could be expanded to cyanolichens. We worked with the cyanolichen Peltigera canina which contain a blue-green alga (a Nostoc sp.). This lichen secreted arginase ti the incubation media, that was labelled with FITC showing lectin activity, since it bind to the cell wall cyanobionts, apparently using a polygalactosylated urease as specific cell wall ligand. Desorption of arginase from the algal cell wall is achieved by 50 mM α- D- galactose, indicating that lectin use the same polygalactosilated ligand that foun in chloroplichens. In addition, other receptors can be developed by both photobionts since concanavalin A, a lectin from Canavalia ensiformis specific from α- D-glucose and α- D-mannose also binds to several cyanobionts and chlorobionts. The nature of the ligand, definided as glycosylated urease, has been confirmed by affinity chromatography using a stationary phase constituted by arginase immobilized on agarose, resulting in the elution of only one fraction enriched in urease activity. Urease was cytochemically located by using recently isolated cyanobionts from P. canina thalli incubated with 40 mM urea and 10mM cobalt choride. Images obtained show that black deposits of cobalt carbonate, revealing urease activity, are mainly located at the cell wall of cyanobionts, although the occurrence of some polydisperse, small granules inside the cells indicates intracellular urease activity. The binding of fungal arginase to glicosylated urease in the algal cell wall implies an affinity mechanism between some specific amino acid residues of the lectin and specific sugar residues of the glycoside moiety of the ligand. In any way, the ability of fungal lectins developing arginase activity seems to be functional not only for chlorobints but also for cyanobiont containing thalli
51 Scald-susceptible cultivars of sugarcane promote signalling to induce the synthesis of a virulence factor in Xanthomonas albilineans
Blanch, B 1, Vicente, C 1, Alarcón, B 1, Díaz, EM 1, Quintana, J 1
1Faculty of Biology. Department of Plant Biology, Complutense University, Madrid, Madrid 28040 E-mail: [email protected]
Leaf scald is a vascular disease of sugarcane plants caused by Xanthomonas albilineans. Scalded leaves show white-yellowish streaks alternating with green zones in parallel to the main veins. The white-yellowish streaks show both phloem and xylem completely occluded by the gum and the overall mesophyll appears to be full of this bacterial secretion, as revealed by scanning electron microscopy. The gum in conducting tissues has been purified from juices obtained from scalded stalks and it was identified as a xanthan-like polysaccharide composed by repeated tetrameric units containing two rests of fructose, one of mannose and one of glucuronic acid. Hydrolysis of xanthan with selective mannosidases and β-1,4-glucanases reveals that the macromolecule consists of a linear, β-1,4- backbone of β-glucose units to which mannose in β-1,3 bonds is linked. Since xanthans contain glucuronic acid, the ability of Xanthomonas to produce an active UDP glucose dehydrogenase is often seen as a virulence factor. X. albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. Kinetics of enzymatic oxidation of NADPH is linearly dependent on the amount of oxygen supplied. N-Terminal sequence has been determined as IQPYNH. X. albilineans axenically cultured does not secrete xanthans to liquid media but they are produced in inoculated sugarcane tissues. This host-dependence can be explained on the basis of the action of bacterial proteases upon the dehydrogenase. In vitro enzymatic assay of UDP-glucose dehydrogenase from X. albilineans requires the addition of a protease-inhibitors cocktail to cell-free extracts, since bacterial proteases rapidly hydrolyses the enzyme in solution. The addition of low amounts of 8- azaguanine and chloramphenicol to the culture medium do not impede the production of the dehydrogenase that requires concentrations higher than 0.3 mM of both antimetabolites to inhibit its synthesis, concentration that is sufficient to inhibit the production of proteases. Glycoproteins from sugarcane, the natural host of the bacterium, that are produced as a response to the secretion of bacterial elicitors, also assure the production of the active enzyme by inhibiting bacterial proteases.
52 Cytology of compatible and incompatible hyphal interactions in arbuscular mycorrhizal fungi
Sbrana, C 1, Giovannetti, M 2
1Istituto di Biologia e Biotecnologia Agraria UO Pisa, CNR, Pisa, 56124 2Dipartimento di Biologia delle Piante Agrarie, Università di Pisa, Pisa, 56124 E-mail: [email protected]
Arbuscular mycorrhizal (AM) fungi are 400 million years old obligate biotrophs, which live symbiotically in the roots of most land plants. Fossil records and DNA sequence data confirmed that these ancestral organisms have coevolved with their host plants since the early colonization of land. Efficient survival strategies are active in AM fungi, i.e. a wide host range, the ability of multiple germination, chemotropic guidance to host roots, hyphal morphogenesis elicited only by host-derived signals. Further mechanisms evolved by AM fungi to increase their fitness involve the phenomenon of hyphal fusion (anastomosis), which occurs widely between hyphal tips belonging to the same individual and to different individuals of the same isolates (Giovannetti et al., 1999; 2003; 2004). Recently, anastomoses were also detected in interactions between hyphae belonging to genetically different isolates of Glomus intraradices (Croll et al., 2009). Living-cell microscopy revealed protoplasmic streaming in anastomosing hyphae, while viability staining showed protoplasmic continuity and nuclear migration in perfect fusions. Hyphal tips of individuals belonging to different species and to geographically different isolates of G. mosseae never underwent anastomoses: most hyphal contacts led to intermingling without interference and a few interactions showed pre-fusion incompatible responses. The main feature of pre-fusion incompatibility was represented by growth arrest followed by protoplasm withdrawal and septa formation in contacting hyphae, prior to or after interactions. Pre-contact tropism and growth reorientation suggested the involvement of specific signals guiding early hyphal recognition events, occurring before incompatible interactions. Analyses of interactions among genetically different isolates of G. intraradices showed some perfect fusions and pre- and post-fusion incompatible responses, characterised by hyphal protoplasm withdrawal following anastomosis, with septa formation between involved hyphae. The experimental system used allowed the detection of cytological changes during compatible or incompatible interactions among AM fungal populations and of their ability to undergo genetic recombination, fundamental for the maintenance of AM fungal diversity.
53 Lectins as determinants of cells recognition in cyanolichens through peltigera canina
Díaz, EM 1, Sacristán, M 1, Legaz, ME 1, Alarcón, B 1, Vicente, C 1
1Faculty of Biology. Department of Plant Biology, Complutense University, Madrid, Madrid 28040 E-mail: [email protected]
Lichen are symbiotic between a fungus and a cyanobacterium (cyanolichens) or green alga (chlorolichens), joined to form a new biological entity different from its individual components. Both bionts appear in nature among a mixture of millions of non-symbiotix microorganisms, and mechanisms of compatible combination are required. Thus, specificity is required for the lichen association. Specificity can be defined in this context as the preferential, but not exclusive, association of a biont with another. Recognition of compatible algal cell is carried out by specific lectins produced and secreted by the potential mycobiont. Some lectins from phycolichens have been characterized as glycosylated arginases which bind to an algal cell wall receptor indentified as an alfa-1.4- polygalactosylated urease. The binding is improved by Ca+2 and Mn+2 in a similar way to that described for legume lectins. Two forms of glycosylted arginases seem to be involved in cell contact between phyco- and mycobiont, whereas a second, secreted arginase produces recruitment of compatible algal cells near the surface of fungal arginase is internalised, increasing the levels of algal putrescine, which promotes chloroplast disorganization, activation of glucanases and breakdown of the cell wall with loss of the prototoplast. Evolution of symbiotic relationships implies then the synchronization of cell division and lectin receptor production, probably as a consequence of the perception of environmental factors. The structural and functional similarities between lectins from cyano- and phycolichens, it could be expected that the recognition mechanism in phycolichens could be expanded to cyanolichens. We worked with the cyanolichen Peltigera canina which contain a blue-green alga (a Nostoc sp.). This lichen secreted arginase ti the incubation media, that was labelled with FITC showing lectin activity, since it bind to the cell wall cyanobionts, apparently using a polygalactosylated urease as specific cell wall ligand. Desorption of arginase from the algal cell wall is achieved by 50 mM α- D-galactose, indicating that lectin use the same polygalactosilated ligand that foun in chloroplichens. In addition, other receptors can be developed by both photobionts since concanavalin A, a lectin from Canavalia ensiformis specific from α- D-glucose and α- D-mannose also binds to several cyanobionts and chlorobionts. The nature of the ligand, definided as glycosylated urease, has been confirmed by affinity chromatography using a stationary phase constituted by arginase immobilized on agarose, resulting in the elution of only one fraction enriched in urease activity. Urease was cytochemically located by using recently isolated cyanobionts from P. canina thalli incubated with 40 mM urea and 10mM cobalt choride. Images obtained show that black deposits of cobalt carbonate, revealing urease activity, are mainly located at the cell wall of cyanobionts, although the occurrence of some polydisperse, small granules inside the cells indicates intracellular urease activity. The binding of fungal arginase to glicosylated urease in the algal cell wall implies an affinity mechanism between some specific amino acid residues of the lectin and specific sugar residues of the glycoside moiety of the ligand. In any way, the ability of fungal lectins developing arginase activity seems to be functional not only for chlorobints but also for cyanobiont containing thalli.
54 Towards functional characterization of plant class II formins: first lessons from outliers.
Cvrčková, F 1, Oulehlová, D 2, Grunt, M 1, Zárský, V 1
1Faculty of Sciences, Department of Plant Physiology , Charles University in Prague, Prague, CZ 128 44 2Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague, CZ 160 00 E-mail: [email protected]
Plant cells are able to diversify their surface by re-structuring their cytoskeleton and directing exocytosis to distinct portions of the cell cortex, which we have recently termed \"activated cortical domains \" (ACDs; Zárský et al., New Phytologist, in press). ACD diversification is a central mechanism of plant cell morphogenesis, contributing also to cell and tissue polarity, which is, in turn, crucial for developmental processes, including but not limited to exploratory growth both on the level of single cells (e.g. root hairs or pollen tubes) and whole organs. A number of proteins that may participate in ACD diversification have been identified, including members of several large protein families such as e.g. Rop GTPases and their interactors, subunits of the exocyst complex, receptor-like kinases, enzymes locally modifying membrane composition, or actin-organizing FH2 proteins. Combinatorial use of paralogs could thus contribute to generation of ACD diversity. We are focusing on members of one such a large protein family – the formins (FH2 proteins). Formins of higher plants can be divided into two clades; according to publicly available data (www.arexdb.org), some representatives of both clades exhibit differential gene expression between arabidiopsis trichoblasts and atrichoblasts, i.e. between cell types that differ by the presence of a single specific ACD. For one of the plant formin clades, Class I, several experimental reports have confirmed a role in actin nucleation and other aspects of cytoskeletal organisation, previously documented for yeast and metazoan formins. However, plants possess also a second, considerably divergent and hitherto uncharacterized, FH2 protein group, the Class II formins, whose members exhibit complex gene structure and usually low expression levels. Since experimental study of formin function is hampered by genetic redundancy within the large gene family, we have focused on two arabidopsis Class II formins that represent outliers of this gene family in terms of either domain structure (AtFH16) or overall sequence divergence (AtFH12). We have cloned a cDNA encoding AtFH16, and verified that AtFH12 is expressed under conditions predicted from publicly available microarray data. Heterologous expression of AtFH16 in budding yeast leads to a phenotype suggesting interference with endogenous formin function. Disruption of the AtFH16 gene does not produce an obvious phenotype, indicating functional redundancy within the Class II formin family. However, T-DNA insertion within AtFH12 inhibits root growth and aggravates the toxic effects of GFP-tagged mouse talin (GFP-mTalin) expression, known to cause extensive actin bundling. Taken together, these observations suggest that, despite substantial sequence divergence, plant Class II formins retained the conserved function in cytoskeletal organisation, shared with other FH2 protein clades.
This work was supported by MSM0021620858 and LC06004 projects.
55 Involvement of auxin-binding proteins and auxin in response of maize seedling to blue light
Čudejková, M 1, Pěnčík, A 2, Rolčík, J 2, Fellner, M 1
1Laboratory of Molecular Physiology, Palacky University in Olomouc, Faculty of Science, Olomouc, 78371 2Laboratory of Growth Regulators, Palacky University in Olomouc and Institute of Exp. Botany, Olomouc, 78371 E-mail: [email protected]
Auxin-Binding Protein 1 (ABP1), a putative auxin receptor has been extensively studied. It was found that in Arabidopsis, ABP1 is essential for embryo development, and it participates in auxin-mediated cell elongation in different species. In maize, several ABPs have been identified but their roles are still not understood. The aim of this study is to contribute to the understanding of the role of maize ABP1 and ABP4 during growth and development, with special reference to seedlings developed in blue light (BL). We have observed, that BL decreases the level of free IAA in maize aerial organs. Using maize abp1 and abp4 single mutants, and the abp1abp4 double mutant we have found that ABP1 and/or ABP4 regulate this BL-induced response. However, extent of the elongation of coleoptile and mesocotyl in BL does not correlate with the levels of free IAA. Interestingly, we observed that BL inhibits root elongation in WT plants, but not in abp1, abp4 single and double mutants. Our results indicate that in maize, ABPs positively influence elongation growth of etiolated seedlings, and that ABP1 and ABP4 are involved in BL-signaling pathway that regulates auxin accumulation. Additionally, the data suggest that ABP1 and ABP4 are engaged in BL-induced inhibition of root elongation.
This work was supported by grant from Ministry of Education of the Czech Republic to MF (grant no. 1P05ME792).
56 Light alters plant elongation responses to auxin
Fellner, M 1, Čudejková, M 1, Juricić-Knezev, D 1, Plotzová, R 1, Pěnčík, A 2, Rolčík, J 2, Bořucká, J 1, Vaclová, T 1, Řehulka, J 1, Zalabák, D 1, Bergougnoux, V 1
1Laboratory of Molecular Physiology, Palacky University in Olomouc, Olomouc, 78371 2Laboratory of Growth Regulators, Palacky University in Olomouc and Institute of Exp. Botany, Olomouc, 78371 E-mail: [email protected]
Despite the recent advances, many fundamental issues of interaction between light and hormone signaling pathways involved in plant growth remain to be uncovered. In model plants Arabidopsis, tomato and maize we investigated effects of light on plant growth responses to exogenous auxins. In conditions in vitro and in the dark, blue light (BL) and red light (RL), NAA and 2,4-D in concentration-dependent manner reduced elongation of hypocotyl in intact Arabidopsis and tomato plants, and reduced coleoptile growth in intact maize seedlings. When Arabidopsis WT plants developed in BL, hypocotyl responsiveness to auxin strongly diminished, whereas in RL, the hypocotyls responded to auxin similarly like in the dark. Weak responses to exogenous auxin under BL were also observed in Arabidopsis mutants, cry2 and hy2. Unlikely, mutant cry1 grown in BL responded to auxin in the same extent like in the dark. These data indicate that functional photoreceptor CRY1 is involved in BL-induced reduction of hypocotyl responsiveness to exogenous auxin. In tomato, NAA inhibited hypocotyl elongation significantly more in etiolated plants than in BL- and RL-grown seedlings. Low seedling responsiveness to NAA in BL and RL was associated with dramatically higher levels of free IAA in light-grown seedlings in comparison with etiolated plants. Compared to corresponding WT, etiolated seedlings of 7B-1 mutant affected in BL responses exhibited resistance to NAA-induced inhibition of hypocotyl elongation. The response was not affected by BL or RL, and it was associated with weak BL- or RL-induced augment in the level of endogenous IAA. Other analyses suggest that BL- and RL-induced reduction of sensitivity to the inhibitory effect of NAA is mediated by photoreceptor CRY1. Surprisingly, BL amplified hypocotyl sensitivity to the inhibitory effect of 2,4-D, and the response was not affected by cry1 mutation. These data suggest that the responsiveness of tomato hypocotyls to the inhibitory effects of NAA and 2,4-D is likely regulated by different mechanisms. In older dense-sensitive maize hybrids, relative inhibition of coleoptile elongation by NAA in intact seedlings was essentially smaller in BL or RL than in the dark. Interestingly, the lower responsiveness of coleoptiles to NAA in BL correlated with distinct decline in the level of free IAA in BL-grown seedlings. In comparison with old hybrids, coleoptile in modern dense-resistant hybrid was less sensitive to NAA in the dark, which corresponded with low level of IAA found in etiolated coleoptiles. In opposite, coleoptile in the modern hybrid grown in BL was more sensitive to NAA than that in the older maize lines. It was associated with higher amount of free IAA in BL-grown coleoptiles in modern than in older hybrids. The data support our hypothesis that in maize, the selection of modern hybrids led to the alteration in interaction between light and auxin signaling involved in elongation growth. Finally, analysis of elm1, a phytochrome-deficient mutant in maize indicated that phytochromes are involved in BL- and RL-induced reduction of coleoptile growth responses to exogenous auxin. Our results confirmed the existence of interaction between light and auxin signaling in plant growth. Analyses also suggest the existence of diverse mechanisms of the cross-talk between light and auxin in different plant species.
This work was supported by grant from Ministry of Education of the Czech Republic (gr. no. 1P05ME792)
57 Shotgun proteomics of protein complexes using mass spectrometry.
Fukao, Y 1
1Plant Science Education Unit, The Graduate School of Biological Scienc, Nara Institute of Science and Technology, Ikoma, 6300101 E-mail: [email protected]
Interactions between proteins are crucial for most of the molecular processes in cells. Therefore, identification of protein-protein interaction will yield a better understanding of the cellular machinery on the molecular level. However, the development of such networks for plant proteins has been slowed, because there was no useful high-throughput technique. To address this challenge, we have developed a powerful method to identify the intracellular protein network using LTQ-Orbitrap XL. In this study, complex proteins were purified using affinity column coupled with antibody. The protein samples were directly digested in solution, and then performed shotgun analysis. We would like to discuss about usefulness of this method.
58 Reaction of a light-induced chloroplast movement to local and systemic stimuli and relation to photoinhibition.
Naus, J. 1, Hlavackova, V. 1, Rolencova, M. 1 1Laboratoty of Biophysics, Faculty of Science, Palacky University, Olomouc, 771 46 E-mail: [email protected]
Light-induced chloroplast movement in leaves is a very conservative and wide spread phenomenon in plant kingdom. Chloroplast movement is assumed to protect chloroplasts against photoinhibition. We have studied this movement in leaves of tobacco (Nicotiana tabacum cv. Samsun) in relation to local and systemic stimuli and to photoinhibition processes. The local stimulus was realized by strong light, the systemic one by strong light or burning. Two methods of chloroplast movement detection have been developed. The first method is based on the application of the SPAD chlorophyll-meter. The other method combines cold white light, optical filters and light guides with spectral radiometer LI-1800. The method is called transmittance in partially collimated light Tc, because only apart of the transmitted light is detected. A stronger light (above 400 µmol photons m-2 s-1) caused the movement of chloroplasts to the anticlinal cell walls and the light of lower intensity (50 µmol photons m-2 s-1) causes reverse changes. Successive application of the two light intensities enabled to study the chloroplast movement in an oscillating regime. The state of photosynthetic apparatus at the thylakoid membranes in the chloroplasts was detected by the fluorescence induction method (O-J-I-P curve) As far as systemic stimuli are concerned, it has been shown that systemic stimuli do not influence the light induced chloroplast periodic movement in a detectable range. The light was an essential signal governing the movement with no detectable influence of long-distance signaling pathways. This indicates a control of this motion at the intracellular level. On the other hand, the local irradiance by light of two intensities (400 and 800 µmol photons m-2 s-1 of PAR) and three spectral regions (blue, white, red) has indicated an effect of light intensity and spectrum on both chloroplast movement and photosynthetic performance. The red light did not induce the chloroplast movement but caused changes in the maximal photochemical efficiency of photosystem II (Fv/Fp) similar to those caused by white and blue light. Concerning the relation of chloroplast movement and photoinhibition processes, we have found no component of changes in Fv/Fp correlating with the chloroplast movement. Two fast phases of Fv/Fp changes, a decrease in strong light and an increase in lower light, were the same for blue (chloroplast movement) and red (no chloroplast movement) lights. Thus, the detected changes of Fv/Fp are of photochemical origin. A part of the Fv/Fp decrease could be ascribed to a photoinhibitory effect. The weak blue light was more effective in a long lasting regeneration phase (100 to 300 minutes) in which no detectable chloroplast movement was registered. Chlorophyll fluorescence gives information only about the state of chloroplasts located close to the illuminated surface. These chloroplasts were exposed to the same photoinhibitory damage independently on their movement. Our data indicate a pivotal role of the intensity and spectral composition of the incident light on chloroplast movement of tobacco instead of an effect of a systemic signal. Furthermore, it seems that chloroplast movement regulates the light distribution in the leaf but does not protect the exposed chloroplasts from photoinhibition.
Acknowledgement. This project has been supported by The Ministry of Education of the Czech Republic (MSM 6198959215).
59 Blebbistatin inhibits chemotactic response of smut teliospores towards high and middle molecular mass glycoproteins present in sugarcane juices
Quintana, J 1, De Armas, R 2, Vicente, C 1, Legaz, ME 1, Santiago, R 1, Sacristán, M 1
1Faculty of Biology. Department of Plant Biology, Complutense University, Madrid, Madrid 28040 2Faculty of Biology. Department of Plant Physiology, Havana University, Havana, Havana 32333 E-mail: [email protected]
Sporisorium scitamineum (Syd.). Piepenbr. & Oberw. (= Ustilago scitaminea Sydow & P. Sydow), wich causes sugarcane smut, is one of the most important pathogens of this crop. It produces important looses in yields. This phytopathogen is a basiodiomycete belonging to the order Ustilaginales. Sugarcane smut was one of the first diseases of sugarcane to be recognized because of the conspicuous whip-like sorus produced by the fungus in infected plants. Sori arise either from the terminal meristem or from side shoots of infected stalks and contain a central core of parenchyma and vascular host tissue around which a thin cylinder of teliospores is produced. The dycariotic mycelium is the pathogenic cell type of the life cycle. It exhibits mycelial growth and colonizes meristematic tissues. Thus, colonization of the stalks takes place and diploid teliospores are formed in the host tissues. The ability of sugarcane glycoproteins to induce teliospore cytoagglutination has been reported yet. This ability is related to de capacity of high and middle molecular mass glycoproteins (HMMG and MMMG) obtained from sugarcane juices to bind selectively to smut teliospore wall receptors. After binding, cell aggregation occurs in parallel to a loss of the germination ability of recruited teliospores. However, the interaction between glycoproteins and receptors present in the smut teliospore wall is different depending on the resistance of the cultivar to smut. HMMG and MMMG from Mayarí, resistant cultivar, show high affinity to bind N- acetyl-D-glucosamine residues in glycoproteins of the spore wall whereas HMMG and MMMG from Barbados, sensitive to infection, interact less efficiently. Therefore, production of HMMG and MMMG and interaction with receptors present in teliospore wall is a plant response to smut disease that proves how specific and complex is the relation between host and pathogen. Chemotaxis is involved in a wide variety of biological processes including host-pathogen recognition. Smut fungus teliospores are chemotactically sensitive to HMMG and MMMG. Spores suspended in distilled water are able to move towards sugarcane juice fractions containing HMMG or MMMG. This phenomenon has been reported in two cultivars, Mayarí, resistant, and Barbados, sensitive to smut. In both cases movement of teliospores occurs and is in Barbados where a higher chemotactic index has been obtained. On the other hand, teliospores are more sensitive to HMMG than MMMG. The aim of this study is to verify a direct role of cytoskeleton in this response of teliospores to chemoattractants such as HMMG and MMMG. To achieve this objective, teliospores were treated with blebbistatin, a specific inhibitor of myosin II-dependent cell processes. A suspension of teliospores on blebbistatin was added to a plate where a sealed capillary containing glycoproteins at different concentrations had been inserted. After 15 hours, capillary content was observed under light microscopy. Photographs were obtained in order to quantify the amount of teliospores that had moved toward HMMG and MMMG fractions. In addition, we have assessed effect of blebbistatin upon cell polarization by fluorescence microscopy to verify a direct role of blebbistatin in the reorganization of cytoskeleton that take place previous chemotactic displacement.
60 Regulation of aquaporin by protein phosphorylation in fruit and flower
Shiratake, K 1, Nakagawa, Y 1, Sakakibara, I 1, Miyashita, K 1, Yamaki, S 1
1Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 E-mail: [email protected]
Aquaporins are responsible for water transport across biomembranes and play a crucial role in the regulation of water status in plants. Recent reports showed that aquaporins transport substances other than water, such as glycerol, urea, ammonium, boron, silicon, arsenate, carbon dioxide, hydrogen peroxide and nitrogen monoxide. Therefore aquaporin function not only for water transport but also for many solute transports. More than 30 aquaporin genes were identified in Arabidopsis and rice. Plant aquaporins are classified into 4 groups: plasma-membrane intrinsic protein (PIP), tonoplast intrinsic protein (TIP), Nodulin 26-like intrinsic protein (NIP) and small basic intrinsic protein (SIP). PIPs and TIPs are responsible for water transport across plasma membrane and vacuolar membrane, respectively. PIPs are further classified into two subgroups, i.e. PIP1 and PIP2. PIP2 has higher water transport activity than PIP1. Our interests are regulation and function of aquaporins in horticultural crops, such as fruits and flowers. Gene expression and protein level of PIPs and TIPs were detected in pear fruit development. Gene expression and protein level of TIPs were especially high in young fruit and dramatically decreased in the middle of fruit development. On the other hand, gene expression and protein level of PIPs were almost constant in fruit development. Therefore we considered that water transport PIPs is regulated by post-translational regulation. Post-translational modification of aquaporins has been suggested, such as phosphorylation, methylation, glycosylation, oligomerisation, vesicle trafficking and non-covalent protein modification by pH and calcium ion. It has been suggested that water transport activity of PIP2 is increased by phosphorylation and decreased by dephosphorylation. So we focused to protein phosphorylation of PIP2 in this study. We tried to prepare phospho-specific antibodies against putative phosphorylation sites, i.e. Ser115 and Ser280. Although the phospho-specific antibody against Ser115 did not react to microsomes from pear fruit, that against Ser280 recognizes phosphorylated Ser280 specifically. We determined phosphorylation state in fruit, suspension cells and flower using this antibody. Phosphorylation state of PIP2 in pear fruit changed dramatically with fruit diurnal growth. Phosphorylation state of PIP2 decreased in pear suspension cells one hour after salt and osmotic stress treatments. Phosphorylated PIP2 increased with flower opening of Japanese morning glory. These results show the presence of phosphorylational regulation of aquaporins in many events of plants, including fruit growth and flower opening.
*This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and Japan Society for the Promotion of Sciences (JSPS).
61 Ozone-inducible glycine-rich peptides as plant prions?
Yokawa, K 1, Kagenishi, T 1, Kawano, T 1
1Faculty and Graduate school of Environmental Engineering, The University of Kitakyushu, Kitakyushu, Fukuoka 808-0135 E-mail: [email protected]
Ozone-inducible (OI) genes have been isolated from Atriplex canescens (saltbush) and shown to be responsive to exposure to ozone, SO2 and water deficit. OI peptides contain repeat sequence (YGHGGG) tandemly repeated for 8-10 times, which tightly binds copper. The putative role for OI has been proposed to be active anti-oxidants by chelating the redox-active metals. Our previous study concerning the action of prion protein has revealed that the copper-binding regions in prion protein (such as VNITKQHTVTTTT) interacts with free tyrosine or tyrosine-containing peptides (including tyrosine-rich sequence within prion protein) in the presence of H2O2, finally leading to robust generation of superoxide. We found that tyrosine-residues on peptide sequences contribute as a substrate for this peroxidative ROS generating reaction. The key common structure among the redox-active peptide sequences found in mammals and chicken is the presence of Cu- anchoring His residue(s) placed at the vicinity of glycine-rich sequences. In the present study, we examined the peroxidative catalytic activity of the OI peptides leading to generation of superoxide and break-down of H2O2. Here the generation of superoxide was assessed with the superoxide a specific chemiluminescence of Cypridina Luciferin Analog (CLA). This result suggests that the OI peptides in plants may play role as both pro-oxidants and anti-oxidants thus the fate of plant cells under oxidative stress.
62 Role of auxin, auxin-binding proteins and light in the development of maize seedlings
Juricić-Knezev, D 1, Čudejková, M 1, Pěnčík, A 2, Rolčík, J 2, Bergougnoux, V 1, Fellner, M 1
1Department of Cell Biology and Genetics, Palacky University in Olomouc, Olomouc, 783 71 2Laboratory of Growth Regulators, Institute of Experimental Botany ASCR, Olomouc, 783 71 E-mail: [email protected]
Modern maize (Zea mays L) hybrids developing erect leaves show less sensitivity to dense planting and reduced responsiveness to auxin and light in comparison to older, density sensitive varieties. In addition it has been found that in modern maize hybrid the expression of Auxin-Binding Protein 4 (ABP4), a hypothetical auxin receptor, is impaired. In this study we investigated the role of auxin, ABPs and light in maize growth and development. In vitro, etiolated maize mutants affected in ABP1 and/or ABP4 genes showed less sensitivity to auxin in comparison to wild type (WT). Analysis of endogenous auxin in etiolated coleoptiles revealed that abp1/abp4 double mutant contained the highest concentration of free IAA, indicating that ABP1 and ABP4 cooperate in decreasing the content of free IAA in this maize organ. Interestingly, we found that in abp4 and abp1/abp4 double mutant red light (RL) strongly promoted elongation of coleoptile whereas in WT and abp1 RL did not have any effect on coleoptile elongation. In the greenhouse (in vivo), abp1 and abp4 single mutants developed more and less erect leaves, respectively, while abp1/abp4 double mutant had leaves less vertical than WT. abp4 plants developed the tallest stature with longer leaves than WT, although abp1 mutant developed the longest and the widest leaves. Our data suggest that auxin regulates development of maize seedlings through ABPs and that ABP4 mediates RL-induced inhibition of coleoptile elongation. The data support the existence of interaction between auxin and light in maize growth and development. In addition, these findings suggest the involvement of ABPs in leaf angle development, as well as an important role of ABP1 in maize leaf morphology.
Key words: maize (Zea mays L), plant development, light, auxin, Auxin-Binding Proteins (ABPs).
This work was supported by an EU FP6 Marie Curie Research Training Network VaTEP (grant No. MRTN-CT-2006-035833) and grant no. 1P05ME792 from Ministry of Education of the Czech Republic to MF.
63 Role of acetylcholine in plant cell elongation
Fornaciari, S 1, Anceschi, E 1, Arru, L 1
1Dept. of Agricultural and Food Sciences, University of Modena and Reggio Emilia, Reggio Emilia, 42100 E-mail: [email protected]
We have investigated the role of acetylcholine (ACh) in cell elongation, and the possible influence of ACh on the action of auxin in its plant cell growth-regulating activity. Hypocotyl segments represent an ideal experimental system to look for the contribute of different factors influencing some plant cell behaviour. Tomato (Solanum lycopersicon) hypocotyls elongate when treated with auxin (acid growth theory), and this auxin induced elongation correlates with the expression of an expansin gene (LeEXPA2) (Catalá et al. 1997, Caderas et al. 2000), member of a multigene family of extracellular proteins that mediate cell wall extension and relaxation during growth (McQueen-Mason et al. 1992; McQueen-Mason and Cosgrove 1994, Cosgrove 2000). We explored the possible signaling role of ACh in mediating the auxin-induced expansin gene expression. We cut 1 cm long hypocotyl segments just below the apical hook of 5 days old etiolated tomatoes, and treated them with different combinations of ACh 50 uM, sucrose, and auxin 5 uM. After 2h of treatment, ACh seems to barely induce expansin mRNA expression (about 1,5 fold change with respect to control), although when ACh is supplied with 2% sucrose, LeEXPA2 transcript expression is clearly induced (about 4 fold). But the most interesting result cames out when ACh and auxin are supplied together. Their action is more than additive: where auxin alone determines a 16- fold expansin transcription, the transcript abundance grows up to 39 times when auxin is supplied with ACh. More than this, when sucrose is present in the medium, there is the major induction registered: about 50-fold change with respect to control. These results deserve to be more carefully investigated because of the potential of their implications, and at the same time they open new perspectives and possibilities in unravelling the role of sugars as signalling molecules.
References
Caderas D, Muster M, Vogler H, Mandel T, Rose JKC, McQueen-Mason S, Kuhlemeier C (2000) Limited correlation between expansin gene expression and elongation growth rate. Plant Physiol 123:1399–1414
Catalá C, Rose JKC, Bennett AB (1997) Auxin regulation and spatial localization of an endo-1,4-B-d- glucanase and a xyloglucan endotransglycosylase in expanding tomato hypocotyls. Plant J 12:417–426
Cosgrove DJ (2000) Loosening of plant cell walls by expansins. Nature 407:321–326
McQueen-Mason S, Durachko DM, Cosgrove DJ (1992) Two endogenous proteins that induce cell wall extension in plants. Plant Cell 4:1425–1433
McQueen-Mason SJ, Cosgrove DJ (1994) Disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension. Proc Natl Acad Sci USA 91:6574–6578
64 The model of cell recognition in phycolichens through a fungal lectin that binds to an algal ligand could be expanded to cyanolichens.
Sacristán, M 1, Vivas, MP 1, Díaz, EM 1, Santiago, R 1
1Faculty of Biology. Department of Plant Biology, Complutense University , Madrid, Madrid 28040 E-mail: [email protected]
Lichens are intimate and long-term symbioses of photosynthetic, unicellular algae or cyanobacteria and heterotrophic fungi joined to form a new biological entity different from its individual components. Specificity required for the lichen association can be defined in this context as the preferential, but not exclusive, association of a biont with another. Recognition of compatible algal cells is carried out by specific lectins produced and secreted by the potential mycobiont. However, lichen phenolics are not involved in the recognition process, in contrast to that found for other plant symbioses, such as mycorrhizal or Rhizobium legume associations. Some lectins from phycolichens have been characterized as glycosylated arginases which bind to an algal cell wall receptor identified as an α-1,4-polygalactosylated urease. The binding is improved by Ca2+ and Mn2+, in a similar way to that described for legume lectins. Two forms of glycosylated arginases seem to be involved in the recognition process, one of them, particulated in the cell wall of fungal hyphae and involved in cell contact between phyco- and mycobiont, whereas a second, secreted arginase produces recruitment of compatible algal cells near the surface of fungal hyphae. When glycosylated urease is lacking from the algal cell wall, fungal arginase is internalized, increasing the levels of algal putrescine, which promotes chloroplast disorganization, activation of glucanases and breakdown of the cell wall with loss of the protoplast. Evolution of symbiotic relationships implies then the synchronization of cell division and lectin receptor production, probably as a consequence of the perception of environmental factors (light and temperature). In this work we attempt to validate this arginase-urease interaction as the universal base not only for phycobionts but also for cyanobionts recognition by using a cyanolichen from the Collemataceae family: Leptogium corniculatum (Hoffm.) Minks, containing Nostoc as cyanobiont. It has been found that recently collected thalli of L. corniculatum secrete arginase after 2h of incubation on 40 mM arginine. The enzyme is efficiently retained by activated agarose beads on which galactosylated urease from the phycolichen Evernia prunastri has previously been attached. Leptogium arginase is completely eluted from the bead using 50 mM D-galactose. This implies that an interaction between the cyanolichen lectin and the phycolichen ligand would justify the recognition process when the cyanobiont was able to synthesize urease and to retain a part of this galactosylated enzyme attached to its cell wall. In addition, Nostoc cells showed intense emission of green fluorescence when they were incubated with secreted arginase from Leptogium labelled with FITC (Fluorescein Isothiocyanate). Similar results were obtained when phycobionts recently isolated from E. prunastri were incubated with FITC- arginase from Leptogium, although fluorescence emission was strongly lower than that observed from the homologous cyanobacteria. Desorption of the lectin from the corresponding cell wall was achieved by incubation of FITC-lectin-labelled cells with 100 mM D-galactose for 1 h, resulting in almost total recovery of fluorescence in the supernatant.
65 Scald-susceptible cultivars of sugarcane promote signalling to induce the synthesis of a virulence factor in Xanthomonas albilineans.
Blanch, M 1, Vicente, C 1, Alarcón, B 1, Díaz, EM 1, Quintana, J 1
1Faculty of Biology. Department of Plant Biology, Complutense University, Madrid, Madrid 28040 E-mail: [email protected]
Leaf scald is a vascular disease of sugarcane plants caused by Xanthomonas albilineans. Scalded leaves show white-yellowish streaks alternating with green zones in parallel to the main veins. The white-yellowish streaks show both phloem and xylem completely occluded by the gum and the overall mesophyll appears to be full of this bacterial secretion, as revealed by scanning electron microscopy. The gum in conducting tissues has been purified from juices obtained from scalded stalks and it was identified as a xanthan-like polysaccharide composed by repeated tetrameric units containing two rests of fructose, one of mannose and one of glucuronic acid. Hydrolysis of xanthan with selective mannosidases and β-1,4-glucanases reveals that the macromolecule consists of a linear, β-1,4- backbone of β-glucose units to which mannose in β-1,3 bonds is linked. Since xanthans contain glucuronic acid, the ability of Xanthomonas to produce an active UDP glucose dehydrogenase is often seen as a virulence factor. X. albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. Kinetics of enzymatic oxidation of NADPH is linearly dependent on the amount of oxygen supplied. N-Terminal sequence has been determined as IQPYNH. X. albilineans axenically cultured does not secrete xanthans to liquid media but they are produced in inoculated sugarcane tissues. This host-dependence can be explained on the basis of the action of bacterial proteases upon the dehydrogenase. In vitro enzymatic assay of UDP-glucose dehydrogenase from X. albilineans requires the addition of a protease-inhibitors cocktail to cell-free extracts, since bacterial proteases rapidly hydrolyses the enzyme in solution. The addition of low amounts of 8- azaguanine and chloramphenicol to the culture medium do not impede the production of the dehydrogenase that requires concentrations higher than 0.3 mM of both antimetabolites to inhibit its synthesis, concentration that is sufficient to inhibit the production of proteases. Glycoproteins from sugarcane, the natural host of the bacterium, that are produced as a response to the secretion of bacterial elicitors, also assure the production of the active enzyme by inhibiting bacterial proteases.
66 System potentials, a novel electrical long distance apoplastic signal in plants induced by wounding
Zimmermann, M.R. 1, Maischak, H. 2, Mithöfer, A. 2, Boland, W. 2, Felle, H.H. 1
1Institute of General Botany, Justus-Liebig-University, Giessen, 35390 2Chemical Ecology, Max-Planck-Institute, Jena, 07745 E-mail: [email protected]
Systemic signalling has been investigated on both a dicot (Vicia faba L.) and a monocot (Hordeum vulgare L.) plant. Stimuli were applied to one leaf (S-leaf) and apoplastic responses were monitored on a distant leaf (target; T-leaf) with micro-electrodes positioned in sub-stomatal cavities of open stomata. Cut-injury of leaves and subsequent addition of a variety of different cations caused voltage transients at the T-leaf, which are neither action potentials nor variation potentials: with respect to the cell interior the initial polarity of these voltage transients is hyperpolarizing; they do not obey the all- or-none rule, but depend on both concentration and type of the added substance, and propagate at 5 to 10 cm/min. It is argued that this response is due to stimulation of the plasma membrane H+-ATPase, a notion supported by the action of fusicoccin which also causes such voltage transients on the T-leaf, whereas ortho-vanadate prevents its propagation. Moreover, apoplastic ion flux analysis reveals that, in contrast to action- or variation potentials, all of the investigated ion movements (Ca2+, K+, H+, Cl-) occur after the voltage change has started. We suggest that these wound-induced ‘system potentials’ represent a new type of electrical long distance signaling in higher plants.
67 Microgravity alters the expression of actin and tubulin genes, of some ROS scavenging factors, and of the auxin transport genes aux1 and eir1 in arabidopsis
Tassone, P. 1, Fortunati, F. 1, Meloni, M.A. 2, Pippia, P. 2, Migliaccio, F. 2
1IBAF, National Research Council (CNR), Monterotondo, Rome 00015 2DPCB, University of Sassari, Sassari, 07010 E-mail: [email protected]
Microtubules are common to all eukaryotic organisms, are higly conserved, and display specialized biological functions. Microtubules are organized in different patterns, relying on a variety of mechanisms for assembly, orientation and function. Recent studies suggest that cells may sense mechanical stimuli, including those due to gravity, through changes in the balance of forces in the cytoskeleton. A study of gene expression in simulated microgravity conditions (Random Positioning Machine, is a laboratory facility equipped to randomly change the gravity vector of an accommodated experiment in a 3-dimensional space), made through qPCR, was performed on seedlings of Arabidopsis thaliana, CV Landsberg, and two auxinic mutants (aux1, eir1) of the same plant. The plants were kept in microgravity for different periods of time: 3, 6 and 24 hours, then stored in RNAlater, and subsequently subjected to mRNA extraction. With the mRNA a qPCR analysis was performed, revealing the expression of the chosen genes involved in actin and tubulin synthesis, in senescence processes and auxin transport. The studied genes were actin (ACT8), tubulin α (TUA4, TUA6), tubulin β (TUB2), FeSOD, CAT1, CAT2, AUX1, EIR1). The results indicated that ACT8, TUA4 TUA6 and TUB2 are notably upregulated by microgravity, even though the effect appeared only after a 3 hour run of the seedlings with fluctuation of their expressions, while AUX1 and EIR1 showed a strong upregulation in the transcript levels after 6 hours. The effect on cell perturbation was seen also on genes notoriously activated by oxidative stress, such as FeSOD, CAT1, and CAT3, which are responsible for ROS scavenging at the chloroplastic and peroxisomal level. These genes were up regulated as the previous ones, but only after 24 hours. This results suggest that when the ultrastructure of the cells starts to be compromised, as it is indicated by the effect on the actin and tubulin genes, also the plant cell metabolism is compromised, and ROS-scavenging proteins are activated.
68 Serotonin and melatonin influence somatic embryogenesis in Coffea canephora P.ex.Fr.
Ramakrishna, A. 1, Giridhar, P. 1, Ravishankar, G.A. 1
1Central Food Technological Research Institute - CSIR, Plant Cell Biotechnology Department, Mysore, 5700 20 E-mail: [email protected]
Melatonin(MEL)and Serotonin (SER) are prominent indoleamines which participate in neural transmission in animals. They are also found to occur in various genera of plants. The precise function of these compounds in plant system at physiological level has not been worked out so far. In the present study, the effect of SER, MEL and calcium channel activator calcium ionophore (A23187) on somatic embryogenesis was investigated in in vitro cultures of Coffea canephora. Embryogenic callus cultured on EG (embryogenic) medium comprising MS basal salts (Murashige & Skooge 1962) along with IAA+BA, only 5% explants responded for embryogenesis. SER/ MEL at 100 µM (without IAA) showed 60% and 65% explants response for somatic embryogenesis, wherein 62±0.1 and 84±2.9 embryos produced from each callus mass respectively. Addition of indoleamine inhibitors viz. p- chlorophenylalanine (p-CPA) at 40µM, and Prozac (fluoexitine hydrochloride) at 20 µM reduced induction of somatic embryogenesis and also endogenous pools of SER, MEL and IAA levels by 40- 70%. EG medium with MEL / SER at 100 µM + 50 µM CaCl2/ 0.1mM calcium ionophore A23187 induced 75% and 85% explants responded for somatic embryogenesis. EG medium with calcium channel blocker verapamil hydrochloride at 1mM and a calcium chelator EGTA at 1mM reduced somatic embryogenesis. The results clearly demonstrated that the endogenous profiles of indoleamines and IAA levels positively influenced somatic embryogenesis response. This response was further substantiated by calcium imaging studies. SER was found to be localized in vascular tissues of the coffee stem, root and somatic embryos, also in endocarp region (husk) of the coffee immature fruits. The outcome of this study will certainly help to further realize the multiple roles of the indoleamines in plant morphogenesis and elucidating SER and MEL dependent cellular signaling mechanisms in plants.
69 Network connectance and autonomy analyses of the photosynthetic apparatus in tropical tree species from different successional groups under contrasting irradiance conditions.
Souza MG1; Ribeiro RV1, De Oliveira RF1, Machado EC1
1ESALQ, Universidade de São Paulo, Piracicaba, São Paulo 13418-900
E-mail: [email protected]
Biological systems are complex dynamical systems whose relationships with environment have strong implications on their regulation and survival. From the interactions between plant and environment can emerge a quite complex network of plant responses rarely observed through classical analytical approaches. The objective of this current study was to test the hypothesis that photosynthetic responses of different tree species to increasing irradiance are related to changes in network connectances of gas exchange and photochemical apparatus, and alterations in plant autonomy in relation to the environment. The heat dissipative capacity through daily changes in leaf temperature was also evaluated. It indicated that the early successional species (Citharexylum myrianthum Cham. and Rhamnidium elaeocarpum Reiss.) were more efficient as dissipative structures than the late successional one (Cariniana legalis (Mart.) Kuntze), suggesting that the parameter DT (T ºCair – T ºCleaf) could be a simple tool in order to help the classification of successional classes of tropical trees. Our results indicated a pattern of network responses and autonomy changes under high irradiance. Considering the maintenance of daily CO2 assimilation, the tolerant species (C. myrianthum and R. elaeocarpum) to high irradiance trended to maintain stable the level of gas exchange network connectance and to increase the autonomy in relation to the environment. On the other hand, the late successional species (C. legalis) trended to lose autonomy, decreasing the network connectance of gas exchange. All species showed lower autonomy and higher network connectance of the photochemical apparatus under high irradiance.
70 β-1,3 glucanase activity as a response to xanthomonas signalling in permeabilized leaf discs from healthy and scald-diseased sugarcane plants
Santiago, R 1, Alarcón, B 1, Blanch, M 1, Quintana, J 1, Legaz, ME 1
1Faculty of Biology. Department of Plant Biology, Complutense University, Madrid, Madrid 28040 E-mail: [email protected]
Sugarcane plants develop different defence mechanisms to combat the invasion by pathogenic organisms. Resistance of plants to disease seems to be a multifactorial process and implies constitutive (structural) and active (biochemical) processes according to their function. Several proteins are involved in these mechanisms by playing a predominant role in the process of resistance against pathogens. Enzymes degrading microorganism walls (chitinases, glucanases), and the production of elicitation compounds (e.g. salicylate, jasmonates), are some of the active plant biochemical defence mechanisms. Production of systemic plant β-1,3-glucanase, effected by bacterial signals, and the action of probable elicitors of these defensive proteins are studied as a process of sugarcane plant defence. Leaf discs from five cultivars of sugarcane exhibiting different degree of susceptibility to leaf scald were used to measure β-1,3-glucanase activity before and after experimental infection with Xanthomonas albilineans. Leaf discs were permeabilized with iso-propanol to facilitate the uptake of the enzyme substrate by intact tissues and to improve the enzyme assay. Sugarcane leaves usually produced the enzyme β-1,3-glucanase and its activity can be in vivo measured by using permeabilized leaf cells. Bacterial infection significantly enhanced β-1,3 glucanase activity of susceptible cultivars whereas a significant decrease was observed for the resistant one. Nevertheless, the degree of increased activity was different according to the cultivar. The major increase of activity after the infection were obtained for cv. Louissiana 55-5 (25 %) and Barbados 42-231 (20 %) by comparing it to the activity measured in the controls obtained of healthy plants. Lower increases could be observed in the cv. Cuba 236-51 (7 %) and Jaronú 60-5 (11 %). Nevertheless, the experimental infection of leaves from the resistant cv. Mayarí 55-14 diminished a 16% glucanase activity with regard to the non-infected controls. Low concentrations of exogenous salicylate increased hydrolase activity whereas exogenous jasmonic acid did not act as an elicitor of the enzyme.
71 Action of neurotoxic peptide in plant cells
Kagenishi, T 1, Yokawa, K 1, Kawano, T 1
1Faculty and Graduate school of Environmental Engineering, The University of Kitakyushu, Kitakyushu, Fukuoka 808- 0135 E-mail: [email protected]
Prion protein (PrP) is the causative agent of the transmissible spongirform encephalopathies. In general, PrPs from mammals have six to seven putative Cu-binding sites consisted of 4 distinct sequences. Among the copper binding regions, the neurotoxic region in the PrP (106-126) is reportedly binds copper to form the toxic complex & damage the cells. However, Among four distinct Cu-binding peptides derived from human PrP (KTNMKHMA) corresponding to a partial sequence of the neurotoxic region was shown to lack the pro- oxidant activity while other three peptides catalyze the robust production of ROS in the presence of some biological components. Therefore, in the present study, we tried to use this sequence for positive purpose in plant biotechnology, or plant environmental studies. In plant cells, induction of cell death by oxidative stresses accompanying the generation of reactive oxygen species (ROS) is often mediated by early signaling events such as calcium influx via ROS-mediated activation of calcium channels on the plasma membrane. Copper is known to be one of such oxidative stress 2+ inducer. This phyto-toxic metal actually induces an increase in cytosolic fee calcium concentration ([Ca ]c) followed by cell death in tobacco cells. In this study, impact of copper on the oxidative and calcium signal transductions leading to cell death in suspension culture tobacco cells (Nicotiana tabacum L., cell line BY-2, expressing the aequorin gene) and the effects of the copper-binding peptide derived from the human PrP as a novel plant-protecting agent were assessed. The role of early oxidative events induced by copper was proven by the action of specific ROS scavengers blocking the calcium responses and the calcium signature was monitored by the aequorin luminescence and the calcium events was blocked in the presence of specific channel blockers. Following these early events completed within 10 min, the development of copper-induced cell death was observed during additional 1 hour in a dose-dependent manner. Addition of synthetic peptide (KTNMKHMA) corresponding to the neurotoxic sequence in human PrP, prior to the addition of copper effectively blocked both the calcium influx and cell death induced by copper. Since the agent tested here is a peptide, genetic modification of plants for overproduction and excretion of this or related peptidic agents is one of the possible choices in order to minimize the phytotoxicities of various metals in the future environments.
72 The 7B-1 mutation in tomato confers a blue light-specific lower sensitivity to coronatine
Bergougnoux, V 1, Hlaváčková, V 2, Plotzová, R 1, Novák, O 3, Fellner, M 1
1Department of Cell Biology and Genetics, Palacky University in Olomouc, Olomouc, 78371 2Department of Experimental Physics, Palacky University in Olomouc, Olomouc, 77146 3Laboratory of Growth Regulators, Institute of Experimental Botany, Olomouc, 78371 E-mail: [email protected]
Male sterility of crop species, spontaneous or induced, is a criterion of importance for breeders. In almost all crop species male-sterile mutants have been reported but their use in breeding programs has been limited as some of them were sensitive to abiotic stresses, such as drought or cold temperature. In tomato (Solanum lycopersicum L.), one of the most important crops worldwide, the spontaneous mutant 7B-1, isolated for its photoperiod-dependent male-sterility, has been described as resistant to various abiotic stresses specifically under blue light. Since this finding improved potential of 7B-1’s use in breeding programs, its susceptibility to coronatine (COR)-induced stress, the phytotoxine produced by several Pseudomonas syringae strains, was assessed in this study. The 7B-1 mutant was found to be less sensitive than the corresponding wild-type (WT) to COR treatment in a blue light dependent manner. Treatment of WT and 7B-1 plants with COR induced a strong accumulation of salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) in hypocotyls. Interestingly, accumulation of ABA and SA in the 7B-1 mutant was distinctly greater than in WT, especially in blue light. Based on the cross-talk between SA- and JA-signaling pathways, expression analysis of NPR1 and COI1 genes, respectively involved in these pathways, was investigated in COR- stressed plants. The blue light-specific lower sensitivity of 7B-1 plants to COR was found to be associated with blue light-specific over-expression of the NPR1 gene. This data suggests that the SA-dependent NPR1- dependent pathway could be involved in the lower sensitivity of the 7B-1 mutant to COR. The role of anthocyanins and ABA accumulation during the response to COR is also discussed.
The work was supported by grant from the Ministery of Education of the Czech Republic (grant no. MSM6198959215).
Key-words: blue light-specific response, COI1, coronatine, growth, NPR1, SA-signaling pathway, 7B-1 mutant, tomato (Solanum lycopersicum L.).
73 Investigation of BL-mediated de-etiolation in the spontaneous 7B-1 mutant in tomato
Bergougnoux, V 1, Plotzova, R 1, Fellner, M 1 1Department of Cell Biology and Genetics, Palacky University in Olomouc, Olomouc, 78371 E-mail: [email protected]
In dark condition, plants grow rapidly in order to place meristem in favorable conditions to ensure autotrophic growth. As soon as plants perceive light, growth rate decreases and cotyledons open and accumulate active chloroplasts. This process, known as de-etiolation, is mostly regulated by blue light (BL), through the photoreceptors cryptochromes (CRY) and phototropins (PHOT). Studies of BL signaling pathway are mainly conducted on the plant model Arabidopsis, whereas less researches are developed on important crop species, like tomato (Solanum lycopersicum L.), one of the most important crop species worldwide. In tomato, the spontaneous 7B-1 mutant has been selected originally for its photoperiod-dependent male sterility. This mutant presents essentially taller phenotype than the corresponding wild-type (WT) and previous results suggest that it is defective either in BL perception or signaling pathway. We investigated the BL-mediated de-etiolation in a comparative study involving the 7B-1 mutant and the cry1-1 mutant, defective in CRY1 photoreceptor. When plants were grown in greenhouse, the taller size of the two mutants compared to the WT was correlated with longer internodes, suggesting a difference in cell expansion or cell number. An analysis of leaf surface demonstrated that 7B-1 developed larger leaves than the WT in opposite to cry1-1, suggesting that the 7B-1 mutation, even affecting BL signaling pathway, is different from the cry1-1 mutation. When grown in vitro, 7B-1 and cry1-1 were less sensitive to BL-induced de-etiolation compared to the corresponding WT. Once again, the higher size of plants was correlated with longer cells. Moreover, cell expansion under BL was driven by a higher osmotic pressure in the mutants compared to the WTs. Expression of LeEXT gene, encoding a xyloglucan endotransglycosylase and involved in cell wall loosening, was investigated by semi-quantitative RT-PCR. We found that in 7B-1, LeEXT expression was less inhibited by BL than in WT; in cry1-1 LeEXT expression was stimulated by BL. Finally, expression of PHOT1 and CRY1 genes was investigated by qRT-PCR. Whereas no significant difference was observed mutants and WT for CRY1 expression, expression of PHOT1 was less reduced by BL in the mutants compared to WT, especially in the 7B-1 mutant. Our results show that the mutation in 7B-1 affects BL-mediated cell expansion but in a different manner compared to the cry1-1 mutation. Results obtained from expression analysis led us to the hypothesis that 7B- 1 is affected in PHOT1 signaling pathway, or in the photoreceptor itself. PHOT1-signaling pathway during de-etiolation process in tomato is under further investigations. The work was supported by grant from the Ministery of Education of the Czech Republic (grant no. MSM6198959215).
Key-words: tomato, Solanum lycopersicum L., 7B-1 mutant, blue light (BL), phototropin (PHOT), cryptochrome (CRY) de-etiolation, cell expansion
74 Changes in abscisic acid distribution in heat-stressed pepper seedlings
Cui, J 1, Liu, TX 1, Zhang, ZS 2
1Institute of Minority Traditional Medical Center, Central University for Nationalities, Beijing, 100081 2School of Biology and Food Engineering, Dalian Polytechnic University, Dalian, 116034
E-mail: [email protected]
The heat stress-responsive changes in both abscisic acid (ABA) subcellular distribution and soluble content in pepper mesophyll and root cap cells were investigated by colloidal gold labeling technique and HPLC technique. The results showed that the ABA was localized in the nucleus and the cytoplasm of two cell types in the seedlings under normal temperature, with a higher accumulation in the root cap cells, relatively. As the seedlings were transferred to 40°C for heat stress, the ABA levels in both mesophyll and root cap cells increased markedly, especially in the later. With a sustained heat stress, the ultrastructure of mesophyll cell was damaged severely, and more ABA accumulated in the nucleus of mesophyll cells; comparably, the root cap cell maintained intact ultrastructurally, and a concomitant drastic increase in ABA in the nucleus of root cap cells was also observed. The HPLC quantization demonstrated that the soluble content of ABA in the tissue of root tip was higher than that of leaves under normal temperature, and with the heating time,the content of ABA in leaves increased, but decreased in the root tissue. The above results imply that ABA might be one of the heat stress signaling members in plant cells, whereas the mechanism by which ABA functions during this process remains poorly understood.
75 List of Participants
Akula Ramakrishna Bisson Mary Central Food Technological Research Institute – CSIR Dept. Biological Sciences, Plant Cell Biotechnology Department University at Buffalo, Mysore, 5700 Buffalo, New York 14260 INDIA USA [email protected] [email protected] Blanch Maria Rojo Alarcon Borja Aguareles Faculty of Biology Faculty of Biology. Dpeartment of Plant Biology Department of Plant Biology Complutense University, Madrid Complutense University, Madrid 28040 SPAIN SPAIN [email protected] [email protected]
Arru Laura Botella Miguel Dept. of Agricultural and Food Sciences Departamento de Biología Molecular y Bioquímica, University of Modena and Reggio Emilia, Universidad de Málaga, Málaga, 29071 Reggio Emilia ITALY SPAIN [email protected] [email protected]
Azzarello Elisa Bouteau Francois University of Florence EA3514, Université Paris Diderot, Paris, 75005 Department of Horticulture FRANCE Viale delle Idee 30, [email protected] 50019 Sesto Fiorentino (FI) ITALY Cvrckova Fatima [email protected] Faculty of Sciences, Department of Plant Physiology Baluska Frantisek Charles University in Prague, Prague, CZ 128 44 IZMB, University of Bonn, CZECH REPUBLIC Bonn GERMANY [email protected] [email protected] Díaz Eva Maria Pena Barlow Peter Faculty of Biology School of Biological Sciences Department of Plant Biology, Complutense University, University of Bristol, Bristol, BS8 1UG Madrid, Madrid28040 UNITED KINGDOM SPAIN [email protected] [email protected]
Beilby Mary Doskočilová Anna The University of NSW, IMB Academy of Sciences CR, Prague, School of Physics, Sydney, NSW 2052 CZECH REPUBLIC AUSTRALIA [email protected] [email protected] Fellner Martin Beom-gi Kim Laboratory of Growth Regulators, National Academy of Agricultural Science Palacky University in Olomouc and RDA, Suwon, 441-707 Institute of Exp. Botany, Olomouc, 78371 REPUBLIC OF KOREA CZECH REPUBLIC [email protected] [email protected]
Bergougnoux Veronique Fukao Yoichiro Department of Cell Biology and Genetics Plant Science Education Unit, Palacky University in Olomouc The Graduate School of Biological Science CZECH REPUBLIC Nara Institute of Science and Technology, Ikoma [email protected] 6300101 JAPAN [email protected]
76 Furch Alexandra JAPAN Plant Cell Biology Research Group [email protected] Institute of General Botany Justus-Liebig-University, Giessen, 35390 Kohoutová Lucie GERMANY CZECH REPUBLIC [email protected] [email protected]
Geisler Markus Legaz Maria Estrella Molecular Plant Physiology Faculty of Biology. Department of Plant Biology University of Zurich, Zurich, 8008 Complutense University, Madrid, Madrid 28040 SWITZERLAND SPAIN [email protected] [email protected]
Giovannetti Manuela Lin Jinxing Department of Crop Plant Biology Key Laboratory of Photosynthesis and University of Pisa, Pisa, 56124 Molecular Environmental Physiology ITALY Institute of Botany, Chinese Academy of Sciences, [email protected] Beijing 100093 CHINA [email protected] Hause Bettina Leibniz Institute of Plant Biochemistry Lupini Antonio Department of Secondary Metabolism, Halle, D-06018 Dipartimento Bio.M.A.A. GERMANY Università degli Studi Mediterranea di Reggio Calabria [email protected] 89124 Reggio Calabria ITALY Hedrich Rainer [email protected] Molecular Plant Physiology and Biophysics Biocenter, University of Würzburg, Würzburg, D-97082 Mancuso Stefano GERMANY University of Florence [email protected] Department of Horticulture Viale delle Idee 30, 50019 Hlavackova Vladimira Sesto Fiorentino (FI) Laboratory of Biophysics ITALY Department of Experimental Physics, Faculty of [email protected] Science, Palacký University, Olomouc CZECH REPUBLIC Masi Elisa [email protected] University of Florence Department of Horticulture Jarvis Paul Viale delle Idee 30, 50019 Department of Biology Sesto Fiorentino (FI) University of Leicester, Leicester, Leics LE1 7RH ITALY UNITED KINGDOM [email protected] [email protected] Masson Patrick H. Juricis-Knezev Dejana Laboratory of Genetics Department of Cell Biology and Genetics University of Wisconsin-Madison, Madison, WI 53706 Palacky University in Olomouc, Olomouc, 783 71 USA CZECH REPUBLIC [email protected] [email protected] Mazars Cristian Kagenshi Tomoko UMR 5546,Surfaces Cellulaires et Signalisation chez les Faculty and Graduate school of Environmental Végétaux CNRS/Université de Toulouse engineering Castanet-Tolosan, 31326 The University of Kitakyushu FRANCE Kitakyushu, Fukuoka 808-0135 [email protected] JAPAN [email protected] Migliaccio Fernando Institute of Agro-Environmental and Forest Biology Kawano Tomonori Consiglio Nazionale delle Ricerche, Monterotondo The University of Kitakyushu 00016, Roma Laboratory of Chemical Biology and Bioengineering, , ITALY 808-0135 [email protected] 77 Mithöfer Axel Pagano Mario Max Planck Institute for Chemical Ecology University of Florence Bioorganic chemistry, Jena, 07745 Department of Horticulture GERMANY Viale delle Idee 30, 50019 Sesto Fiorentino (FI) [email protected] ITALY [email protected] Moreau Flora CRP Sante Pandolfi Camilla 1A-B Rue Thomas Edison L-1445 Strassen, University of Florence Luxembourg Department of Horticulture [email protected] Viale delle Idee 30, 50019 Sesto Fiorentino (FI) ITALY Mugnai Sergio [email protected] University of Florence Department of Horticulture Pollastri Susanna Viale delle Idee 30, 50019 University of Florence Sesto Fiorentino (FI) Department of Horticulture ITALY Viale delle Idee 30, 50019 Sesto Fiorentino (FI) [email protected] ITALY [email protected] Nakamura Yoko Department of Chemistry Quintana Gonzalez Julia Graduate School of Science Faculty of Biology Tohoku University, Sendai, 9808578 Department of Plant Biology JAPAN Complutense University, Madrid, Madrid 28040 [email protected] SPAIN [email protected] Narendra Tuteja Plant Molecular Biology Group Rengel Zed International Centre for Genetic Engineering and Centre for Land Rehabilitation Biotechnology, New Delhi, Delhi 110067 The University of Western Australia (M087) INDIA 35 Stirling Highway CRAWLEY WA 6009 [email protected] AUSTRALIA [email protected] Naus Jan Laboratoty of Biophysics Renna Luciana Faculty of Science, Palacky University, Olomouc, 771 University of Florence 46 Department of Horticulture CZECH REPUBLIC Viale delle Idee 30, 50019 Sesto Fiorentino (FI) [email protected] ITALY [email protected] Navazio Lorella Dipartimento di Biologia Risueno María-Carmen Università di Padova Center of Biological Research, CIB Padova, 35131 National Research Council, CSIC, Madrid, 28040 ITALY SPAIN [email protected] [email protected]
Neumann Joseph Sacristán San Cristobal Mara Department of Plant Sciences &Philosophy Faculty of Biology Tel-Aviv University, Tel-Aviv, 69978 Department of Plant Biology ISRAEL Complutense University, Madrid, Madrid 28040 [email protected] SPAIN [email protected]
Oliveira Ricard Santiago Rocio Tejero ESALQ, Universidade de São Paulo Faculty of Biology Piracicaba, São Paulo 13418-900 Department of Plant Biology BRAZIL Complutense University, Madrid, Madrid 28040 [email protected] SPAIN [email protected]
78 Sbrana Cristiana Timmers Ton Istituto di Biologia e Biotecnologia Agraria UO Pisa, Lab. Plant Microorganism Interactions, CNRS/INRA CNR, 56124 Pisa Castanet-Tolosan, 31326 ITALY FRANCE [email protected] [email protected]
Schachtschabel Doreen Van Bel Aart Max Planck Institute for Chemical Ecology Plant Cell Biology Research Group Bioorganic chemistry, Jena, 07745 Institute of General Botany GERMANY Justus-Liebig-University, Giessen, 35390 [email protected] GERMANY [email protected] Scherer Guenther Institute of Floriculture and Woody Plant Science Van Volkenburgh Elisabeth Molecular Yield Physiology - Herrenhaeuser Str. 2, D- University of Washington 30419 Hannover, GERMANY Biology Department [email protected] 407 Hitchcock Hall, Seattle, WA 98195-5325 USA Shabala Sergey [email protected] School of Agricultural Science University of Tasmania, Hobart, Tasmania 7001 Vicente Cordoba Carlos AUSTRALIA Faculty of Biology [email protected] Department of Plant Biology Complutense University, Madrid, Madrid 28040 Shen Wen-Hui SPAIN CNRS, Institute of Plant Molecular Biology (IBMP), [email protected] Strasbourg, 67084 FRANCE Volkmann Dieter [email protected] University of Bonn Institute of Cellular and Molecular Botany Shiratake Katsuhiro Kirschallee 1, D-53115 Bonn Graduate School of Bioagricultural Sciences GERMANY Nagoya University, Nagoya, 464-8601 [email protected] JAPAN [email protected] Yokawa Ken Faculty and Graduate school of Environmental Staves Marc Engineering Biology Department The University of Kitakyushu, Kitakyushu, Fukuoka Grand Valley State University, Allendale, Michigan 808-0135 49401 JAPAN USA [email protected] [email protected] Yoon In Sun Stefano Giovanni Department of Agricultural Bio-resources University of Florence National Academy of Agricultural Sciences Department of Horticulture Suwon, Kwonsun-Gu, Seodun-Dong 441-707 Viale delle Idee 30, 50019 Sesto Fiorentino (FI) REPUBLIC OF KOREA ITALY [email protected] [email protected] Zhang Zong Shen Tassone Paola Institute of Minority Traditional Medical Center IBAF, National Research Council (CNR), Central University for Nationalities, Beijing, 100081 Monterotondo, 00015 Rome CHINA ITALY [email protected] [email protected] Thomas Clement Zimmerman Mathias Rudi Laboratory of Plant Molecular Biology Institute of General Botany CRP-Santé, Luxembourg, L-1526 Justus-Liebig-University, Giessen, 35390 LUXEMBOURG GERMANY [email protected] [email protected]
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