Transcriptional and phenotypic plasticity of Solanum nigrum: Can it defend against both competitors and herbivores?
Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.)
vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller Universität Jena
von Diplom-Biologe
Dominik D. Schmidt
geboren am 01.03.1974 in Hamm
Gutachter
1. Prof. Dr. Ian T. Baldwin
2. Prof. Dr. Don Cipollini
3. Prof. Dr. Ralf Oelmüller
Tag der Doktorprüfung: 25. Oktober 2004
Tag der öffentlichen Verteidigung: 22. November 2004
Table of contents
Table of contents
Table of contents ...... I Manucript overview...... II 1. General introduction ...... 1 2. Manuscript 1 – Solanum nigrum - a model ecological expression system ...... 9 3. Manuscript 2 - Competition- and MeJA-elicited responses of S. nigrum...... 43 4. Manuscript 3 - Biodiversity and transcriptional defense responses...... 65 5. Manuscript 4 – Solanaceous responses to herbivory...... 83 6. General discussion ...... 109 7. Summary...... 121 8. Zusammenfassung ...... 122 9. References ...... 126 10. Appendix...... 133 10.1 Supplementary data ...... 133 10.2 Curriculum vitae...... 134 10.3 Publication list...... 135 10.4 Oral and poster presentations...... 136 10.5 Danksagung ...... 137 10.6 Eigenständigkeitserklärung...... 138
I Manuscript overview
Manuscript 1 – authors’ contribution
Solanum nigrum: a model ecological expression system
Dominik D. Schmidt, André Kessler, Danny Kessler, Silvia Schmidt, Michelle Lim, Klaus Gase and Ian T. Baldwin
Molecular Ecology 13: 981-995 (2004)
Under the supervision of Ian T. Baldwin I was responsible for developing the concept of a ecological expression system and for planning, realization and analysis of all field and greenhouse experiments. André Kessler supported me in the realization of the VOC experiment. Danny Kessler was responsible for determining the insects and helped with the field work. Silvia Schmidt contributed to the Southern Blot analysis for the characterization of the asRuBPCase line. Michelle Lim transformed Solanum nigrum with pRESCRUB. Klaus Gase provided the pRESCRUB vector.
Dominik D. Schmidt André Kessler Danny Kessler Silvia Schmidt Michelle Lim Klaus Gase Ian T. Baldwin II Manuscript overview
Manuscript 2 – authors’ contribution
The effect of competition on transcriptional responses elicited by methyl jasmonate in Solanum nigrum—a glasshouse and field comparison
Dominik D. Schmidt and Ian T. Baldwin
Submitted to Molecular Ecology
I was responsible for planning, realization and analysis of the glasshouse and field experiments presented in this manuscript to study the effect of competition on jasmonate-mediated defense responses. I developed the outline and the first version of the manuscript, which then subsequently was optimized by Ian T. Baldwin and me.
Dominik D. Schmidt Ian T. Baldwin
III Manuscript overview
Manuscript 3 – authors’ contribution
The canopy height of competitors explains the influence of biodiversity on jasmonate-induced transcriptional responses in field-grown Solanum nigrum
Dominik D. Schmidt and Ian T. Baldwin
Submitted to Ecology Letters
The experiment was designed to broaden the view of Manuscript 2 with regard to the type of competition. I developed the design of the field experiment in discussion with Ian T. Baldwin and was responsible for the realization and transcriptional, chemical and statistical analysis of the experiments. I developed the outline and the first version of the manuscript, which then subsequently was optimized by Ian T. Baldwin and me.
Dominik D. Schmidt Ian T. Baldwin
IV Manuscript overview
Manuscript 4 – authors’ contribution
Specificity in ecological interactions: Attack from the same Lepidopteran herbivore results in species-specific transcriptional responses in two Solanaceous hostplants
Dominik D. Schmidt, Claudia Voelckel and Ian T. Baldwin
Submitted to Plant Physiology
In this comparison of the transcriptome of Solanum nigrum and Nicotiana attenuata, both attacked from Manduca sexta, we present evidence for profound differences in Solanaceous defense responses. Claudia Voelckel and I were responsible for realization of the experiment, whereby Claudia Voelckel worked on N. attenuata and I worked on S. nigrum. We conducted the data analysis together. Claudia Voelckel and I equally contributed to the first draft of the manuscript, which in turn was optimized under the supervision of Ian T. Baldwin.
Dominik D. Schmidt Claudia Voelckel Ian T. Baldwin
V
VI 1. General Introduction
1. General introduction When I looked out of the window of my partner´s apartment last autumn I noticed a bunch of sycamore trees that as deciduous trees had abscised most of their leaves except for some branches, which still had retained green leaves on them. Obviously, those leaves had responded to an environmental cue preventing them from abscising. The cue was likely the street lamps that bathed those leaves in light while the other leaves of the canopy remained in darkness during the night. Such plastic responses are common among plants that are able to express different phenotypes depending on their biotic or abiotic environment. A given genotype has the capacity to change its chemistry, physiology, morphology, or development in response to environmental cues, a phenomenon known as phenotypic plasticity (Figure 1-1; Sultan 2000). Phenotypic plasticity allows for adaptive responses in species interactions and reciprocal phenotypic changes in the organisms influence interactions in ecological and evolutionary time scales (Agrawal 2001).
Figure 1-1. Solanum nigrum (inbred line Sn30) at the same developmental stage (approx. 3-4 weeks old) growing in Jena, Germany, (A) on an agricultural field or (B) on a driveway (bars equal 5 cm).
1 1. General Introduction
The interaction among plants, namely competition for space and the suite of similar limited resources that plants require, is likely the most important biotic stress for plants (Goldberg 1990). The intensity of competition is determined by the resource demands of the competing species. In comparison to interspecific competition, intraspecific competition is assumed to be the more intense, because individuals of one species most likely exhibit the same resource demands (Begon et al. 1996). Together with plants, invertebrate herbivores account for the majority of all higher species (Strong et al. 1984) and competitive outcomes are frequently altered by attack from herbivores (Cipollini 2004; Herms & Mattson 1992). The interaction of these selective factors is thought to have resulted in trade-offs between growth and defense which is most clearly seen in the cost-saving strategy of inducible defense traits (Heil & Baldwin 2002). Few studies have examined how competition influences the expression of defense traits. Intraspecific competition reduced levels of anti-digestive acting proteins (trypsin proteinase inhibitors, TPIs) in Brassica napus (Cipollini & Bergelson 2001). In A. thaliana, jasmonic acid (JA)-induced TPI production was associated with reduced plant fitness, but intraspecific competition had no effect on the magnitude of fitness costs (Cipollini 2002). To date several studies have addressed the effect of herbivory and interspecific competition on fitness correlates of plants (Bonser & Reader, 1995; Fowler & Rausher, 1985; McEvoy et al. 1993), but I am aware of no studies that have examined the direct effects of interspecific competition on the expression of defense traits. A central question is whether the biodiversity of competitors specifically modifies defense responses of a plant and if so, which are the factors driving those specific interactions. The synergistic effect of competition and herbivory on plant performance may be driven by resource allocation trade-offs or may result from direct hormonal interactions that mediate growth- and defense-related traits (Cipollini 2004; Fig. 1-2). Three phytohormones are thought to play a central role in the signaling pathways responsible for plant resistance [salicylic acid, SA, (Delaney et al. 1994); ethylene, ET (O’Donnell et al.1996); JA, (Creelman & Mullet 1997)]. JA and related
2 1. General Introduction oxylipins mediate the majority of insect-induced responses (Kessler & Baldwin 2002; Li et al. 2004; Walling 2000), but through synergism or antagonism with SA and ET the plant fine-tunes its response to specific herbivores (Reymond & Farmer 1998; Winz & Baldwin 2001). In competitive interactions light and ET signaling are important for the plant (Ballaré 1999; Pierik et al. 2003, 2004a,b). Phytochromes and cryptochromes enable the plant to perceive red and blue light signals
Figure 1-2. Overview over signals important for plant responses to herbivory and competition. Jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) play a central role in herbivore-induced responses. Far-red and blue (FR/B) light, ethylene and additional signals (?), such as soil- or airborne cues, mediate competitive interactions. that depen d on the structure of the competing plant community (Ballaré 1999). ET sensing has been very recently shown to be involved in the competition-induced shade avoidance syndrome; ET-insensitive tobacco plants showed a delayed shade avoidance reaction when grown in the presence of ET-sensitive wildtype plants (Pierik et al. 2003). Further chemical signals, such as volatile airborne compounds or root exudates, are thought to be involved in plant-plant communication and hence may play roles in plant competition (Dicke & Bruin 2001; Bais et al. 2004). In
3 1. General Introduction addition there is increasing evidence that signals involved in responses to biotic stress are also involved in responses to abiotic stress such as ozone, drought, or UV-B radiation. The variety of signals illustrates that plant responses to stresses are mediated by complex signaling networks rather than by linear signal cascades (Genoud et al. 2001). Gene expression profiling using microarrays enhanced the understanding of signaling networks mediating plant resistance to herbivores and pathogens (e.g. Glazebrook 2001; Schenk et al. 2000). The knowledge of differentially regulated genes in response to herbivore attack provides the information for the powerful approach of transformation, which can be used to specifically silence genes and thus to study their function. Despite the great impact of the genomics revolution for plant biology, ecologists have not yet fully adopted molecular tools to test hypotheses of adaptive phenotypic responses. This is mostly due to the lack of appropriate model systems for which a suite of molecular tools is available. Genomic tools have been developed mostly for economically important species, but since agricultural plants have long been under intense selection for particular yield-enhancing traits, genetic associations mediating adaptive traits are likely to have been altered during agricultural selection and hence are difficult to interpret in these plants. I present the establishment of molecular tools for the native plant Solanum nigrum that should facilitate the identification and manipulation of the genes that mediate complex environmental responses. S. nigrum (Fig. 1-3) was not only chosen because of its phylogenetic proximity to the crop species potato and tomato for which a substantial molecular knowledge base exists, but also because its particular natural history makes it ideal for studying the interaction of competition and herbivore resistance. S. nigrum, also known as Black, Garden or Common Nightshade, is one of the most variable species groups of the genus Solanum and therefore often referred to as S. nigrum complex (Beg & Khan 1988; Edmonds & Chweya 1997; Dehmer & Hammer 2004). The Eurasian species is the hexaploid S. nigrum s.str., which comprises two subspecies and several varieties; S. americanum is the neotropical diploid
4 1. General Introduction
Figure 1-3. Solanum nigrum growing in its characteristic habitat, a field to the north of Jena. Top left inset: The pedunculate inflorescenses bear usually 4 to 8 pentamerous flowers with a cone of anthers. Bottom right inset: The almost black fleshy berries inspired the name: Black Nightshade. counterpart. A s an annual herbaceous pioneer plant, Black Nightshades colonize nitrogen-rich agricultural and disturbed habitats and can be obnoxious weeds in crop growing, e.g. in potato or vegetable fields (Edmonds & Chweya 1997). S. nigrum is predominantly self-pollinating, but also attracts bees, which hang from the anther cone and harvest pollen by vibrating their wings, in a process called “buzz-pollination” (Buchmann 1983). The fleshy berries of S. nigrum implicate various
5 1. General Introduction strategies for seed dispersal, especially due to the high content of secondary metabolites (Cipollini & Levey 1997). These compounds are mainly steroid alkaloids (glycoalkaloids) and sapogenins (Sharma & Chan 1979; Ridout et al. 1989; Dopke et al. 1987; Benidze 1994), which are also distributed in other plant parts at different levels during particular periods of development (Mathe et al. 1979; Eltayeb et al. 1997). Glycoalkaloids play an important role in plant-insect interactions as feeding deterrents and feeding stimulants and hence provide a target for manipulative studies (Sanford et al. 1996; del Campo et al. 2001; Muller & Renwick 2001; Singh et al. 2001;). The diversity of plant secondary metabolites, that are important in ecological interactions, is reflected in the taxonomic diversity of plants. On the other hand, among plants only a handful of signals appear to be recruited for mediating plant-plant-herbivore interactions (Fig. 1-2). Mainly two model pathways used by plant biologists to characterize the activation of defense responses, namely the Brassicaceous (Arabidopsis) and Solanaceous (tomato) model (Leon et al. 2001; Walling 2000). Both models recruit common signals, namely JA, ET, and SA, yet in tomato the Solanaceae-specific peptide systemin plays an essential role in local and systemic wound-activated responses (Ryan 2000; Ryan & Pearce 2003). However, there is evidence of large variability of defense responses even within the Solanaceae. A comparison of closely related Nicotiana species showed significant differences in defense signaling and secondary metabolites in response to the same Lepidopteran herbivore (Lou & Baldwin 2003). Hence, detailed comparisons among members of the same plant family are needed to reveal further insights in the complexity of plant responses to biotic stress. In the collection of manuscripts (MSs) presented in this study I investigate the plant-plant-herbivore interactions between herbivore- or elicitor-induced S. nigrum plants and intraspecific or interspecific competitors, and contrast S. nigrum-specific defense responses to another Solanaceous species, Nicotiana attenuata. To facilitate these analyses it was necessary to establish tools for S. nigrum that allow for transcriptional and
6 1. General Introduction
Figure 1-4. A plant of a given genotype expresses its phenotype in response to the abiotic and biotic environment. The responses of the plant can be monitored and manipulated on different levels. Only the integrated view will allow for a deep understanding of ecological interactions. phenotypic analysis of ecologically relevant traits (Fig. 1-4). I chose oligonucleotide microarrays, enriched in genes relevant for plant- herbivore interactions, to monitor gene expression. Additionally, I measured the production of direct (TPI) and indirect (volatile organic compounds, VOCs) defenses and fitness correlates to determine the maturation of the transcriptome into a relevant phenotype. In conclusion, I address the following questions with the methods developed for the ecological model system S. nigrum.
• Are molecular biological tools applicable for ecological field experiments?
7 1. General Introduction
• Do laboratory-based results of S. nigrum’s transcriptional and phenotypic responses reflect the plant’s behavior in its native environment?
• Which are the characteristics of S. nigrum’s response to herbivory? How similar is the defense strategy of S. nigrum to other Solanaceous species?
• How does S. nigrum respond to competition?
• Are defense- and growth-related traits of S. nigrum altered by competition and if so, does it depend on the species of the competitor or the type of competing species present?
• Which are the mechanisms of the competition interaction?
8 MS 1 – S. nigrum – an ecological model system
Manuscript 1
Molecular Ecology 13 (2004): 981-995
Solanum nigrum L.: a model ecological expression system and its tools
Dominik D. Schmidt, André Kessler, Danny Kessler, Silvia Schmidt, Michelle Lim, Klaus Gase and Ian T. Baldwin*
Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany
Key words: indirect and direct defenses, proteinase inhibitor, Agrobacterium-mediated transformation, oligonucleotide micro- array, herbivore-induced volatile production, plant fitness
Correspondence: Ian T. Baldwin, Max Planck Institute for Chemical Ecology, Hans-Knoell-Str. 8, D-07745 Jena, Germany, Fax: 03641 571102, E-mail: [email protected]
Running title: Solanum nigrum - an ecological model system
8605 words; 6 figures; 1 table; 2 supplementary tables
9 MS 1 – S. nigrum – an ecological model system
Abstract
Plants respond to environmental stresses through a series of complicated phenotypic responses, which can be understood only with field studies because other organisms must be recruited for their function. If ecologists are to fully participate in the genomics revolution and if molecular biologists are to understand adaptive phenotypic responses, native plant ecological expression systems which offer both molecular tools and interesting natural histories are needed. Here, we present Solanum nigrum, a Solanaceous relative of potato and tomato for which many genomic tools are being developed, as a model plant ecological expression system. To facilitate manipulative ecological studies with S. nigrum, we describe: 1) an Agrobacterium-based transformation system and illustrate its utility with an example of the anti-sense expression of RuBPCase, as verified by Southern gel blot analysis and real-time quantitative PCR (qPCR); 2) a 789- oligonucleotide microarray and illustrate its utility with hybridizations of herbivore-elicited plants, and verify responses with RNA gel blot analysis and real-time qPCR; 3) analyses of secondary metabolites that function as direct (proteinase inhibitor [PI] activity) and indirect (herbivore- induced volatile organic compunds [VOCs]) defenses; and 4) growth and fitness-estimates for plants grown in under field conditions. With these tools, we demonstrate that attack from flea beetles elicits: 1) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signaling and 2) increases in PI transcripts and activity, and VOC releases. Both flea beetle attack and JA elicitation increased PIs and decreased fitness in field-grown plants. Hence, PIs and JA-signaling are targets for manipulative studies.
10 MS 1 – S. nigrum – an ecological model system
Introduction
Molecular techniques are widely used in all of biology, but their incorporation into ecological studies has largely been confined to the characterization of population structure and species distributions. As a consequence, the extraordinary advances that molecular techniques have permitted most biological disciplines, namely the ability to identify the genetic basis of a biological phenomenon and manipulate it, have yet to be realized in ecology. Many reasons underlie the non-participation of ecologists in the genomics revolution, but the limited availability of appropriate model systems has played an important role. Molecular tools developed for one model system can be difficult to transfer to other systems without substantial investment in technique development. Most techniques have been developed for agronomically and economically important organisms and cannot be readily applied to wild relatives. Transformation systems, in particular, can be difficult to use with near relatives. Agrobacterium-mediated gene transfer protocols are available for a number of higher plants and fungi, and allow the manipulation of the expression of genes mediating ecological interactions if these transformation systems have been adapted for native species. Plants and herbivores account for the majority of all higher species (Strong et al. 1984), and their interactions structure many of the planet’s biological processes. The transformation of autotrophs allows for the ‘bottom-up’ manipulation of ecological interactions and thereby provides a powerful tool for studying community and ecosystem processes. Once the ability to manipulate the expression of individual genes in a native species is available, the next task is to decide which genes to manipulate and how to interpret the responses to the manipulations. Microarrays allow biologists to examine the expression of hundreds of genes simultaneously, and their use in combination with elicitation studies provides a powerful means of identifying ‘suspect’ genes relevant for ecological interactions (Hui et al. 2003; Korth 2003). Alternatively, various differential display procedures (Voelckel & Baldwin 2003) allow researchers to ‘ask the organism’ to identify transcripts relevant in a
11 MS 1 – S. nigrum – an ecological model system given ecological interaction. Once a gene or a suite of genes has been selected for manipulation, the next challenge lies in interpreting the fitness consequences of the manipulation. Laboratory bioassays are not likely to provide a full functional understanding of many traits elicited by biotic interactions as is illustrated by the traits mediating plant-herbivore interactions. Plants are known to recruit components of their community, as is graphically illustrated by the elicitation of indirect defenses (volatile organic compound [VOC] emissions, extrafloral nectar production, etc.) that plants use to enlist the natural enemies of herbivores in their defense against herbivores. A functional understanding of these complex responses can be understood only in the context of the selective forces under which these responses evolved, namely their natural environments. Moreover, ecologists have long known that competition from other plants and herbivore pressure represent the two most important selective forces determining relative plant fitness in natural habitats (Begon et al. 1996). As a consequence, traits mediating competitive ability and herbivore resistance are likely intertwined, and understanding the genetic basis of these responses will require experimental manipulations in natural environments. Last, since environmental performance is a whole-plant trait best measured by various surrogates of Darwinian fitness (seed set, male reproductive success), an understanding of how the expression of a gene product contributes to a plant’s Darwinian fitness is required. The choice of an ecologically relevant organism is crucial for the study of plant-environment interactions. Adaptive responses are mediated by complex polygenic traits (Simms & Rausher 1992), and since agricultural plants have long been under intense selection for particular yield-enhancing traits, genetic associations mediating adaptive traits are likely to have been altered during agricultural selection and hence are difficult to interpret in these plants. We introduce a suite of molecular tools for the native plant Solanum nigrum that should facilitate the identification and manipulation of the genes that mediate these complex environmental responses. S. nigrum was selected not only
12 MS 1 – S. nigrum – an ecological model system because of its phylogenetic proximity to the agricultural species tomato and potato for which substantial genetic tools are available, but also because its particular natural history makes it ideal for studying the interaction of competition and herbivore resistance. S. nigrum is attacked by various herbivores from different feeding guilds and grows in association with many other species. As an annual, it colonizes nitrogen- rich agricultural and disturbed habitats at a wide range of altitudes throughout its pan-arctic distribution (Edmonds & Chweya 1997). If S. nigrum is to become a model ecological system, a minimum number of molecular tools are necessary. Most important, it must be readily transformable so that hypotheses about the ecological function of particular genes can be falsified. Here, we present such a transformation system for S. nigrum. cDNA libraries of environmentally elicited plant tissues provide a means of cloning genes and microarrays allow biologists to examine the expression of hundreds of genes simultaneously. Here we present such a library and a 789 oligonucleotide microarray, representing 558 genes of ecological interest. We analyze traits thought to be important for plant performance that are quantifiable in complex environments and offer a means of measuring their correlation with S. nigrum’s Darwinian fitness. We have selected a direct defense and an indirect defense for the analysis. Proteinase inhibitors (PIs) are among the best-studied induced direct defense chemicals in plants (Heath et al. 1997; Jongsma et al. 1994; Koiwa et al. 1997; Ryan 1990), which function by inhibiting particular digestive proteinases of herbivores, such as chymotrypsin and trypsin. High PI content has been found to reduce herbivore growth in plants that were transformed with heterologous PI genes (Rahbe et al. 2003; Xu et al. 1996). Xu et al. (2001) characterized two PI s of the pin2 family (SaPIN2a and SaPIN2b) in Solanum americanum, a species belonging to the taxonomically diverse Solanum nigrum group (Edmonds & Chweya 1997). We isolated the SaPIN2b homologue from S. nigrum (SnPIN2b) and used it to verify the responses observed on the array with a RNA-gel blot and real-time qPCR. We present a technique for quantifying the herbivore-induced VOC emissions from plants and illustrate its use with measurements from field-grown plants. VOCs are
13 MS 1 – S. nigrum – an ecological model system known to attract predators to the herbivore-damaged plant and therefore play a key role in plant-insect interactions (Baldwin et al. 2002; Dicke et al. 2003; Kessler & Baldwin 2001).
Materials and Methods
Plant growth Solanum nigrum L. [inbred line, Sn30, of seeds collected from the field site in Jena, Germany; voucher specimens of the Sn30 line are deposited in the Max Planck Institute for Chemical Ecology branch of the Herbarium Haussknecht (JE), Jena] seeds were incubated in 3.5mM
Ca(NO3)2 overnight at 4°C and germinated in a peat-based substrate with clay additions (Tonsubstrat, Klasmann, Geeste-Groß Hesepe, Germany). Plants were grown in individual 400-mL pots for three weeks in the greenhouse (26 °C / 16 h light; 25 °C / 8 h dark). After 5 days of acclimatization to outside conditions, plants were randomly planted into monoculture plots at the experimental field site. The site is a former agricultural field with alluvial loam as substrate and located north of Jena, Germany. For the greenhouse experiments, plants were grown in 2 L pots with supplemental lighting from 400W Na-vapor HID lamps and watered once a day.
Agrobacterium-based transformation A fragment of the Nicotiana attenuata gene for RuBPCase was PCR (Hermsmeier et al. 2001) PCR amplified. After digestion with XhoI and BstEII the resulting fragment (323 bp) was cloned in pRESC20 (Zavala et al. 2004), yielding the transformation vector pRESC2RUB (10.0 kb) which was used for the transformation of S. nigrum. S. nigrum seeds (inbred line Sn30) were used for transformation. Seeds were sterilized for 5 min in a 5-mL aqueous solution of 0.1 g dichloroisocyanuric acid (Sigma, St. Louis, MO, USA) with 50 µL of 0.5 % (v/v) Tween-20 (Merck, Darmstadt, Germany). Seeds were washed 3 times with sterile water and incubated for 3 days at 4 ºC in an aqueous
14 MS 1 – S. nigrum – an ecological model system
solution of 3.5mM Ca(NO3)2.4H2O (Merck, Darmstadt, Germany). Subsequently, seeds were washed 3 times with sterile water and transferred onto a germination medium containing Gamborg’s B5 with minimal organics (Sigma) and 0.6% (w/v) phytagel (Sigma). The plates were maintained in a growth chamber (Percival, Perry, Iowa, USA) at 26 ºC/16h light with 155 µm/m2/s PAR at shelf height and 24 ºC/8h dark. A. tumefaciens strain 4404 was maintained and cultivated for transformation as described in (Krügel et al. 2002). Hypocotyls of sterile 1-week-old seedlings were excised with a scalpel dipped into the Agrobacterium suspension, and co-cultivated as described in (Krügel et al. 2002). Within a month, explants on callus induction media developed calli followed by green shoot primordia and shoots, so that sub-culture onto maturation media (described in Krügel et al. 2002) was necessary only until plantlets formed. Plantlets were subsequently sub-cultured onto maturation media, which also served as rooting media in this case, until roots appeared. Further selection of the putatively transformed plants, including segregation analysis of T1 plants using germination bioassays on hygromycin-containing media, is described in (Krügel et al. 2002).
Microarray hybridization and analysis Pooled leaf samples were ground under liquid nitrogen and total RNA was extracted with TRI REAGENTTM (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. The herbivore- infested test samples were labeled with Cy3 and the corresponding control (reference) samples with Cy5 according to the procedure described in Halitschke et al. (2003). The labeled samples were hybridized to the microarray (789 50-mer oligonucleotides spotted onto an epoxy-coated glass slide; Quantifoil Microtools, Jena, Germany) according to the published procedure (Halitschke et al. 2003). An Affymetrix 428™ Array Scanner (Affymetrix, Santa Clara, CA, USA) was used to scan the hybridized microarrays with sequential scanning for Cy5- cDNA and then for Cy3-labeled cDNA at a maximum resolution of 10 µm/pixel with a 16-bit depth. The images
15 MS 1 – S. nigrum – an ecological model system were evaluated with the AIDA Image Analyzer (Raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany) software. Each image was overlaid with a grid to assess the signal strength (quantum level = QL) for both dyes from each spot. The background correction was calculated with the ‘non spot’ mode of the AIDA software package. The microarray-specific normalization factor was calculated based on the Cy5- / Cy3- total fluorescence ratio (Halitschke et al. 2003). The ratios of normalized fluorescence values for Cy3 and Cy5 of each individual spot (expression ratio = ER) and the mean of the four replicate spots for each cDNA were calculated. A transcript was defined as being differentially regulated, if the following three criteria were fulfilled: 1) the average expression ratio for the 4 spots exceeded the thresholds (0.67 and 1.5); 2) the individual expression ratios were significantly different from 1 as determined by a t-test; 3) the combined signal fluorescent intensity from both Cy3 and Cy5 averaged over the 4 spots was greater than 1000 QL. A complete list of all signal ratios (± SE) and details on all spotted genes can be found in Supplementary Table 2. To evaluate these criteria, we hybridized two microarrays with the same cDNA pools and found that 84% of the genes had the same regulation (Heidel & Baldwin 2004).
RNA gel blot analysis RNA samples (20µg) were size-fractionated by 1.2% (w/v) agarose formaldehyde gel electrophoresis and capillary blotted onto a nylon membrane (GeneScreenPlus; NEN-DuPont, Boston) as described in the manufacturer’s instructions. Ethidium bromide staining of the gel prior to blotting revealed rRNA bands, which served as the loading control. After blotting and UV-crosslinking, 32P-labeled probes specific for PIN2b were used for detection. The probe for PIN2b was obtained by PCR of S. nigrum cDNA with primers specific for SaPIN2b from S. americanum (Xu et al. 2001). This fragment was used to screen a S. nigrum leaf cDNA library (Lambda ZAP II kit, Stratagene, LaJolla, CA, USA), and the longest resulting PIN2b sequence (679 bp; SnPIN2b; AY422686) was used as a probe to detect PIN2b transcripts.
16 MS 1 – S. nigrum – an ecological model system
Real-time quantitative PCR (qPCR) Total RNA was reverse transcribed into cDNA using SuperScriptTM II RNaseH- Reverse Transcriptase (Invitrogen, Groningen, The Netherlands) according to the manufacturer’s instructions. The amount of cDNA template used per well was reverse transcribed from 10 ng total RNA; each sample was replicated thrice. The following sequences were used for the design of primers specific for PR-1, SnPIN2b, psbA and RuBPCase: Lycopersicon esculentum PR-1 (Tornero et al. 1997), S. nigrum PIN2b (see above), S. nigrum photosystem II D1 protein (Zhu et al. 1989) and L. esculentum RuBPCase LESS17 (McKnight et al. 1986). 18S RNA (template for primers: S. tuberosum gene for 18S RNA, GenBank Accession No. X67238) was used for quantitative normalization. The ABI PRISM® 7700 Sequence Detection System (Applied Biosystems, Darmstadt, Germany) was used for the SYBR Green I based assay. The qPCRTM Core Kit for SYBR® Green I (Eurogentec, Seraing, Belgium) was used according to the manufacturer’s instructions with the following cycler conditions: 10 min 95°C; 40 cycles: 30 sec 95°C, 30 sec 60°C. To ensure the specificity of the PCR reaction, a melting curve analysis was conducted using the ABI PRISM® 7700 Dissociation Curve Software. To detect asRuBPCase transcripts amplicons specific for the as-construct were designed (Halitschke & Baldwin 2003). The assay using a double dye-labeled probe was performed on an ABI PRISM® 7700 Sequence Detection System (qPCRTM Core Kit, Eurogentec) with 18S RNA for normalization (TaqMan® Ribosomal RNA Control Reagents, Applied Biosystems).
Isolation and blotting of genomic DNA Plant genomic DNA was prepared from leaves of S. nigrum using cetyltrimethylammonium bromide (Reichhardt & Rogers 1994). DNA samples were restriction digested with EcoRV, size-fractionated by 0.8 % agarose gel electrophoresis, and Southern blotted onto a nylon membrane with high-salt buffer (Brown 1995). The blot was analyzed with 32P-labelled probe specific for the hygromycin resistance gene (hph).
17 MS 1 – S. nigrum – an ecological model system
Plant treatments MeJA-induction: We applied 250 µg of methyl jasmonate (MeJA; Sigma) in 20µL lanolin (Sigma) to the stem of the 5-week-old plants (n = 62) above the third leaf node. To exclude possible lanolin effects, we treated control plants (n = 55) with 20µL lanolin. Flea beetle damage: Flea beetles (Epitrix pubescens) were abundant at our field site. To compare uninfested control plants with flea-beetle- infested plants, we sprayed the controls (n = 28) with the pyrethroid- based insecticide Spruzit (0.1 %, Neudorff, Emmerthal, Germany) and the infested plants (n = 31) with water directly after planting each day until tissue was harvested for microarray and PI analysis. The average flea beetle load of the water-sprayed plants the duration of the experiment was approximately 40 adults per plant compared to 1-5 for pyrethroid-sprayed plants. For the microarray analysis, we harvested and pooled fully expanded leaves of 8 individual plants 48h after exposure to flea beetles, flash-froze them in liquid nitrogen, and stored the leaf samples at –80°C until RNA extraction. For the PI analysis, a systemic leaf near the youngest node of all control and elicited plants was harvested 3 days after induction (MeJA or flea beetle), flash-frozen in liquid nitrogen, and stored at –80°C until protein extraction. Herbivore comparison: Leptinotarsa decemlineata originated from wild populations on S. tuberosum, Acherontia atropos, from a laboratory population reared on S. nigrum. To achieve approximately the same leaf area damage among herbivore treatments, we placed 2 L. decemlineata adults or two second-instar A. atropos larvae on individual 5-week-old plants (5 replicates per treatment). After 24h of continuous feeding we collected VOCs from the differentially treated plants (see VOC analysis).
Trypsin-PI analysis Harvested leaves were ground in liquid nitrogen. Proteins were extracted according to the protocol used for Nicotiana attenuata (Van Dam et al. 2001) and protein content was measured by the method of Bradford (1976) with immunoglobulin G (Sigma) as standard. We determined the
18 MS 1 – S. nigrum – an ecological model system activity of trypsin inhibitors by the radial immunodiffusion assay (Jongsma et al. 1993). A series of soybean trypsin inhibitor (STI, Sigma) solutions was used to obtain a reference curve. Trypsin-PI activity is expressed as nmol/mg of total protein.
VOC analysis In an open-flow trapping system, VOCs of one fully-expanded stem leaf were collected (see Fig. 3A). To confine insects to a single leaf and to trap volatiles from the same leaf, leaf and insects were enclosed in 400-mL polystyrene chambers fitted with holes at both ends. Air was pulled through the chamber at 450-500 mL min-1 (measured by a mass flow meter: Aalborg Instruments, Orangeburg, NY, USA) and subsequently through a charcoal air sampling trap (ORBOTM-32; SUPELCO, State College, PA, USA) by a portable vacuum pump. Each charcoal trap was spiked with 300ng tetraline as an internal standard
Fig. 1 Herbivores on Solanum nigrum: (A) Colorado potato beetle (Leptinotarsa decemlineata Say); (B) flea beetle (Epitrix pubescens Koch); (C) cicada (Macrosteles sexnotatus Fallèn); (D) mirid bug (Lygus wagneri Remane); (E) pentatomid bug (Dolycoris baccarum Linnè); (F) bean aphids (Aphis fabae Scopoli) and syrphid fly.
19 MS 1 – S. nigrum – an ecological model system
(ISTD) for quantification, eluted with 750µL dichloromethane, and analyzed by GC-MS according to Halitschke et al. (2000).
Results
Herbivore community During the 2002 and 2003 growing seasons, we sampled different native and planted populations of S. nigrum near Jena for insects. In 2002, the main collection sites were a planted population in Jena- Isserstedt (~100 plants) and native and planted populations in Jena-Nord (~500 plants); 2003 specimens were collected mainly from a planted population (~140 plants) on the Jena/Beutenberg-Campus. Populations were sampled at least once a week from May to September of each year. We classified insects as being herbivores on S. nigrum only if the adult insects or their larvae were repeatedly observed to feed on S. nigrum in both years (Table 1). Many additional insect species were observed on the plants, but only a small subset were observed to consistently feed on this plant. For example, only 3 of at least 24 Coleopteran species repeatedly found on the plants were classified as S. nigrum herbivores. The phytophagous insects belong to different groups according to their feeding behavior and host-plant specialization. Two Solanaceous specialists, both leaf-chewing beetles, namely the Colorado potato beetle L. decemlineata and the flea beetle E. pubescens, were repeatedly found on S. nigrum (Fig. 1). The flea beetles infested the plantation and native S. nigrum plants heavily throughout 2002 in Jena-Nord. L. decemlineata appeared in August, having dispersed from small potato fields. In addition to the leaf-chewing species, we found a variety of piercing- sucking insects, including bugs, cicadas, and aphids. Aphis fabae colonized S. nigrum plants from May until September. Later in the season (July/August) mirid bugs of the genus Lygus were the most abundant herbivores. The rich herbivore fauna attracted a large variety of parasitoids and predatory species (syrphids [Fig. 1F], chysopids, coccinellids, etc).
20 MS 1 – S. nigrum – an ecological model system
Table 1 List of herbivore species observed feeding on Solanum nigrum in experimental field plots in Jena, Germany, during two growing seasons. Species are sorted by order (Coleoptera, Lepidoptera, Heteroptera, Auchenorrhyncha, Sternorrhyncha) and are classified by feeding guild (C- leaf chewing, PP-piercing sucking on phloem, PM- piercing sucking on mesophyll) and whether they are specialized on Solanaceous plants.
Feeding Speciali- Species Order Family guild zation Epitrix pubescens C Chrysomelidae C Specialist Leptinotarsa decemlineata C Chrysomelidae C Specialist Barypeithes pellucidus. C Curculionidae C Generalist
Plutella xylostella L Plutellidae C Generalist?
Lygus pratensis H Miridae PM Generalist Lygus rugulipennis H Miridae PM Generalist Lygus wagneri H Miridae PM Generalist Stenodema sericans H Miridae PM Generalist Dolycoris baccarum H Pentatomidae PM Generalist Holocostethus vernalis H Pentatomidae PM Generalist Corizus hyoscyami H Rhopalidae PM Generalist
Philaenus spumarius A Cercopidae PP Generalist Balclutha punctata A Cicadellidae PP Generalist Evacanthus interruptus A Cicadellidae PP Generalist Empoasca spec. A Cicadellidae PP Generalist Eupteryx aurata A Cicadellidae PP Generalist Macrosteles sexnotatus A Cicadellidae PP Generalist
Aulacorthum solani langei S Aphididae PP Generalist Aphis fabae S Aphididae PP Generalist
21 MS 1 – S. nigrum – an ecological model system
Fig. 2 Characterization of asRuBPCase S. nigrum lines. (A) DNA gel-blot analysis of three as-RuBPCase T lines (as1, as2, as3, as4; three replicates 1 each) and one wildtype, untransformed line (wt). Genomic DNA was digested with EcoRV and the blot was hybridized with a probe specific for the hygromycin resistance gene (hph). The lines as1 and as2 have single copy insertions of the transgene, while as3 has two copies and as4 has multiple copies. The wt DNA is shown as a negative control. (B) Summary of transformation efficiency. Twenty-two independent lines were examined in detail, 19 were found to be transformed as verified by antibiotic selection and PCR. (C) Relative expression of asRuBPCase (lower panel) and RuBPCase (upper panel) in as-lines (as1-as4) and wt, as determined by real-time qPCR. The as-lines showed expression of the as- transcript in different amounts and had reduced amounts of RuBPCase transcripts in comparison to the amounts measured in wt plants (r = - 0.549, p = 0.0326). Transformation clearly resulted in differential silencing of the endogenous RuBPCase transcripts.
Transformation We developed an Agrobacterium-based transformation procedure for S. nigrum, which requires approximately 5-6 months from the transformation to the production of T0 plants bearing mature fruit. Transformation and regeneration of plantlets (2 cm in size) that can be transferred into soil requires 2 months. Callus generation in S. nigrum
22 MS 1 – S. nigrum – an ecological model system occurred at a lower rate than it does in Nicotiana attenuata, but the calli that did generate, did so at a higher rate (M. Lim unpublished results). Short tissue culture times are helpful in reducing somaclonal variation, which limits the production and utility of transgenic plants (Beaujean et al. 1998). In comparison to the published S. nigrum protoplast transformation procedure using A. rhizogenes (Wei et al. 1986), our protocol proved to be more efficient in the regeneration of plants. Additionally, the use of Ri-based vectors increases the frequency of phenotypically abnormal and infertile plants in comparison to the T- DNA based vectors used here (Davey et al. 1987). Hygromycin resistance (hph) proved to be a reliable selectable marker that could be incorporated into the germination media and subsequently allowed seedlings to be rapidly selected. The efficiency of the procedure is high (Fig. 2 B inset): 19 of the 22 haphazardly selected asRuBPCase lines (86 %) could be verified as harboring the transgene by means of antibiotic selection and
PCR. For further characterization, we selected 10 T1 lines and examined them by DNA gel blot analysis (4 lines are shown in Fig. 2A). All lines contained the transgene, and 2 of the 10 lines tested contained the transgene as a single copy insertion. Real-time qPCR supported the successful incorporation of the transgene into S. nigrum´s genome (Fig. 2C). As expected no antisense (asRuBPCase) transcripts were detected in wildtype (wt) plants, whereas these transcripts were abundant in the transformed lines. The quantity of asRuBPCase-transcripts in transformed lines correlated negatively with the quantity of RuBPCase-transcripts (Pearson´s correlation coefficient r = -0.549, p = 0.0327), demonstrating that the transformation had successfully reduced the expression of this important photosynthetic gene.
23 MS 1 – S. nigrum – an ecological model system
24 MS 1 – S. nigrum – an ecological model system
Fig. 3 (p. 24) Expression of S. nigrum genes in response to attack from flea beetles, Epitrix pubescens, by microarray, RNA gel-blot, and real-time quantitative PCR analysis (C = control, FB = flea-beetle-infested). Microarray analysis revealed gene regulation in different categories and independent gel-blot and real-time PCR analyses verified expression patterns of individual genes from the array. The expression ratios (normalized mean Cy3/Cy5 ratio) for three categories (defense, photosynthesis, signaling) from an array hybridized with fluorescently labeled cDNA from attacked and unattacked plants are depicted. Inset table (A) summarizes the numbers of up- and down-regulated genes in the categories (see Supplemetary Table 1 for a complete list of regulated genes and gene descriptions). Arrows identify genes used for verification of the expression data (panel B-E). (B) RNA gel blot analysis hybridized with an SnPIN2b probe (18S RNA is shown as a control for equal loading). (C-E) Real-time qPCR for the genes (C) SnPIN2b, (D) PR1, and (E) psbA. The expression level in the control sample equals 1.
Microarray analysis Results of the oligonucleotide microarray analysis are summarized in Fig. 3 and the complete list of all regulated genes, their expression ratios, and annotations can be found in Supplementary Table 1. The hybridization compared Epitrix pubescens-infested plants with plants treated with insecticide to protect from damage by flea beetles. We found a total of 155 genes to be significantly regulated (27 % of 568 genes on the microarray). The regulated genes were assigned to different putative functional categories (Fig. 3A). Several genes involved in the biosynthesis of defense-related secondary metabolites were down- regulated (tropinone reductase, TRII; phenylalanine ammonia lyase, PAL). A suite of PI genes were strongly up-regulated (SaPIN2a, SaPIN2b, pin2, PI-WuSP), which correlated with the observed increase of Trypsin- PI activity in flea beetle-infested plants (Fig. 4C). Amongst defense- related genes, we found an α−dioxygenase (PIOX; cv57.4) to be up- regulated, in addition to several pathogenesis-related proteins (PR-1; PR-2; PRP4; PRP5; PRP-6; PRp27). Photosynthesis-related genes were generally down-regulated (e.g. different subunits of RuBPCase). Genes
25 MS 1 – S. nigrum – an ecological model system involved in defense-signaling processes were strongly up-regulated in flea beetle-infested plants (octadecanoid pathway: lipoxygenases LOX; alleneoxide synthase AOS; 12-oxophytodienoate reductase opr). The microarray results were verified by RNA gel blot analysis with an SnPIN2b probe and real-time qPCR for the genes SnPIN2b, PR-1, and psbA (Fig. 3 B-E).
Proteinase inhibitors We assayed systemic leaves of MeJA- and flea beetle (Epitrix pubescens)- infested plants for their Trypsin-PI content. Greenhouse- grown plants showed a significant increase in Trypsin-PI activity (Student´s t-test, t = -15.239, p = 0.001) after treatment with 250µg MeJA (Fig. 3A). A similar increase was found in field-grown plants (Fig. 3B; Student´s t-test, t = 5.301, p < 0.001), but the constitutive Trypsin-
Fig. 4 Trypsin-PI protein levels (mean ± SE) in MeJA-treated (250 µg/plant) and flea beetle-infested plants. (A) Greenhouse-cultivated S. nigrum plants, MeJA-treated; (B) field-grown S. nigrum, MeJA-treated; (C) field-grown S. nigrum, flea beetle-infested. All treatments significantly increased Trypsin-PIs in the leaves of S. nigrum. Trypsin- PI-levels correlated with expression of Trypsin-PI genes (Fig. 3A, B, G). 26 MS 1 – S. nigrum – an ecological model system
PI-levels of field plants were higher than those of greenhouse plants at a similar developmental stage. Trypsin-PI-levels in field-grown S. nigrum plants were significantly higher in response to attack from the naturally occurring herbivore, E. pubescens (Fig. 3B; Student´s t-test, t = 2.397, p = 0.0199). The increase of PIs was similar in MeJA-elicited and flea beetle- infested plants.
Fig. 5 Volatile organic compounds (VOCs) released in response to attack from different herbivore species. (A) VOCs were collected from 15 field- grown plants, using an open-flow trapping system. (B) Representative Total Ion Chromatograms of the headspace volatiles eluting from a GC column of an undamaged leaf from an undamaged Solanum nigrum plant (control), a leaf damaged by Leptinotarsa decemlineata, and a leaf damaged by an Acherontia atropos hornworm (from separate plants). The labels represent an unknown monoterpene (1), 3-carene (2), β-myrcene (3), +/-limonene (4), unknown compound (5), cis-3-hexenyl acetate (6) cis-3-hexen-1-ol (7), longifolene (8), trans-β-caryophyllene (9), unknown sesquiterpene 1 (10), unknown sesquiterpene 2 (11).
27 MS 1 – S. nigrum – an ecological model system
Volatile organic compounds In a field experiment, we collected VOCs emitted from plants in response to attack from phytophagous insects. We used an open-flow trapping system (Fig. 5A), which allowed us to synchronously sample all experimental replicates and to maintain the sampled leaves under physiological conditions. We allowed two herbivore species (L. decemlineata and A. atropos) to feed separately on S. nigrum for 24h and compared the composition of VOCs trapped with those trapped from uninfested control plants. Emissions of several compounds, ranging from monoterpenes (e.g. 3-carene, β-myrcene, +/-limonene) to green-leaf volatiles (cis-3-hexenyl acetate, cis-3-hexen-1-ol) and sesquiterpenes (longifolene, trans-β-caryophyllene), was increased in insect-attacked plants (Fig. 5B).
Fitness We evaluated the fitness consequences of MeJA-elicitation under field conditions in the plants previously assayed for Trypsin-PIs. S. nigrum is extremely plastic in its growth form and is able to adjust its morphology to diverse conditions. In botanically precise terminology, the growth shape varies from decurrent with plagiotropic to fastigiate branching to excurrent with orthotropic branching. Plant size at reproductive maturity varies from 5 cm to over 1 m. We manipulated plant size in a greenhouse experiment by planting S. nigrum into different sized pots and found that the dry biomass correlated strongly with the number of fruits produced (R2 = 0.665). In the field, we did not find morphologically detectable changes in response to a single elicitation with MeJA, nor was the number of flowers produced (data not shown) significantly influenced by elicitation. However, fruit number and seed production differes significantly between induced and uninduced plants. MeJA-treated plants produced approximately 40 % fewer fruits than did control plants (Fig. 6), suggesting that MeJA-elicited responses result in large fitness costs for an individual plant.
28 MS 1 – S. nigrum – an ecological model system
Fig. 6 Fitness estimates of S. nigrum. (A) Fruit number: Comparison of MeJA-elicited vs. control plants. Fruit numbers were significantly lower in plants that received a single treatment of 250 µg MeJA (Student´s t- test, t = 3.256, p = 0.0038). One fruit contained 77 ± 1.45 (SEM, n = 33) seeds; hence control plants produced approximately 130,000 seeds, while elicited plants produced 80,000 seeds per plant. (B) Fruit number is linearly correlated with the above-ground dry mass of greenhouse-grown plants. Different plant sizes were obtained by growing plants in (1) 1-L pots, (2) 2-L pots, or (3) 3-L pots.
29 MS 1 – S. nigrum – an ecological model system
Discussion To facilitate the identification of genes mediating responses to ecological interactions, and to allow for the manipulation of their expression, we present the following tools for the S. nigrum expression system: 1) an Agrobacterium-based transformation system; 2) an oligonucleotide microarray, enriched with ecologically relevant genes; 3) measures of direct (PIs) and indirect defenses (VOC emission) under both field and laboratory conditions; and 4) measures of Darwinian fitness. These tools have been optimized to analyze responses that are rapidly elicited by ecological interactions. We illustrate their utility by analyzing responses to attack by a native herbivore of S. nigrum and compare the elicited responses with those elicited by MeJA treatment. The analysis links changes in transcript abundance with phenotypic changes, which, in turn, are correlated with changes in the fitness of plants grown under field conditions. An initial survey of the differential gene expression of S. nigrum revealed a large-scale change in the plants’ transcriptome in response to flea beetle attack: 27% of the monitored genes showed a significant change in expression pattern. Genes involved in important primary processes, such as carbon fixation and metabolism, were largely down- regulated, while defense genes, including genes involved in defense- related signaling, were largely up-regulated. The octadecanoid signaling cascade with its key compound, jasmonic acid (JA), is known to play an important role in triggering many of the insect-induced responses of a plant (Blee 2002; Farmer et al. 2003). Consistent with a regulatory role for oxylipins in the elicitation of defense-related genes in S. nigrum, oxylipin biosynthetic enzymes (AOS, allene oxide synthase; LOX, lipoxygenase; OPR, 12-oxophytodienoate reductase) were strongly up- regulated, as were the transcripts of the JA-elicited PI defense genes (e.g. SaPIN2a, SaPIN2b, pin2). PIs are known to adversely affect the performance of herbivorous insects (Rahbe et al. 2003; Xu et al. 1996), and the causal associations among JA signaling, PI elicitation, and insect resistance were recently established with N. attenuata plants in which JA signaling was silenced by expressing LOX-H3 in an antisense orientation
30 MS 1 – S. nigrum – an ecological model system
(Halitschke & Baldwin 2003). In this study herbivore performance was enhanced on asLOX plants most likely due to attenuated PI and nicotine defense responses. Several pathogenesis-related (PR) proteins (PR-1; PR-2, β-1,3- glucanase; PR3, PR4 both chitinases; PRP6; Prp27), which are known to be elicited by pathogen attack or salicylic acid (SA) treatment, were among the strongest up-regulated transcripts. From this, we deduce that herbivore attack to S. nigrum may elicit both SA- and JA-related signaling and the commonly invoked tradeoff between systemic resistance of plants to microorganisms and resistance to insect herbivores (Felton et al. 1999; Thaler et al. 2002) may not apply to this plant species. Some transcripts may be co-regulated by both insects and microorganisms. For example, α-dioxygenase (α-DOX; formerly PIOX, pathogen-induced oxygenase), an enzyme which catalyzes the conversion of linolenic acid to its 17-hydroperoxy-derivative (Hamberg et al. 1999), is up-regulated in S. nigrum in response to flea beetle feeding. In N. tabacum, α-DOX can be induced by bacterial elicitors (de Leon et al. 2002; Sanz et al. 1998), and in N. attenuata, by attack from Manduca sexta larvae (Hermsmeier et al. 2001). While the biological function of α-DOX remains unclear, its transcriptional behavior illustrates the likely interactions of pathogen and herbivore signaling under natural conditions. Adaptive responses are likely to be the result of complex interacting signal networks rather than single signal cascades (Genoud et al. 2001; Reymond & Farmer 1998), and transcription factors may play an important role in coordinating the responses from these many signal cascades. In plants attacked by flea beetles, a transcription factor of the WRKY family (WRKY3) was strongly up-regulated, as it was shown to be in N. attenuata attacked by M. sexta larvae (Hui et al. 2003). WRKY transcription factors occur in large gene families and are known to regulate numerous stress-related genes, including those responsive to pathogens and wounding (Eulgem et al. 2000). Transcription factors may coordinate large-scale patterns of transcriptional changes and deserve more attention in the regulation of environmental responses.
31 MS 1 – S. nigrum – an ecological model system
The coordination of transcriptional responses to flea beetle attack extends beyond the up-regulation of stress-responsive transcripts to include a coordinated down-regulation of growth-related transcripts, which is most clearly seen in the negative correlation between the expression of photosynthetic and that of defense genes (Hermsmeier et al. 2001; Schittko et al. 2001). The herbivore-induced suppression of RuBPCase transcripts (pDH64.7, RUB inas, rbcL) and of additional elements of the photosynthetic machinery (e.g. photosystem proteins: N. tabacum PSI, N.t. PSII precursor, S. nigrum PSII D1) might benefit the plant by redirecting carbon flux toward the production of defenses. RuBPCase activase (rca), a stromal protein catalyzing the dissociation of inhibitory sugar bisphosphates from uncarbamylated and carbamylated RuBPCase in an ATP-requiring process (Portis 1995; Robinson & Portis 1989), may play an important role in regulating RuBPCase transcripts and perhaps other photosynthetic proteins, and was down-regulated in attacked plants. It has been shown that the light-dependent regulation of RuBPCase is controlled by rca (Zhang et al. 2002) but recently, Voelckel & Baldwin (2003) demonstrated that the expression of rca in N. attenuata increased in response to attack by the mirid bug, Tupiocoris notatus. Hence it is possible that rca participates in the herbivore-induced down- regulation of photosynthetic metabolism. In addition to the down- regulation of photosynthetic genes, genes involved in cell wall metabolism (XTH4, xyloglucan endotransglycosylase), glycolysis (DH63, homologous to Petunia hybrida triosephosphate isomerase; DH123 and cGap, both homologous to N. tabacum glyceraldehyde-3-phosphate dehydrogenase), and nitrogen metabolism (nir, nitrate reductase; GOGAT, glutamine oxoglutarate aminotransferase; etc.) were also down- regulated. These alterations suggest many hypotheses about the regulation of primary metabolism in response to herbivore attack and require additional work. Even though only a few of the sequences on this ‘Solanaceous’ microarray were designed from S. nigrum specific-sequences, it is clear from the verifications of selected array responses by RNA gel blot analysis and real-time qPCR (Fig. 3B-E) that the microarray provided
32 MS 1 – S. nigrum – an ecological model system valuable information about differential gene expression in S. nigrum. Several studies have established the utility of using sequence information of related species for monitoring gene expression. Kane et al. (2000) demonstrated that expression patterns derived from oligonucleotide (50- mer) microarrays reflected those from cDNA (~300-400bp PCR products) microarrays and that 50-mer oligonucleotides are specific, if the target sequences shared 80% or more with the oligonucleotide. Izaguirre et al. (2003) analyzed the transcriptome of N. longiflora with a cDNA microarray consisting of N. attenuata sequences and Held et al. (2004) used the same microarray to characterize transcriptional responses in N. clevelandii and N. quadrivalvis. Girke et al. (2000) compared gene expression in developing seeds of Arabidopsis thaliana and Brassica napus with an Arabidopsis-specific microarray. Regardless of whether the array is designed from homologous or heterologous sequences, responses should always be verified before a hypothesis about the functional significance of a gene is pursued. The need for verification is particularly acute when microarray studies suggest a lack of response, as negative results can be caused by small sequence differences between the oligonucleotides and the targeted transcripts. When a microarray produces signals, the spotted oligonucleotides will likely function as probes for screening S. nigrum cDNA libraries, thus facilitating the verification procedure. Once a transcriptional response is verified, the next step in an ecological analysis is to determine if the response correlates with a change in phenotype, not only in the laboratory but also in the field. The hypothesis from the microarray analysis that flea beetle attack elicited JA signaling and thereby increased PI production was supported by the observations with both field- and greenhouse-grown plants that beetle attack and MeJA treatment significantly increased PI levels. The absolute levels of PI activity were higher in field-grown plants (Fig. 4), suggesting that plants growing in the rough-and-tumble of the natural world may be partially induced in comparison to the coddled plants grown in the glasshouse. PIs are probably elicited by exposure to UV-B, as shown by Izaguirre et al. (2003), who used phenotypic
33 MS 1 – S. nigrum – an ecological model system measures and microarrays to compare transcriptional patterns in N. longiflora elicited by UV-B with the pattern elicited by Manduca sexta herbivory. In this study, UV-B exposure not only increased PI transcripts and activity but also down-regulated photosynthesis-related transcripts in a manner similar to herbivore-elicited responses, suggesting that common regulatory elements had been recruited by this pair of abiotic and biotic stressors. In response to herbivore attack, plants frequently activate indirect defense responses that complement the function of the direct defenses (Kessler & Baldwin 2002). By releasing VOCs in response to herbivore attack, plants attract predators and parasitoids of herbivorous insects (Dicke et al. 2003; Kessler & Baldwin 2001; Turlings et al. 1990). When S. nigrum plants were attacked by herbivores from two different Orders of insects in the field (Fig. 6), the composition and the quantities of the VOCs trapped from the headspace of leaves significantly differed from those of unattacked plants. The fact that an induced response was observed in field-grown plants is significant, particularly in light of recent reports on Zea mays demonstrating just how significantly abiotic factors such as soil nutrition, air humidity, temperature, and light can influence the herbivore-induced VOC response (Gouinguene & Turlings 2002; Schmelz et al. 2003).The large and diverse predator community (syrphids, chrysopids, coccinellids and braconid wasps) we observed during our field studies of S. nigrum could respond to these VOC emissions. Whether any of these potential predators actually respond to increased VOCs can be readily tested by adding components of or the entire herbivore-induced volatile blend to plants containing a ‘predator- monitor’ (an herbivorous insect or egg used to score predation events: Kessler & Baldwin 2001). Whether or not a trait can be formally considered to be a defense depends on whether it increases a plant’s Darwinian fitness in environments with aggressors. The best surrogate measures for Darwinian fitness are determined by a plant’s life history, but for selfing annual plants such as S. nigrum, lifetime seed production is likely to be an adequate measure. Fruit and seed production were found to be strongly
34 MS 1 – S. nigrum – an ecological model system correlated with plant above-ground dry mass (Fig. 6B), and these are an important measures for the resource partitioning to growth, reproduction and defense in response to biotic interactions (Bazzaz et al. 1987; Givnish 1986; Mauricio et al. 1993). The reproductive output of plants that have been under strong artificial selection for particular yield components will frequently be strongly buffered from variations in canopy. For example, tomato plants are strongly buffered from leaf area loss from herbivores and do not decrease fruit number in response to herbivore attack (Thaler 1999). We found that jasmonate elicitation of S. nigrum did not reduce plant size, a conclusion Thaler et al. (1996) had reached with regard to jasmonate treatment of tomato. Moreover, we found that jasmonate elicitation in S. nigrum significantly reduced lifetime fruit and seed production, although this is not observed in tomato (Thaler 1999), which likely reflects differences between agronomic and native species in their selective history. Jasmonate elicitation is known to significantly decrease lifetime seed production in N. attenuata (Baldwin 1998), and a large fraction of these fitness costs can be attributed to the induced production of PIs (Zavala et al. 2004). The underlying mechanisms of S. nigrum’s fitness costs remain to be explored. Direct manipulation of the genetic basis of an observed response is the most powerful means of falsifying functional hypotheses available to biologists. Agrobacterium-based transformation systems provide the means to produce stably transformed lines in which particular genes are silenced or over-expressed. Independently transformed lines are typically variable in their phenotypes due to the random insertion of the transgene into the genome, which, in turn, leads to differences in transcriptional activity (‘positional’ effects). Krügel et al. (2002) demonstrated that N. attenuata lines, transformed with an asLOX construct exhibited up to 71% reduction in wound-induced JA accumulation. This genetically determined phenotypic variation is enormously useful, because it allows the fitness consequences of a trait to be quantitatively analyzed. The S. nigrum asRuBPCase lines showed a clear reduction in RuBPCase transcripts (Fig. 2C), yet they did not reveal differences in their growth phenotype in comparison to wildtype plants
35 MS 1 – S. nigrum – an ecological model system in the glasshouse. Whether this lack of growth phenotype persists when these plants are grown under field conditions will be interesting to determine. Experiments in realistic environments may reveal why plants appear to be ´over-engineered` with respect to their RuBPCase pools (Matt et al. 2002; Quick et al. 1991). The rapid development of new transformation vectors that allow for more efficient silencing of endogenous genes (RNAi with inverted repeat elements: Waterhouse & Helliwell 2003), will make the process of producing transformants with fully silenced genes more efficient, thereby facilitating the search for phenotypes of plants that are grown in complex environments. Most of the tools presented in this paper have been used only in controlled laboratory experiments. The results obtained from latter experiments can be different from what is seen in plants growing in nature. For example, while constitutive PI levels were higher in field- grown plants than in greenhouse-grown plants, field-grown plants (Fig. 4) were still inducible. The combination of several stresses often provokes a potentiation of a response, leading either to an increase or a decrease of subsequent responses to the same or other stresses (Zimmerli et al. 2000). Therefore the plant may recruit similar fundamental cellular reactions in response to various stresses, which may be what is happening in response to UV-B irradiation and herbivory (Izaguirre et al. 2003) or cold and drought stress in comparison to disease resistance (Singh et al. 2002). Experimentation with field-grown plants in which a variety of stresses are factorially manipulated will elucidate both the amount of cross-talk that occurs among environmental responses as well as the fitness consequences of the different selective forces for plants whose ability to respond is selectively impaired. In summary, while molecular biology has provided the ability to manipulate the expression of individual genes, understanding the functional consequences of these manipulations will require additional ecological tools to dissect the complex interplay of selective forces that all organisms face in nature. Multivariate and path analysis provide a means to examine correlations among the different levels of analysis that occur from gene expression to the formation of a phenotype with a given
36 MS 1 – S. nigrum – an ecological model system
Darwinian fitness. Ecologists have been successful in using such approaches to evaluate complex correlations and to recognize interrelationships among the biotic and abiotic factors that structure ecosystems. The challenge remains to find a way to incorporate the powerful manipulative and descriptive molecular methods into this “big picture” analysis so as to harvest the fruits of the molecular revolution.
Acknowledgments: We thank Susan Kutschbach and Thomas Hahn for invaluable assistance in microarray processing and data analysis, Dr. Jörg Perner for assistance in determining beetles, Dr. Wolfgang Weisser for assistance in determining aphids, Dr. Tamara Krügel for valuable support and advice and the Max-Planck-Gesellschaft and the DFG (project BA2138/1-1) for financial support.
References Baldwin IT (1998) Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences of the United States of America 95, 8113-8118. Baldwin IT, Kessler A, Halitschke R (2002) Volatile signaling in plant- plant-herbivore interactions: what is real? Current Opinion in Plant Biology 5, 351-354. Bazzaz FA, Chiariello NR, Coley PD, Pitelka LF (1987) Allocating Resources to Reproduction and Defense. Bioscience 37, 58-67. Beaujean A, Sangwan RS, Lecardonnel A, Sangwan-Norreel BS (1998) Agrobacterium-mediated transformation of three economically important potato cultivars using sliced internodal explants: an efficient protocol of transformation. Journal of Experimental Botany 49, 1589-1595. Begon M, Harper JL, Townsend CR (1996) Ecology: Individuals, Populations and Communities Blackwell Scienctific Publications, Oxford. Blee E (2002) Impact of phyto-oxylipins in plant defense. Trends in Plant Science 7, 315-321. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Analytical Biochemistry 72, 248-254. Brown T (1995) Southern Blotting. In: Short Protocols in Molecular Biology (ed. Struhl K). John Wiley & Sons, New York.
37 MS 1 – S. nigrum – an ecological model system
Davey MR, Mulligan BJ, Gartland KMA, et al. (1987) Transformation of Solanum and Nicotiana species using an Ri-plasmid vector. Journal of Experimental Botany 38, 1507-1516. de Leon IP, Sanz A, Hamberg M, Castresana C (2002) Involvement of the Arabidopsis alpha-DOX1 fatty acid dioxygenase in protection against oxidative stress and cell death. The Plant Journal 29, 61-72. Dicke M, van Poecke RMP, de Boer JG (2003) Inducible indirect defence of plants: from mechanisms to ecological functions. Basic and Applied Ecology 4, 27-42. Edmonds JM, Chweya JA (1997) Black nightshades, Solanum nigrum L. and related species, Gatersleben, Rome. Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends in Plant Science 5, 199-206. Farmer EE, Almeras E, Krishnamurthy V (2003) Jasmonates and related oxylipins in plant responses to pathogenesis and herbivory. Current Opinion in Plant Biology 6, 372-378. Felton GW, Korth KL, Bi JL, et al. (1999) Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory. Current Biology 9, 317-320. Genoud T, Santa Cruz MBT, Metraux JP (2001) Numeric simulation of plant signaling networks. Plant Physiology 126, 1430-1437. Girke T, Todd J, Ruuska S, et al. (2000) Microarray analysis of developing Arabidopsis seeds. Plant Physiology 124, 1570-1581. Givnish TJ (1986) Economics of biotic interactions. In: On the economy of plant form and function (ed. Givnish TJ), pp. 667-679. Cambridge University Press, Cambridge. Gouinguene SP, Turlings TCJ (2002) The effects of abiotic factors on induced volatile emissions in corn plants. Plant Physiology 129, 1296-1307. Halitschke R, Baldwin IT (2003) Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata. The Plant Journal in press. Halitschke R, Gase K, Hui D, Schmidt DD, Baldwin IT (2003) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VI. Microarray analysis reveals that most herbivore- specific transcriptional changes are mediated by fatty acid-amino acid conjugates. Plant Physiology 131, 1894-1902.
38 MS 1 – S. nigrum – an ecological model system
Halitschke R, Kessler A, Kahl J, Lorenz A, Baldwin IT (2000) Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata. Oecologia 124, 408-417. Hamberg M, Sanz A, Castresana C (1999) Alpha-oxidation of fatty acids in higher plants - Identification of a pathogen-inducible oxygenase (PIOX) as an alpha-dioxygenase and biosynthesis of 2- hydroperoxylinolenic acid. Journal of Biological Chemistry 274, 24503-24513. Heath RL, McDonald G, Christeller JT, et al. (1997) Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests. Journal of Insect Physiology 43, 833-842. Heidel AJ, Baldwin IT (2004) Analysis of defense responses in Nicotiana attenuata to herbivores from different feeding guilds. Plant, Cell and Environment in press. Held M, Gase K, Baldwin IT (2004) Array technology meets ecology: Sources of variance, statistical analyses and applications of a cDNA microarray for plant-herbivore interactions. Ecological Applications in press. Hermsmeier D, Schittko U, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. I. Large-scale changes in the accumulation of growth- and defense-related plant mRNAs. Plant Physiology 125, 683-700. Hui D, Iqbal J, Lehmann K, et al. (2003) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata V. Microarray Analysis and Further Characterization of Large-Scale Changes in Herbivore- Induced mRNAs. Plant Physiology 131, 1877-1893. Izaguirre MM, Scopel AL, Baldwin IT, Ballare CL (2003) Convergent responses to stress. solar UV-B radiation and Manduca sexta herbivory elicit overlapping transcriptional responses in field- grown plants of Nicotiana longiflora. Plant Physiology 132, 1755-1767. Jongsma MA, Bakker PL, Stiekema WJ (1993) Quantitative determination of serine proteinase inhibitor activity using a radial Diffusion Assay. Analytical Biochemistry 212, 79-84. Jongsma MA, Bakker PL, Visser B, Stiekema WJ (1994) Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack, wounding and virus- infection. Planta 195, 29-35. Kane MD, Jatkoe TA, Stumpf CR, et al. (2000) Assessment of the sensitivity and specificity of oligonucleotide (50-mer) microarrays. Nucleic Acids Research 28, 4552-4557. 39 MS 1 – S. nigrum – an ecological model system
Kessler A, Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature. Science 291, 2141-2144. Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: The emerging molecular analysis. Annual Review of Plant Biology 53, 299-328. Koiwa H, Bressan RA, Hasegawa PM (1997) Regulation of protease inhibitors and plant defense. Trends in Plant Science 2, 379-384. Korth KL (2003) Profiling the response of plants to herbivorous insects. Genome Biology 4, 221.1-4. Krügel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002) Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology 12, 177-183. Matt P, Krapp A, Haake V, Mock HP, Stitt M (2002) Decreased Rubisco activity leads to dramatic changes of nitrate metabolism, amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants. Plant Journal 30, 663-677. Mauricio R, Bowers MD, Bazzaz FA (1993) Pattern of leaf damage affects fitness of the annual plant Raphanus sativus (Brassicaceae). Ecology 74, 2066-2071. McKnight TD, Alexander DC, Babcock MS, Simpson RB (1986) Nucleotide sequence and molecular evolution of 2 tomato genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. Gene 48, 23-32. Portis AR (1995) The regulation of Rubisco by Rubisco activase. Journal of Experimental Botany 46, 1285-1291. Quick WP, Schurr U, Fichtner K, et al. (1991) The impact of decreased Rubisco on photosynthesis, growth, allocation and storage in tobacco plants which have been transformed with antisense Rbcs. The Plant Journal 1, 51-58. Rahbe Y, Deraison C, Bonade-Bottino M, et al. (2003) Effects of the cysteine protease inhibitor oryzacystatin (OC-I) on different aphids and reduced performance of Myzus persicae on OC-I expressing transgenic oilseed rape. Plant Science 164, 441-450. Reichhardt M, Rogers S (1994) Preparation of plant DNA using CTAB. In: Current Protocols in Molecular Biology (ed. Struhl K), pp. 2.3.3- 2.3.7. John Wiley & Sons, New York. Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene expression. Current Opinion in Plant Biology 1, 404- 411.
40 MS 1 – S. nigrum – an ecological model system
Robinson SP, Portis AR (1989) Adenosine triphosphate hydrolysis by purified Rubisco activase. Archives of Biochemistry and Biophysics 268, 93-99. Ryan CA (1990) Protease inhibitors in plants - genes for improving defenses against insects and pathogens. Annual Review of Phytopathology 28, 425-449. Sanz A, Moreno JI, Castresana C (1998) PIOX, a new pathogen- induced oxygenase with homology to animal cyclooxygenase. Plant Cell 10, 1523-1537. Schittko U, Hermsmeier D, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. II. Accumulation of plant mRNAs in response to insect-derived cues. Plant Physiology 125, 701-710. Schmelz EA, Alborn HT, Engelberth J, Tumlinson JH (2003) Nitrogen deficiency increases volicitin-induced volatile emission, jasmonic acid accumulation, and ethylene sensitivity in maize. Plant Physiology 133, 295-306. Simms EL, Rausher MD (1992) Quantitative genetics. In: Ecology and Evolution of Plant Resistance (ed. Simms EL), pp. 42-68. University of Chicago Press, Chicago. Singh KB, Foley RC, Onate-Sanchez L (2002) Transcription factors in plant defense and stress responses. Current Opinion in Plant Biology 5, 430-436. Strong DR, Lawton JH, Southwood R (1984) Insects on Plants Blackwell Scientific Publications, Oxford. Thaler JS (1999) Induced resistance in agricultural crops: Effects of jasmonic acid on herbivory and yield in tomato plants. Environmental Entomology 28, 30-37. Thaler JS, Fidantsef AL, Bostock RM (2002) Antagonism between jasmonate- and salicylate-mediated induced plant resistance: Effects of concentration and timing of elicitation on defense- related proteins, herbivore, and pathogen performance in tomato. Journal of Chemical Ecology 28, 1131-1159. Thaler JS, Stout MJ, Karban R, Duffey SS (1996) Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field. Journal of Chemical Ecology 22, 1767-1781. Tornero P, Gadea J, Conejero V, Vera P (1997) Two PR-1 genes from tomato are differentially regulated and reveal a novel mode of expression for a pathogenesis-related gene during the
41 MS 1 – S. nigrum – an ecological model system
hypersensitive response and development. Molecular Plant-Microbe Interactions 10, 624-634. Turlings TCJ, Tumlinson JH, Lewis WJ (1990) Exploitation of herbivore-induced plant odors by host-seeking parasitic wasps. Science 250, 1251-1253. Van Dam NM, Horn M, Mares M, Baldwin IT (2001) Ontogeny constrains systemic protease inhibitor response in Nicotiana attenuata. Journal of Chemical Ecology 27, 547-568. Voelckel C, Baldwin IT (2003) Detecting herbivore-specific transcriptional responses in plants with multiple DDRT-PCR and subtractive library procedures. Physiologia Plantarum 118, 240-252. Waterhouse PM, Helliwell CA (2003) Exploring plant genomes by RNA-induced gene silencing. Nature Reviews Genetics 4, 29-38. Wei ZM, Kamada H, Harada H (1986) Transformation of Solanum nigrum L protoplasts by Agrobacterium rhizogenes. Plant Cell Reports 5, 93-96. Xu DP, Xue QZ, McElroy D, et al. (1996) Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests. Molecular Breeding 2, 167-173. Xu ZF, Qi WQ, Ouyang XZ, Yeung E, Chye ML (2001) A proteinase inhibitor II of Solanum americanum is expressed in phloem. Plant Molecular Biology 47, 727-738. Zavala JA, Patankar AG, Gase K, Baldwin IT (2004) Constitutive and inducible protease inhibitor production incurs large fitness costs in Nicotiana attenuata. Proceedings of the National Academy of Sciences of the United States of America 101, 1607-1612. Zhang N, Kallis RP, Ewy RG, Portis AR (2002) Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger Rubisco activase isoform. Proceedings of the National Academy of Sciences of the United States of America 99, 3330-3334. Zhu L, Hu N, Zhai W, Li X (1989) The nucleotide sequence of the chloroplast psbA gene from Solanum nigrum atrazine resistant biotype and its relevant analysis. I Chuan Hsueh Pao 16, 381-388. Zimmerli L, Jakab G, Metraux J-P, Mauch-Mani B (2000) Potentiation of pathogen-specific defense mechanisms in Arabidopsis by beta- aminobutyric acid. Proceedings of the National Academy of Sciences of the United States of America 97, 12920-12925.
42 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Manuscript 2
Submitted to Molecular Ecology
The effect of competition on transcriptional responses elicited by methyl jasmonate in Solanum nigrum—a glasshouse and field comparison
Dominik D. Schmidt and Ian T. Baldwin*
Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745 Jena, Germany
Key words: Solanum nigrum, competition, defense, microarray analysis, methyl jasmonate, field and glasshouse experiments
Correspondence: Ian T. Baldwin, Department Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans- Knöll-Strasse. 8, D-07745 Jena, Germany, Fax: 03641 571102, E- mail: [email protected]
Running title: Competition- and MeJA-elicited responses of S. nigrum
4651 words; 2 Figures; 3 Tables
43 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Abstract
Herbivory and competition are among the most important selective forces for plants and their interaction is known to decrease fitness more than either stress alone. Treatment with methyl jasmonate (MeJA) elicits herbivore resistance and defense-related traits in Solanum nigrum and provides an easily standardized elicitation. We used oligonucleotide microarrays to study how competition influences the MeJA-elicited transcriptome of S. nigrum plants. In both glasshouse (GH) and field experiments, plants were grown alone, with conspecifics, or with 3 naturally co-occurring interspecific competitors (Lolium perenne, Plantago lanceolata, Trifolium pratense). RNA was extracted from pooled leaf samples of 9 replicate plants per treatment and used to hybridize a 60- gene microarray to analyze the effect of competition on MeJA-elicited responses (each with 3 biological replicates). This analysis revealed that elicited plants grown in the GH and field had largely the same transcriptional response except that field-grown showed less regulation, but the analysis could not distinguish plants grown with and without competitors. Analysis with a 568-gene array clearly identified a transcriptional signature of competition that was distinguished from that elicited by MeJA. While genes involved in photosynthesis and glycolysis were downregulated by both competition and MeJA elicitation (separately and in combination), genes involved in ethylene and gibberellin signaling, which were upregulated by competition, were inhibited when competing plants were elicited by MeJA. Moreover, MeJA and competition had opposing effects on genes involved in nitrate metabolism and cell wall biosynthesis. We conclude that transcriptional analysis of plants grown in natural environments could reveal the basis of the often-invoked trade-off between growth- and defense-related traits and that ethylene and gibberellin signaling are promising targets for future research.
44 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Introduction
Competition for space and the suite of similar, limited resources that plants require is likely the most important biotic stress for plants (Goldberg 1990). After competition, attack from herbivores and pathogens is thought to be the next most important selective factor and competitive outcomes are frequently altered by attack from herbivores and pathogens (Cipollini 2004; Herms & Mattson 1992). The interaction of these selective factors is thought to have resulted in trade-offs between growth- and defense-related traits. Plants must grow sufficiently fast to compete, yet produce the defenses required for survival in environments that contain attackers (Herms & Mattson 1992). A recent meta-analysis of 39 multifactorial field experiments confirmed that competitors and predators often interactively affect fitness (Gurevitch et al. 2000). The synergistic effect of competition and herbivory on plant performance may be driven by resource allocation trade-offs or may result from direct hormonal interactions that mediate growth- and defense-related traits (Cipollini 2004). Three phytohormones are thought to play a central role in the signaling pathways responsible for plant resistance to insect and pathogen attack [salicylic acid, SA, (Delaney et al. 1994); ethylene, ET (O'Donnell et al. 1996; Penninckx et al. 1996); jasmonic acid, JA, (Creelman & Mullet 1997)]. JA and related oxylipins [e.g., methyl-JA (MeJA), 12-oxophytodienoic acid, phytoprostanes, etc.] mediate many insect-induced responses (Kessler & Baldwin 2002; Li et al. 2004; Walling 2000), but through synergism or antagonism with SA and ET, plants are thought to fine-tune their responses to specific herbivores (Reymond & Farmer 1998; Winz & Baldwin 2001). The signaling that mediates competitive interactions has been much less studied and is thought to be mediated by light and ET (Ballaré 1999; Pierik et al. 2003, 2004). Similarly poorly studied are the processes that mediate the interaction between competitive ability and herbivore resistance. MeJA elicitation, which increases herbivore resistance, also decreases competitive ability (Baldwin 1998; Cipollini 2002; Van Dam & Baldwin 2001). This 45 MS2 – Competition- and MeJA-mediated responses of S. nigrum functional tradeoff could result from the large resource requirements of herbivore-resistance traits (Baldwin & Ohnemeiss 1994; Zavala et al. 2004) or other JA-mediated responses. Constitutive and wound-induced proteinase inhibitor (PI) production in Brassica napus was reduced by competition in a density-dependent manner, but could be restored in plants growing under high nutrient conditions (Cipollini & Bergelson 2001), which is consistent with the expectations of a resource-based tradeoff. However, the molecular mechanisms of this tradeoff remain obscure (Cipollini 2004) and we are aware of no studies that have examined the transcriptional responses to competition and their interaction with elicited defense responses. Due to the multiple roles of signals in plant responses, it has become abundantly clear that responses to stresses are mediated by complex signaling networks rather than by linear signal cascades (Genoud et al. 2001). Gene-expression profiling with microarrays has been profoundly useful in unraveling such networks and has demonstrated the interrelatedness of JA, SA, and ET signaling in response to biotic stresses (Glazebrook 2001; Reymond et al. 2000; Schenk et al. 2000). However, these microarray studies have been largely conducted on plants grown in laboratories or glasshouses. Given that plants grown in the field are subject to a great diversity of biotic and abiotic elicitors, it is unclear whether the transcriptional imprints of different biotic stresses will be discernible in the rough and tumble of nature. For example, oxylipin signaling, which is clearly involved in herbivore resistance is also elicited by abiotic stresses such as UV-B radiation (Conconi et al. 1996; Izaguirre et al. 2003), suggesting that MeJA-elicited transcriptional responses of field-grown plants may be confounded. To date, microarrays have not been used to analyze the transcriptional responses to competition or the interaction between competition and elicited defense responses against herbivore attack. Here we use microarrays to determine if transcriptional responses elicited by MeJA that are discernible in glasshouse (GH)-grown plants are also detectable in field-grown Solanum nigrum plants and whether
46 MS2 – Competition- and MeJA-mediated responses of S. nigrum these responses are altered when plants compete with a selection of naturally occurring competitors. We chose to work with S. nigrum (Schmidt et al. 2004) because as a native plant its responses to herbivore attack and competition have not been confounded by artificial selection. To standardize the elicitation of defense and competitive responses across the different environments, we elicited plants with MeJA applications and grew plants in GH and field mesocosms, with conspecifics, or with 3 naturally co-occurring interspecific competitors (Lolium perenne, Plantago lanceolata, Trifolium pratense).
Materials and methods
Plant growth and treatments Solanum nigrum L. (inbred line Sn30) seeds were incubated overnight at 4°C in 3.5mM Ca(NO3)2 and germinated in a peat-based substrate augmented with clay (Tonsubstrat, Klasmann). Plants for field and GH experiments were grown individually in multi-pot flats (Teku) for two weeks in the GH (26-28 °C / 16 h light; 22-24 °C / 8 h dark) before being planted together in the mesocosms. Plantago lanceolata (field- collected seeds from agricultural and roadside meadows in Jena), Lolium perenne, and Trifolium pratense (both purchased from Greenfield, Krefeld, Germany) were grown under the same conditions as S. nigrum for six weeks. Nine replicate mesocosms were established for each MeJA and competition treatment. When the mesocosms were established, all plants (S. nigrum and the competitors) were of approximately the same size (15-20 cm). For the GH experiments, individual S. nigrum plants were transplanted into 5 L pots alone or in combination with three competitors, either of the same species (S. nigrum or P. lanceolata) or one of each of three species (P. lanceolata, L. perenne, T. pratense; Table 1). For the field experiment, we used a plot with calcareous soil at the Max Planck Institute for Biogeochemistry (Jena, Germany). We used a randomized block design with a subunit size of 1 m2 for each field mesocosm and planted the target plant, S. nigrum, in the middle of three competitors on
47 MS2 – Competition- and MeJA-mediated responses of S. nigrum
June 18, 2003; the distance between focal plant and each competitor was approximately 15 cm. Day length (average for June 2003 16 h light; http://www.calsky.com) and temperature conditions (average temperature for June 2003 20.1°C, minimum 9.6°C, maximum 33.8°C; http://wetter.mb.fh-jena.de/station) were comparable to the GH conditions (see above). Mesocosms were weeded once a week. The field mesocosms were sprayed with the pyrethroid-based insecticide Spruzit (0.1 %, Neudorff) every third day until the start of the experiment to prevent herbivore damage. We elicited both the field and the GH experiment two weeks after planting to ensure interaction between the focal plants and the competitors. To elicit the focal S. nigrum plant, we applied 250 µg of MeJA (Sigma) in 20µL lanolin (Sigma) to the stem (n=9 for each competition treatment) above the third leaf node. To control for possible lanolin effects, we treated control plants (n=9 for each competition treatment) with 20µL pure lanolin. Two fully expanded stem leaves above the fourth leaf node were harvested 24 h after treatment, frozen in liquid nitrogen and stored at –80°C until RNA extraction. Microarray hybridization and analysis Each microarray was hybridized from RNA extracted from a pooled leaf sample consisting of two leaves from all 9 replicate mesocosms for each competition and MeJA treatment (Table 1). To maximize the information obtained from the experiment and provide biological replicates for the competition treatment, we analyzed the three different competition regimes (intraspecific competition, competition with Plantago lanceolata and competition with Lolium perenne, P. lanceolata and Trifolium pratense) as three biological replicates for the competition treatment (Table 1). At the time of the MeJA elicitation and sample harvest, all plants were in an early stage of competition and height and canopy structure were similar across all mesocosms. To facilitate comparisons across microarrays used in both field and GH experiments, we chose an hybridization design, in which all treatment samples were hybridized against the same control sample (Table 1).
48 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Non-elicited S. nigrum plants, grown without competitors either in the field or the GH, served as the control sample for the respective field and GH experiments. We extracted total RNA using TRI REAGENTTM (Sigma) according to the manufacturer’s instructions. The test samples were labeled with Cy3 and the corresponding control samples with Cy5 according to the procedure described in Halitschke et al. (2003). We used two different microarrays for the hybridizations, a 120-oligonucleotide microarray (60 genes) for all comparisons in GH mesocosms, and a 789- oligonucleotide microarray (568 genes), which included the 120 oligonucleotides of the smaller microarray for the field mesocosms. On each microarray, each oligonucleotide was spotted four times on the epoxy-coated glass slide (Quantifoil Microtools). The hybridized microarrays were scanned with an Affymetrix 428™ Array Scanner (Affymetrix) and the pictures analyzed as described in (Schmidt et al. 2004). We used the Genespring software (Silicon Genetics) to calculate expression ratios (ERs) by dividing the signal of the test sample by the signal of the control sample. Each microarray was normalized to the 50th percentile; raw signals below 500 were excluded. We defined a transcript as being regulated, if it met the following criteria: (1) The ER (mean of three biological replicates; the mean of each microarray was calculated from the ER of four oligonucleotides) differed significantly from 1 as determined by a Student’s t-test. (2) The ER exceeded the threshold of 0.5 for down regulation or 2.0 for upregulation. Annotated lists of all transcripts are available from the authors. To verify the expression data obtained from the microarrays, RNA gel blot analysis and real-time quantitative PCR (qPCR) were conducted to determine the relative expression of PIN2b. Procedures were used as described in Schmidt et al. (2004) with the only modification that the S. nigrum elongation factor 1a gene (SnEF1a, AY574951) was chosen as endogenous control in the real-time qPCR experiment using the comparative 2-ΔΔCt method (Livak & Schmittgen 2001).
49 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Table 1 List of hybridized microarrays and the corresponding experiment (Exp.). Competition treatments were treated as biological replicates; there was no morphological evidence of species-dependent differences in competition at the time of the MeJA elicitation. To facilitate comparisons among treatments within glasshouse (GH) and field environments, all GH and field arrays were hybridized against the same sample taken from lanolin-treated plants grown without competitors in GH or field, respectively.
Micro- Exp. Biological Treatment sample Control sample array # replicate (Cy3) (Cy5) 1 GH 1 MeJA; competition1 2 GH 2 MeJA; competition2 Lanolin-treated 3 GH 3 MeJA; competition3 plants without 4 GH 1 Competition1 competition 5 GH 2 Competition2 6 GH 3 Competition3 7 Field 1 MeJA; competition1 8 Field 2 MeJA; competition2 Lanolin-treated 9 Field 3 MeJA; competition3 plants without 10 Field 1 Competition1 competition 11 Field 2 Competition2 12 Field 3 Competition3 1Intraspecific competition 2Competition with Plantago lanceolata 3Competition with Lolium perenne, P. lanceolata and Trifolium pratense
50 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Results
MeJA-elicited transcriptional response is influenced by the experimental environment We compared the transcriptional response of competing S. nigrum to MeJA in the GH and the field. Analysis of expression of 60 genes revealed large overlap in regulated genes (Fig. 1A, Table 2). MeJA elicitation of competing plants increased the expression of genes involved in the biosynthesis of oxylipins (lipoxygenase C, allenoxide synthase).
Fig. 1 Venn diagrams of the numbers of overlapping and non- overlapping significantly upregulated (↑; ER>2.0) or downregulated (↓; ER<0.5) genes based on analysis of 60 genes (A-C) and 568 genes (D). Genes whose expression is significantly regulated are listed in Tables 1 and 2. (A) Comparison of MeJA- plus competition-regulated genes in the GH and field experiment. (B) Comparison of MeJA- plus competition- regulated genes and competition-regulated genes in GH mesocosms. (C/D) MeJA- plus competition-regulated genes and competition- regulated genes in field mesocosms comparing ERs of 60 (C) or 568 (D) genes. 51 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Table 2 ERs (mean of three biological replicates) of regulated genes based on the 60-gene microarray. In competing plants that were elicited with MeJA (COM + MJ) more genes are differentially regulated than in competing control plants (COM), both in the GH and the field experiment. ERs of significantly upregulated genes (t-test p<0.05, ER>2.0) are shaded in dark grey; ERs of significantly downregulated genes (t-test p<0.05, ER<0.5) are shaded in light grey.
GH field COM COM COM COM Gene Function GenBank # + MJ + MJ DEFENSE/STRESS RESPONSE Tomato allenoxide AF230371 3.68 1.04 5.67 1.39 AOS synthase Tobacco luminal X60057 2.52 2.40 1.13 1.13 BiP binding protein CAT1 Tomato catalase M93719 1.19 1.94 2.08 1.70 LOX C Tomato lipoxygenase U37839 1.82 1.25 2.08 1.07 pin2 Tomato PIN2 K03291 4.97 1.12 6.48 1.17 Potato polyphenol A27686 0.44 0.59 0.83 0.93 PPO oxidase SaPIN2a S. americanum PIN2a AF209709 2.19 0.83 1.90 1.02 SaPIN2b S. americanum PIN2b AF174381 4.61 1.31 5.33 1.22 PHOTOSYNTHESIS S. nigrum PSII D1 U25659 0.26 0.47 0.37 0.49 psbA protein Tomato rubisco L14403 0.31 0.51 0.56 0.70 rbcL large subunit L. pennelii rubisco AF037361 0.37 0.61 0.41 0.42 rca activase OTHER FPS1 Tomato FPP synthase AF048747 8.11 2.03 2.81 1.29 RALF Tobacco RALF AF407278 0.35 0.65 0.72 0.75 Mint (E)-b-farnesene 0.42 0.72 0.59 0.74 Tspa11 synthase AF024615 Tomato xyloglucan 0.25 0.50 0.63 0.75 XTH4 ET/H AF186777
52 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Genes known to be downstream of the oxylipin signaling cascade were concomitantly upregulated, such as genes involved in the biosynthesis of defense proteins (PIs) and a farnesyl pyrophosphate synthase gene that is important in the biosynthesis of terpenoids. Photosynthesis-related genes were generally downregulated in GH and field mesocosms (Table 2). However, only in GH-grown plants, transcripts of two defense/stress-related genes, namely PPO (polyphenyl oxidase) and RALF (rapid alkalinization factor), and a gene involved cell wall metabolism (XTH, xyloglucan endotransglucosylase/hydrolase) were downregulated. Together with an upregulation of catalase in field- grown plants, these results demonstrate that field-grown plants had more stress-responsive genes upregulated than GH-grown plants probably due to the greater intensity and diversity of environmental elicitation experienced by plants grown in the field. MeJA influences the competition-induced signaling in S. nigrum With the 60-gene array we could detect effects of competition only in GH-grown plants (Fig. 1B). However, those changes were not different from competing plants that were elicited with MeJA (Table 2) and were not detected in field-grown plants (Fig. 2C). To increase the resolution for the detection of competition-specific transcriptional changes, we used a 568-gene array and found 15 genes that responded only to competition, 34 genes that responded only to the combination of competition and MeJA elicitation, and 5 genes that were commonly regulated in both treatments (Fig. 1D, Table 3). Among the MeJA- induced genes were additional TPI genes, the key enzyme of the mevalonate pathway, 3-hydroxymethyl-3-glutaryl coenzyme A reductase (HMGR), and 5 genes of unknown function (Table 3). Both MeJA- elicited competing plants and unelicited competing plants downregulated photosynthesis-related genes coding for different subunits of RUBISCO, photosystem proteins, and glycolysis-related genes (Table 3). The most striking changes elicited in competing plants were related to signaling processes. Competition specifically elicited the
53 MS2 – Competition- and MeJA-mediated responses of S. nigrum expression of genes involved in ethylene biosynthesis (1- aminocyclopropane-1-carboxylic acid oxidase, ACO) and ethylene perception (ETR2). In MeJA-elicited competing plants, these genes were either not regulated (ACO) or downregulated (ETR2). Similarly a DELLA transcription factor that mediates gibberellin signaling [RGL2; (Gomi & Matsuoka 2003)] was only upregulated in competing plants. A gene that is part of molybdenum cofactor biosynthesis (molybdopterin synthase sulfurylase; (Campbell 1999) was upregulated in competing plants without MeJA elicitation. The molybdenum cofactor is essential for of the activity of nitrate reductase (nia) and transcripts of nia were significantly upregulated in competing plants, but the ER was below the threshold for significant expression. A suite of genes, all of which are thought to be involved in stress responses [a lipid transfer protein, a germin-like protein and a wound- induced protein kinase (Bernier & Berna 2001; Denecke et al. 1991; Yang et al. 2001)] were found to be upregulated only in S. nigrum plant that were competing with P. lanceolata. However as replicate hybridizations were not performed, these responses need to be verified.
Table 3 ERs (mean of three biological replicates) of regulated genes based on the 568-gene microarray. See Table 2 for details of abbreviations and of the shading.
COM COM Gene Function GenBank # + MJ OXILIPIN SIGNALING NaLOX2 N. attenuata lipoxygenase 2 AY254348 5.05 1.05 LOX C Tomato lipoxygenase C U37839 2.62 1.04 NaAOS N. attenuata allene oxide AJ295274 7.30 1.93 out synthase AOS Tomato allenoxide synthase AF230371 2.70 1.13 LeDES Tomato divinyl ether synthase AF317515 0.92 7.79 HORMONES / SIGNALING NaACO1 Tobacco ACC oxidase AY426756 1.42 2.98 out LeETR2 Tomato ETR2 AF043085 0.13 12.82
54 MS2 – Competition- and MeJA-mediated responses of S. nigrum
COM COM Gene Function GenBank # + MJ RGL2C A. thaliana RGL2C AC009895 0.50 6.24 DEFENSE PI inas N. attenuata proteinase inhibitor AF542547 8.88 1.35 PI-WuSP N. attenuata proteinase inhibitor AY426751 8.95 1.22 pin2 tomato PIN2 K03291 9.70 1.18 pin2 tomato PIN2 K03291 5.36 1.08 SaPIN2a S. americanum PIN2a AF209709 5.91 0.71 SaPIN2b S. americanum PIN2b AF174381 6.18 1.15 SaPIN2b S. americanum PIN2b AF174381 5.12 1.19 DH60 Tomato Prg1 CA591878 5.21 1.82 pin1 Tomato PIN1 K03290 3.54 1.44 TERPENOID BIOSYNTHESIS HMGR2 N. attenuata HMGR2 AF542543 7.67 1.36 FPS1a Tomato FPP synthase AF048747 3.02 1.11 FPS1b Tomato FPP synthase AF048747 3.02 1.28 DH120 N. sylvestris HMGR CA591813 4.35 1.69 STRESS RESPONSE germin Tomato germin 484262 AI484262 0.39 0.56 CAT1a Tomato catalase M93719 2.23 1.55 CAT1b Tomato catalase M93719 2.23 1.66 PHOTOSYNTHESIS DH108 Tobacco photosystem II CA591793 0.36 0.44 protein pDH56.1 Tobacco RUBISCO CA591705 2.49 0.24 pDH61.1 Tomato lhbC1 AW191805 0.38 0.45 pDH64.7 N. sylvestris RUBISCO ssu AW191829 0.19 0.23 rca inas N. attenuata RUBISCO activase BU494545 0.39 0.33 rca out N. attenuata RUBISCO activase BU494545 0.39 0.34 Tobacco RUBISCO RF071 CA591784 0.19 0.19 pseudogene RUB inas N. attenuata RUBISCO AW191829 0.21 0.19 rbcL Tobacco RUBISCO lsu NC_001879 0.55 0.50 psbA S. nigrum PS II D1 protein U25659 0.37 0.46
55 MS2 – Competition- and MeJA-mediated responses of S. nigrum
COM COM Gene Function GenBank # + MJ rca_a L. pennelii RUBISCO activase AF037361 0.48 0.45 rca_b L. pennelii RUBISCO activase AF037361 0.40 0.38 DH149 S. oleracea photosystem I psaL AF542546 0.47 0.64 S.oleracea photosystem II RN032 CA591773 2.41 0.01 polypeptide GLYCOLYSIS frk(1) Tomato fructokinase (Frk1) U64817 0.24 0.39 pGap Tobacco GAPDH M14418 0.58 0.48 pPGK Tobacco phosphoglycerate Z48977 0.48 0.49 kinase OTHER Tobacco molybdopterin pDH55.7 AW191825 1.45 2.31 synthase sulfurylase Na-XTH1 N. attenuata xyloglucan AY422687 0.49 0.81 out endotransglycosylase Tobacco clp16S-23S rRNA cv81.4 BU494529 0.23 0.29 IGS UNKNOWN cv11.4 unknown BU494517 0.74 2.98 cv74.2 unknown BU494526 3.08 0.48 cvs17 unknown BU494562 2.76 5.21 cvs20 unknown BU494564 1.00 4.77 DH135 unknown CA591797 0.73 0.32 DH30 unknown CA591848 5.38 1.11 DH38 unknown CA591856 1.46 3.40 DH71 unknown CA591889 0.58 4.59 pDH25.1 unknown CA591690 0.41 3.69 pDH5.2 unknown CA591687 2.99 1.15 RB304 unknown CA591720 3.40 0.84
56 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Verification of microarray data We conducted real-time qPCR and RNA gel blot analysis for the PIN2b gene to verify the results of the microarrays (Fig. 2). Analysis of RNA used in the hybridizations of 15 arrays, revealed that the three different methods delivered the same result. Remarkably, even the pattern of quantitative differences in PIN2b expression was verified by
Fig. 2 Comparison of PIN2b expression in glasshouse- or field-grown S. nigrum, which were subjected to MeJA-elicitation and competition or competition only. The bars depict the relative PIN2b expression for the corresponding microarrays (see Table 1) based on real-time qPCR; -ΔΔ the expression was calculated with the comparative 2 Ct method using SnEF1a as an external standard and the control treatment (Table 1) as calibrator; each bar represents the mean of three replicate measurements (± SE). The stars indicate the ER (mean of four spots) obtained from the microarray hybridizations. For samples used to hybridize microarrays 1-6, a RNA gel blot is displayed with 18S-rRNA as a loading control.
57 MS2 – Competition- and MeJA-mediated responses of S. nigrum the real-time qPCR analysis, but the microarray data tended to underestimate the expression values determined by real-time qPCR (correlation of the real-time qPCR and microarray expression data: R2=0.6899).
Discussion
We examined MeJA-elicited transcriptional responses of S. nigrum plants grown with competitors in the GH or the field to examine two questions that are germane to plant responses to multiple biotic stresses in general and to the utility of transcriptional analysis for field-based ecological analysis: 1) Does S. nigrum respond to MeJA elicitation similarly in GH and natural environments? And if so, 2) does competition have an effect on MeJA-elicited transcriptional responses? The experimental design which included three biological replicates for each treatment allowed for the analysis of transcriptional effects common to intra- and interspecific competition regimes. Moreover, the analysis of transcriptional responses to particular competitors, such as P. lanceolata, provided hints for unique competitive interactions but as these were not replicated, they require further verification. In general, we found the relative expression patterns observed on the microarrays to be reproducible by other transcript quantification procedures; when the same samples were analyzed by RNA gel blots and real-time qPCR for PIN2b transcripts, the patterns observed on the microarrays were verified (Fig. 2). MeJA treatment elicited the upregulation of defense-related genes concomitantly with a downregulation of photosynthesis-related genes, a pattern consistent with a herbivore-induced switch from growth- to defense-related gene expression (Hermsmeier et al. 2001; Herms & Mattson 1992). The comparison of MeJA-elicited transcriptional responses in GH- and field-grown plants revealed that GH-grown plants exhibited responses that were not observed in field-grown plants most likely because particular genes were constitutively expressed in field- grown plants. For example, a touch-inducible gene that encodes a 58 MS2 – Competition- and MeJA-mediated responses of S. nigrum xyloglucan endotransglucosylase/hydrolase (XTH) that is involved in cell wall remodeling (Rose et al. 2002) was downregulated in GH-grown plants. Field-grown plants are more exposed to more mechanical stresses from wind or rain and therefore are unlikely to downregulate XTHs in response to MeJA. The upregulation of catalase, the suppression of both a wound-inducible polyphenyl oxidase gene and a gene putatively involved in stress-signaling (RALF) in GH-grown plants, but not in field- grown plants, are additional examples. However this analysis of 60-genes did not provide sufficient resolution to distinguish responses mediated by competition from those elicited by MeJA. To increase the resolution of the analysis, we analyzed the interaction with an array containing 568 genes, and discovered that S. nigrum consistently downregulated photosynthesis- and glycolysis-related genes in response to the combination of MeJA and competition and in response to competition alone. However, competing plants upregulated genes related to ethylene- and GA-signaling only when they were not elicited by MeJA, suggesting that the responses mediated by these hormones could account for changes in growth and defense processes in competing plants. ET is known to have multiple and diverse effects in all aspects of plant life (Wang et al. 2002). For herbivore resistance traits, ET is known to positively and negatively interact with oxylipin-mediated responses. For example, JA and ET synergistically activate defense- related genes (O'Donnell et al. 1996; Penninckx et al. 1996) and many genes induced by ET are also elicited by MeJA in A. thaliana (Schenk et al. 2000). ET and light signaling are important regulators in competitive interactions. ET-insensitive tobacco plants, which are unresponsive to blue light signals from a competitor’s canopy, perform poorly when competing against wildtype neighbors (Pierik et al. 2003, 2004). Plants use these blue light signals in addition to the alterations in red:far-red ratios (R:FR) to adjust their growth responses and increase competitive ability (Ballaré 1999). Biosynthesis and catabolism of GA, which profoundly alters stem growth and competitive ability, is also influenced by light signals (García-Martinez & Gil 2002). The molecular mechanisms by which a plant responds to external signals that signify
59 MS2 – Competition- and MeJA-mediated responses of S. nigrum competitive environments, such as light and possibly ET, to trigger endogenous hormone cascades that finally alter the phenotype in an adaptive manner remain to be determined. However, it is clear from this analysis that microarrays can fruitfully identify signals that are activated across a range of different competitive regimes. The analysis not only identified hormones that appear to mediate significant transcriptional responses, common to all competition regimes, but it also pointed to responses that are likely to be specific to growth with particular competitors. The most strikingly different response was observed when S. nigrum competed with P. lanceolata. Under this competitive regime, an additional suite of genes was elicited, which are thought to be involved in stress responses. It is therefore possible that S. nigrum is responding to particular signals from P. lanceolata. P. lanceolata emits a rich bouquet of volatile organic compounds (Fons et al. 1998) which includes eugenol that is thought to be responsible for the allelopathic characteristics of Sassafras albidum (Gant & Clebsch 1975). With the recent discovery that certain regulatory promoter elements respond to plant secondary metabolites [e.g. the diterpenoid sclareol [Grec et al. 2003)], it is possible that compounds emitted by P. lanceolata activate or suppress specific genes in S. nigrum. Hence it is likely that species-specific signals modify the more general competition signals to tailor plant responses to particular competitive environments. The transcriptional analysis provided here suggests that oxylipin, ET and GA signaling interact to mediate the defense-competition tradeoff in a variety of different competitive regimes. It is remarkable that such complex signal interactions could be observed in plants growing in complex natural environment. Ecologists frequently manipulate complicated biotic interactions in the field, but rarely consider using the established array technology to characterize the transcriptional responses to these interactions. Microarray platforms that have been publicly funded (e.g. http://www.tigr.org/tdb/potato /microarray2.shtml) offer ecologists a means of rapidly identifying molecular targets that could be manipulated in complex interactions. As has been illustrated by the recent advances in understanding the role of
60 MS2 – Competition- and MeJA-mediated responses of S. nigrum
ET (Pierik et al. 2003, 2004) and R:FR signaling (Ballaré 1999) in competitive interactions, rapid progress in establishing the relevance of a given signal can be made when a plant’s ability to perceive a signal is manipulated genetically. Transcriptional analysis provides a list of candidate genes that could be targeted for genetic manipulation.
Acknowledgments
We thank Susanne Kutschbach, Wibke Kröber and Thomas Hahn for assistance in microarray processing and data acquisition; Danny Kessler and Celia Diezel for invaluable support with the fieldwork; the Max-Planck-Gesellschaft and the Deutsche Forschungsgemeinschaft (FOR. 456; BA2138/1-1) for financial support.
References Baldwin IT (1998) Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences of the United States of America 95, 8113-8118. Baldwin IT, Ohnmeiss TE (1994) Coordination of photosynthetic and alkaloidal responses to damage in uninducible and inducible Nicotiana sylvestris. Ecology 75, 1003-1014. Ballaré CL (1999) Keeping up with the neighbours: phytochrome sensing and other signaling mechanisms. Trends in Plant Science 4, 97-102. Bernier F, Berna A (2001) Germins and germin-like proteins: Plant do- all proteins. But what do they do exactly? Plant Physiology and Biochemistry 39, 545-554. Campbell WH (1999) Nitrate reductase structure, function and regulation: Bridging the gap between biochemistry and physiology. Annual Review of Plant Physiology and Plant Molecular Biology 50, 277-303. Cipollini DF (2004) Stretching the limits of plasticity: Can a plant defend against both competitors and herbivores. Ecology 85, 28-37. Cipollini DF, Bergelson J (2001) Plant density and nutrient availability constrain constitutive and wound-induced expression of trypsin inhibitors in Brassica napus. Journal of Chemical Ecology 27, 593-610.
61 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Cipollini DF (2002) Does competition magnify the fitness costs of induced responses in Arabidopsis thaliana? A manipulative approach. Oecologia 131, 514-520. Conconi A, Smerdon MJ, Howe GA, Ryan CA (1996) The octadecanoid signaling pathway in plants mediates a response to ultraviolet radiation. Nature 383, 826-829. Creelman RA, Mullet JE (1997) Biosynthesis and action of jasmonates in plants. Annual Review of Plant Physiology and Plant Molecular Biology 48, 355-381. Delaney TP, Uknes S, Vernooij B, et al. (1994) A central role of salicylic acid in plant disease resistance. Science 266, 1247-1250. Denecke J, Goldman MHS, Demolder J, Seurinck J, Botterman J (1991) The tobacco luminal binding protein is encoded by a multigene family. Plant Cell 3, 1025-1035. Fons F, Rapior S, Gargadennec A, Andary C, Bessiere JM (1998) Volatile components of Plantago lanceolata (Plantaginaceae). Acta Botanica Gallica 145, 265-269. Gant RE, Clebsch EEC (1975) Allelopathic influences of Sassafras albidum in old-field succession in Tennessee. Ecology 56, 604-615. Garcia-Martinez JL, Gil J (2001) Light regulation of gibberellin biosynthesis and mode of action. Journal of Plant Growth Regulation 20, 354-368. Genoud T, Santa Cruz MBT, Metraux JP (2001) Numeric simulation of plant signaling networks. Plant Physiology 126, 1430-1437. Glazebrook J (2001) Genes controlling expression of defense responses in Arabidopsis - 2001 status. Current Opinion in Plant Biology 4, 301- 308. Goldberg DE (1990) Components of resource competition in plant communities. In: Perspectives on plant competition (eds. Grace JB, Tilman D), pp. 27-49. Academic Press, San Diego. Gomi K, Matsuoka M (2003) Gibberellin signaling pathway. Current Opinion in Plant Biology 6, 489-493. Grec S, Vanham D, de Ribaucourt JC, Purnelle B, Boutry M (2003) Identification of regulatory sequence elements within the transcription promoter region of NpABC1, a gene encoding a plant ABC transporter induced by diterpenes. Plant Journal 35, 237-250. Gurevitch J, Morrison JA, Hedges LV (2000) The interaction between competition and predation: A meta- analysis of field experiments. American Naturalist 155, 435-453. Halitschke R, Gase K, Hui D, Schmidt DD, Baldwin IT (2003) Molecular interactions between the specialist herbivore Manduca 62 MS2 – Competition- and MeJA-mediated responses of S. nigrum
sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VI. Microarray analysis reveals that most herbivore- specific transcriptional changes are mediated by fatty acid-amino acid conjugates. Plant Physiology. 131, 1894-1902. Herms DA, Mattson WJ (1992) The Dilemma of Plants - to Grow or Defend. Quarterly Review of Biology 67, 283-335. Hermsmeier D, Schittko U, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. I. Large-scale changes in the accumulation of growth- and defense-related plant mRNAs. Plant Physiology 125, 683-700. Izaguirre MM, Scopel AL, Baldwin IT, Ballaré CL (2003) Convergent responses to stress. Solar Ultraviolet-B radiation and Manduca sexta herbivory elicit overlapping transcriptional responses in field-grown plants of Nicotiana longiflora. Plant Physiology 132, 1755- 1767. Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: The emerging molecular analysis. Annual Review of Plant Biology 53, 299-328. Li L, Zhao YF, McCaig BC, et al. (2004) The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development. Plant Cell 16, 126-143. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCt method. Methods 25, 402-408. O'Donnell PJ, Calvert C, Atzorn R, et al. (1996) Ethylene as a signal mediating the wound response of tomato plants. Science 274, 1914-1917. Penninckx I, Eggermont K, Terras FRG, et al. (1996) Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 8, 2309-2323. Pierik R, Visser EJW, De Kroon H, Voesenek L (2003) Ethylene is required in tobacco to successfully compete with proximate neighbours. Plant Cell and Environment 26, 1229-1234. Pierik R, Whitelam GC, Voesenek L, de Kroon H, Visser EJW (2004) Canopy studies on ethylene-insensitive tobacco identify ethylene as a novel element in blue light and plant-plant signalling. Plant Journal 38, 310-319.
63 MS2 – Competition- and MeJA-mediated responses of S. nigrum
Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene expression. Current Opinion in Plant Biology 1, 404- 411. Reymond P, Weber H, Damond M, Farmer EE (2000) Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 12, 707-719. Rose JKC, Braam J, Fry SC, Nishitani K (2002) The XTH family of enzymes involved in xyloglucan endotransglucosylation and endohydrolysis: Current perspectives and a new unifying nomenclature. Plant and Cell Physiology 43, 1421-1435. Schenk PM, Kazan K, Wilson I, et al. (2000) Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proceedings of the National Academy of Sciences of the United States of America 97, 11655-11660. Schmidt DD, Kessler A, Kessler D, et al. (2004) Solanum nigrum: A model ecological expression system. Molecular Ecology 13, 981-995. Van Dam NM, Baldwin IT (2001) Competition mediates costs of jasmonate-induced defenses, nitrogen acquisition and transgenerational plasticity in Nicotiana attenuata. Functional Ecology 15, 406-415. Walling LL (2000) The myriad plant responses to herbivores. Journal of Plant Growth Regulation 19, 195-216. Wang KLC, Li H, Ecker JR (2002) Ethylene biosynthesis and signaling networks. Plant Cell 14, S131-S151. Winz RA, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-induced ethylene reduces jasmonate- induced nicotine accumulation by regulating putrescine N- methyltransferase transcripts. Plant Physiology 125, 2189-2202. Yang KY, Liu YD, Zhang SQ (2001) Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco. Proceedings of the National Academy of Sciences of the United States of America 98, 741-746. Zavala JA, Patankar AG, Gase K, Baldwin IT (2004) Constitutive and inducible protease inhibitor production incurs large fitness costs in Nicotiana attenuata. Proceedings of the National Academy of Sciences of the United States of America 101, 1607-1612.
64 MS3 – Biodiversity and defense responses of S. nigrum
Manuscript 3
Submitted to Ecology Letters
The canopy height of competitors explains the influence of biodiversity on jasmonate- induced transcriptional responses in field- grown Solanum nigrum
Dominik D. Schmidt and Ian T. Baldwin*
Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745 Jena, Germany
Key words: interspecific competition, above-ground interaction, plant- herbivore interaction, plant fitness, microarray analysis
*Correspondence: Ian T. Baldwin, Department Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse. 8, D-07745 Jena, Germany, Fax: 03641 571102, E-mail: [email protected]
Running title: Biodiversity and transcriptional defense responses
5208 words; 2 figures; 1 table; 1 supplemental table
65 MS3 – Biodiversity and defense responses of S. nigrum
Abstract
How best to integrate the hundreds of individual species-level interactions is a central challenge to all studies that examine the influence of biodiversity on ecosystem function. Induced resistance of plants is known to influence material and energy flow among trophic levels, but how biodiversity affects its expression is unknown. To study the influence of plant biodiversity on the induced defenses of Solanum nigrum, we transplanted plants of an isogenic line into 12 plots of the Jena Biodiversity Experiment (Roscher et al. 2004) in which 60 grassland species were factorially manipulated. After 8 days of growth, plants were elicited with methyl jasmonate (MeJA) and their transcriptome was compared with a 60-gene microarray enriched in genes relevant in plant-herbivore interactions to the transcriptome of unelicited plants growing without competitors. Across all competitive environments, defense-related genes were up-regulated in response to MeJA elicitation. Principal component analysis (PCA) of the expression ratios of all microarrays revealed that MeJA-up-regulated genes accounted for most of the variation in the dataset and no pattern could be attributed to the number of competing species. However, the PCA identified a pattern in the MeJA-induced transcriptome when the microarrays were classified by the canopy height classes of the competing plant species. Photosynthesis- and defense-related genes were differentially regulated in plants grown with short canopy competitors as compared to those grown with medium or tall canopy competitors. The concentration of defense proteins trypsin proteinase inhibitors (TPIs) were correlated with the expression of TPI transcripts and increased significantly in response to MeJA elicitation; however, the increase in TPI levels was attenuated in plants grown with short or tall competitors. The attenuation of this nitrogen- intensive defense was associated with increases in the expression of genes involved in the biosynthesis of C-based defense metabolites (sesquiterpenes). We conclude that the morphological characteristics of interspecific competitors have a stronger effect on S. nigrum’s transcriptional and TPI responses to MeJA elicitation than the number of competing species,
66 MS3 – Biodiversity and defense responses of S. nigrum suggesting that functional categorizations are useful in characterizing the influence of biodiversity on plant-herbivore interactions.
67 MS3 – Biodiversity and defense responses of S. nigrum
Introduction
Understanding the ecological consequences of biodiversity loss has become a central mission of ecology (Chapin et al. 2000) and major advances have been made in describing the relationship between plant species diversity and ecosystem processes (Schulze and Mooney 1993; Loreau et al. 2002). Most studies that have experimentally altered diversity have focused on the effects of plant taxonomic diversity or plant functional- group diversity on primary production in grassland ecosystems (Loreau et al. 2001). Several experiments using randomly assembled communities have found that primary production is positively correlated with plant diversity, yet the processes responsible for this correlation remain largely unknown (Loreau et al. 2002). The interaction between plants and their herbivores is an important determinant of primary productivity (Strong et al. 1984), and this interaction is known to be strongly influenced by the ability of plants to launch defense responses after herbivore attack. The molecular basis of induced resistance is being rapidly elucidated (Kessler and Baldwin 2002) and it is clear that these conditional responses are strongly influenced by the plant’s physiological condition (Baldwin 1998; Cipollini 2004; Lou and Baldwin 2004; Schmelz et al. 2003). The jasmonic acid (JA) signaling cascade mediates many of the induced responses found in plants, for example when plants are attacked by herbivores or pathogens (Farmer et al. 2002; Turner et al. 2003). It is known that exogenously applied methyl JA activates the JA signaling cascade and can be easily employed as a standardized elicitation to induce defense responses in plants without actually putting herbivores on the plants (Baldwin 1998; Thaler et al. 1996). Hence, the MeJA-elicited changes in the transcriptome (‘defensome’) offer a fast response variable to monitor environmental effects on plant traits and, hence, transcriptional patterns can be used to monitor higher-level interactions. To address the role of plant biodiversity for trophic interactions and element cycling, the Jena Biodiversity Experiment was set up in 2002 based on a pool of 60 plant species typical of Central European grasslands (Roscher et al. 2004). The species combinations are factorially manipulated
68 MS3 – Biodiversity and defense responses of S. nigrum with regard to species richness and functional diversity. The species were assigned to functional groups based on morphological, phenological and physiological traits (Roscher et al. 2004). Below-ground traits, such as root architecture or the ability of nitrogen fixation, play an important role in mineral nutrient acquisition of a plant and influence the productivity of a plant community (Mulder et al. 2002; Rajaniemi et al. 2003). Above-ground traits define the architecture of the photosynthesizing plant organs and hence canopy space occupancy of each species. As a consequence, manipulation of the functional diversity results in communities with distinct morphological characteristics. We have developed a suite of molecular tools, such as customized microarrays, for the native plant S. nigrum to study the genes that mediate complex environmental responses. The particular natural history of S. nigrum makes it ideal for studying the interaction of competition and herbivore resistance. It deploys various direct [e.g. trypsin proteinase inhibitors (TPIs)] and indirect defenses against a wide range of herbivores from different feeding guilds (Schmidt et al. 2004 ). As an annual, S. nigrum colonizes nitrogen-rich disturbed habitats throughout its panarctic distribution (Edmonds and Chweya 1997) and grows in association with many other plant species. In the Jena experimental arena of diversity we examine the influence of changing diversity on the MeJA-elicited defensome of S. nigrum.
69 MS3 – Biodiversity and defense responses of S. nigrum
Materials and methods
Field plots The experimental site is located on the floodplain of the Saale river at the northern edge of Jena (Thuringia, Germany) and was established by joint efforts of the groups involved in the project “The role of biodiversity for element cycling and trophic interaction” (funded by Deutsche Forschungsgemeinschaft). A total pool of 60 grassland species was used and the exact design of the plots is described in (Roscher et al. 2004; see also http://www2.uni-jena.de/biologie/ecology/biodiv/speciespool.html). Four functional groups of plants were assigned to the 60 species: Grasses, short herbs, tall herbs and legumes (Roscher et al. 2004). In August 2002, we transplanted S. nigrum into 12 plots of different species richness and functional groups (see next paragraph and Table 1). The establishment of the sown species varied and the functional groups (Table 1) were mainly represented by the following species; canopy class “short”: Plantago lanceolata, Taraxacum officinale, Bellis perennis, and grasses such as Trisetum flavescens, Dactylis glomerata and Poa spp.; canopy class “medium”: there are the combinations with the highest species diversity, namely up to 60 species, creating a even utilisation of the canopy space; canopy class “tall”: Onobrychis viciifolia, Knautia arvensis, and weed species such as Rumex obtusifolius. The assignments of single plot to either category were verified by photographic documentation taking into account that some weed species were present in addition to the sown species combinations (Roscher et al. 2004).
Plant growth and treatments Solanum nigrum L. (inbred line Sn30, seeds formerly collected at the field site) seeds were incubated in 3.5mM Ca(NO3)2 overnight at 4°C and germinated in a peat-based substrate with clay additions (Tonsubstrat, Klasmann). Plants were grown in multipot flats (Teku) for three weeks in the glasshouse (24 °C / 16 h light; 20 °C / 8 h dark) and the flats were kept outside the glasshouse for five days to acclimatize the plants to the field
70 MS3 – Biodiversity and defense responses of S. nigrum conditions prior to transplanting. We planted 18 S. nigrum plants into each of the 12 plots and started the experiment one week after transplanting to ensure an interaction between target plants and interspecific competitors. We applied 250 µg of MeJA (Sigma) in 20µL lanolin (Sigma) to the stem of the S. nigrum target plants (n=9) above the third leaf node, which is known to elicit a systemic defense response (S. Schmidt unpublished results). To control for lanolin effects, we treated control plants (n=9) with 20µL pure lanolin. Samples for the microarray analysis were harvested after 24h. Lanolin-treated control plants grown without competitors in 3L-pots outside the greenhouse at the Max Planck Institute for Chemical Ecology (Jena, Germany), were used as standard control in the microarray experiment. The height and weight of the target plants were determined 60 days after MeJA elicitation (Table 1).
Table 1 Summary of plot details and plot classification. Microarray hybridizations were conducted for all plots except II03 and II06.
Plot # II01 II03 II14 II18 II02 II06 II09 II13 II17 II12 II15 II16
Species numbers 4 60 8 16 2 4 4 1 8 8 1 4
Grasses 1 16 2 4 2 ------
Short herbs 1 12 2 4 - 2 4 1 4 - - 2
Tall herbs 1 20 2 4 - - - - - 8 - 1
Legumes 1 12 2 4 - 2 - - 4 - 1 1 Functional All plant functional Short herbs / short legumes / Tall herbs / tall diversity groups present. grasses dominant. legumes dominant. Canopy class medium short tall
61.33 59.67 68.86 49.00 43.96 54.82 59.10 45.32 52.06 48.43 79.96 65.91 S. nigrum (0.79) (1.80) (1.94) (1.56) (1.67) (2.23) (1.06) (1.23) (3.22) (2.13) (2.19) (1.96) mean height (SEM) [cm] 58.62 (1.57) 51.74 (1.40) 64.82 (1.62)
S. nigrum 7.45 6.28 11.46 20.83 10.52 4.54 8.70 8.98 8.57 9.48 19.31 6.88 mean above- (0.54) (1.32) (1.49) (2.60) (1.72) (0.39) (0.89) (0.99) (1.89) (1.17) (2.99) (0.67) ground dry mass (SEM) 11.45 (1.21) 8.15 (0.57) 11.66 (1.31) [g]
71 MS3 – Biodiversity and defense responses of S. nigrum
Microarray hybridization and analysis 24 h after MeJA elicitation, two fully expanded leaves above the fourth leaf node of nine replicate plants per treatment were harvested and shock frozen. The leaves of the 9 replicates were ground under liquid nitrogen and total RNA was extracted with TRI REAGENTTM (Sigma) according to the manufacturer’s instructions. We performed standard two- dye microarray hybridizations with cDNA of MeJA-elicited test samples labeled with Cy3 and the corresponding control cDNA (reference) with Cy5 according to the procedure described in Halitschke et al. (2003). To facilitate comparisons across microarrays we chose an hybridization design, in which we hybridized all treatment samples against the same control sample. Non- elicited S. nigrum plants served as the control sample in all hybridized microarrays. We used a 120-oligonucleotide (60 genes) microarray that is described in Schmidt and Baldwin (submitted) and aquired and analysed the raw data as described in Schmidt et al. (2004). We used the software GeneSpring 6.1 (Silicon Genetics) to perform Principal Component Analysis (PCA) with the genes or the treatments as criteria. The statistical analyses facilitated the identification of commonly or differentially regulated genes between treatments. We defined a transcript as being regulated, if it met the following criteria: (1) The ER of the biological replicates (ER of each microarray was calculated from 6 spots) differed significantly from 1 as determined by a Student’s t-test. (2) It exceeded the threshold ER of 0.67 for down- or 1.5 for up-regulation. Thresholds and reproducibility of microarray results have been tested and approved elsewhere (Halitschke et al. 2003; Heidel and Baldwin 2004). A complete list comprising all raw data is available from the authors.
Trypsin proteinase inhibitors 72h after MeJA elicitation a young systemic leaf of each replicate (n=9 for elicited and non-elicited plants) per plot was harvested and shock frozen in liquid nitrogen. Trypsin proteinase inhibitors (TPIs) were analyzed as described in Schmidt et al. (2004). For statistical analysis (JMP 5.01; SAS Institute Inc.) we log-transformed the data and used ANOVA with a Tukey- Kramer HSD post hoc test to correct for multiple comparisons.
72 MS3 – Biodiversity and defense responses of S. nigrum
Results
Microarray analysis PCA is an unconstrained ordination technique we used to localize samples (= different treatments represented by 16 microarrays; Fig. 1A) in ordination space so that the distances between the samples reflect the dissimilarities of the elements composing a sample. These elements are the genes with their treatment-dependent ERs. A PCA reduces the dimensionality of the dataset by defining variables (= principal components, ordination axes) that explain the largest proportion of total variance in the smallest number of dimensions. Principal components 1 and 2 accounted for more than 80% of the variance in the data and the two treatments, MeJA and control, are clearly distinguished by these axes. There was no apparent correlation to the number of sown species; however, the two axes revealed subtle effects of differences in functional group composition on the MeJA- induced transcriptional pattern. For subsequent analysis, we used the microarrays of plants, grown in the same canopy class (Table 1) as biological replicates to find the significant transcriptional differences between plants that were grown in different competitive environments (Fig. 1B; Supplementary Table 1). Among the up-regulated genes that respond to MeJA there are genes mainly involved in defense signaling (allenoxide synthase; wound-induced protein kinase) or the biosynthesis of defensive compounds such as TPIs or farnesyl pyrophosphate (FPP), an important precursor of terpenoids, namely sesquiterpenes and sterols (Chappell 1995). Additional genes of terpenoid metabolism show opposite regulation, e.g. a 3-hydroxymethyl-3-glutaryl- coenzyme A reductase 1 (HMGR1) is down-regulated in all competing plants, but especially in plants with tall competitors; HMGR1, that has been shown to be induced by MeJA in potato, is involved in the biosynthesis of sterols and, hence, provides metabolites for biosynthesis of steroidal alkaloids (Choi et al. 1994; Yang et al. 1991). The concomitant down- regulation of HMGR1 and up-regulation of FPP synthase argue for fine- tuned coordination of MeJA-induced terpenoid biosynthesis in response to competition.
73 MS3 – Biodiversity and defense responses of S. nigrum
Fig. 1 Description see next page.
74 MS3 – Biodiversity and defense responses of S. nigrum
Genes of primary metabolism were down-regulated by MeJA, for example acid invertase and RuBPCase activase. Pathogenesis-related (PR) proteins exhibited up-regulation (PR-1) or down-regulation (PR-5) in response to MeJA. The MeJA-driven transcriptional of S. nigrum pattern was influenced by the level of interspecific competition. This was most clearly seen in the down-regulation of additional photosynthesis-related genes, a RALF precursor gene and a polyphenyl oxidase in MeJA-elicited plants growing with competitors of short canopies. A gene coding for a glutamate synthase, an essential enzyme of nitrogen assimilation in plants, was down-regulated in MeJA-elicited plants grown with tall competitors. Interestingly, S. nigrum grown in plots with short or tall competitors showed overlapping gene regulation (e.g. up-regulation of PR-1 and down-regulation of a signaling gene; Fig. 1B, Supplementary Table 1).
Trypsin proteinase inhibitors (TPIs) We assayed systemic leaves of MeJA-elicited and non-elicited S. nigrum plants for their TPI content. The significantly higher TPI levels in response to MeJA-elicitation were affected by the competitive environment of the plant (Fig. 2). The species richness had no significant effect on induced TPI-levels (Fig. 2A), however the canopy structure significantly influenced MeJA-induced TPI accumulation. Tall competitors attenuated the increase in TPIs significantly in comparison to medium competition; short competitors led to an intermediate TPI level. TPI-levels of plants
Fig. 1 (p. 73) Gene expression analysis. (A) The PCA biplot incorporates the expression data of all 16 microarrays. MeJA-elicitation is responsible for most of the variation in the gene expression and in the group of MeJA- elicited plants an effect of differential competition is discernible. This effect is dependent on the canopy height of the community (assignment of classes see Table 1) and no effect of species numbers is evident (species numbers are indicated next to the symbols). CON = Lanolin-treated control plants (open symbols); MeJA = MeJA-elicited plants (closed symbols). (B) The Venn diagram shows overlapping and non-overlapping gene regulation of MeJA- elicited plants. The expression ratios (ERs; mean of 6 replicate spots per microarray) of significantly regulated transcripts (t-test; p<0.05) for each functional category were calculated from 2 or 3 biological replicates; ↑ = up-regulated transcripts (ER>1.5); ↓ = down-regulated transcripts (ER<0.67)7. 5
MS3 – Biodiversity and defense responses of S. nigrum under short and tall competition were comparable to those of non- competing plants (Schmidt et al. 2004).
Fig. 2 TPI levels (mean ±SE) in non-elicited S. nigrum (open bars; Control) and MeJA-elicited S. nigrum (black bars; MeJA) in response to interspecific competition. (A) MeJA-induced increases in TPI-levels are significantly different in all plots (Student’s t-test, all p’s<0.01), however, species numbers showed no correlation to TPI-levels. (B) MeJA-induced TPI-levels differed significantly in correlation the plant functional categories (ANOVA with Tukey-Kramer HSD post-hoc test).
76
MS3 – Biodiversity and defense responses of S. nigrum
Discussion
The links between species diversity, functional diversity, and ecosystem functioning remain controversial (Körner 1993), but there is growing consensus that the functional diversity, or the species traits, rather than the species richness per se, strongly influence ecosystem level processes (Díaz and Cabido 2001). We chose an “ask the plant” approach to study the significance of plant diversity at an organism level and examined two questions at the interface of plant responses to competition and herbivory: 1) Is the MeJA-elicited “defensome” different in plants grown in plots with different competitors? And if so, 2) Is the defensome influenced more by the biodiversity of the plot (number of species with which the focal plant interacts) or some functional characteristic of the interacting species? We discuss our findings in the context of how the MeJA-elicited transcriptome of S. nigrum reflects the biotic environment and how this information can be related to what is known from synergistic effects of competition and herbivory. Our results showed no effect of species numbers, but provide evidence that competitors belonging to diverse functional groups alter MeJA-induced gene expression that is furthermore reflected by changes TPI levels. Tall canopy competitors significantly reduced MeJA-elicited TPI levels in S. nigrum. Reduced TPI levels are most likely the effect of resource competition as has been demonstrated for Brassica napus and Nicotiana attenuata. The decrease of TPIs in B. napus in response to intraspecific competition could be buffered if plants were grown under conditions of high nutrient availability (Cipollini and Bergelson 2001). In N. attenuata lower N availability was correlated to lower TPI production in response to MeJA or herbivory (Lou and Baldwin 2004). That N metabolism is influenced by different competitive regimes is indicated by the suppression of Fd-GOGAT (Ferredoxin-dependent glutamate synthase) in competing plants, with a significant down-regulation only observed in MeJA-elicited S. nigrum competing with tall competitors. Fd-GOGATs are important enzymes involved in N assimilation, photorespiration, and amino acid synthesis (Lam et al. 1996). It is known that Fd-GOGAT activity is induced by light and
77 MS3 – Biodiversity and defense responses of S. nigrum nitrate (Stitt et al. 2002) and hence suggests least favorable light and nitrate conditions in communities with tall competitors. Decreases in TPIs, which are N-based defense compounds, appear to be buffered in S. nigrum by expression of C-based compounds, such as sesquiterpenes. Our transcrptional data is consistent with previous results that showed a flea beetle-induced up-regulation of HMGR2 (Schmidt et al. 2004), which produces the precursors for sesquiterpene biosynthesis, in contrast to HMGR1 that provides substrates for sterol biosynthesis (Yang et al. 1991). With the notion that S. nigrum emits rich bouquets of terpenoids in response to herbivory (Schmidt et al. 2004) that are widely recognized as important indirect defense measures in plant-insect interactions (e.g. Kessler and Baldwin 2001), it seems that the shift of competing S. nigrum from N- to C-based defense compounds is accompanied by buffering decreased direct defenses through deployment of indirect defenses. This shift is most evident in S. nigrum that compete with tall competitors, however, S. nigrum’s interaction with short competitors was characterized by different transcriptional effects. The down-regulation of a RALF (rapid alkalinization factor) precursor gene in MeJA-elicited S. nigrum plants grown in short canopies indicates interaction on the root level. As light never becomes limiting (Rajaniemi et al. 2003), root competition might be more efficient in communities with short canopies, because Plantago lanceolata and grasses that were present in the respective plots are known to have dense rooting systems (refs). RALF is a 5-kDa polypeptide and was isolated from tobacco leaves as a signaling compound involved in defense and development (Pearce et al. 2001; Haruta and Constabel 2003). The purified polypeptide if applied to germinating seeds of tomato or Arabidopsis thaliana, inhibited root growth of the seedlings, hence a down-regulation of RALF might have the opposite effect and facilitate root development and therefore foraging for nutrients. Evidence that N availability may influence the expression of RALF is emerging from results of Lou and Baldwin (2004). They found that in non- competing plants of N. attenuata grown under high N availability the caterpillar-induced up-regulation of RALF was significantly higher than in plants grown under low N conditions.
78 MS3 – Biodiversity and defense responses of S. nigrum
The assimilation of nitrate is strongly linked to photosynthesis in plants and decreased expression of important photosynthetic genes such as RuBPCase is correlated to inhibition of nitrate accumulation, to decreased amino acid levels, and as a consequence to a large decrease in N-containing defense metabolites such as chlorogenic acid and nicotine in tobacco plants carrying a transgene to suppress the expression of RuBPCase (Matt et al. 2002). The lower induced TPI protein levels and the down-regulation of a gene coding for a further defense protein, namely polyphenyl oxidases (PPOs), in MeJA-elicited plants in short canopies is correlated to a significant down-regulation of photosynthesis-related genes. In general it is known that herbivory or MeJA reprograms the plant’s transcriptome from growth- to defense-related traits (Hermsmeier et al. 2001; Voelckel and Baldwin 2003) which is clearly seen in the down-regulation of RuBPCase activase (rca) among competing plants that were elicited with MeJA. Rca is an important regulator of RuBPCase activity (Portis 1995). Strikingly, only in plants with short competitors MeJA lead to a significant down-regulation of additional genes coding for proteins of the photosynthetic machinery (RuBPCase large subunit, photosystem II D1 protein), which may be a negative effect of stronger root competition. In conclusion, the functional type of competition, or the morphological characteristics of competitiors, significantly influenced growth- and defense-related genes in MeJA elicited S. nigrum. While the phenotypic measures describe the resulting effects of competition from diverse environments on important defense- and growth related traits, the gene expression analysis points towards regulatory mechanisms of the interactive effects of competition and herbivory on plants and reflects the effects much earlier than they are seen in a relevant phenotype. To dissect the underlying mechanisms, the direct manipulation of the genetic basis is the most powerful tool. Genes related to nitrate metabolism, stress-related signaling and terpenoid biosynthesis are potential candidates to study questions related to competitive and defensive abilities of plants in the complexity of nature. While molecular biology has provided the ability to manipulate the expression of individual genes, understanding the functional consequences of these manipulations will require additional ecological tools
79 MS3 – Biodiversity and defense responses of S. nigrum to dissect the complex interplay of selective forces that all organisms face in nature.
Acknowledgements
We thank Susan Kutschbach, Thomas Hahn and Klaus Gase for assistance in microarray work; Danny Kessler for help with TPI analysis and fieldwork; the Max Planck Society and the Deutsche Forschungsgemeinschaft (FOR. 456; BA2138/1-1) for financial support.
References
Baldwin I.T. (1998) Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences of the United States of America, 95, 8113-8118 Chapin F.S., Zavaleta E.S., Eviner V.T., Naylor R.L., Vitousek P.M., Reynolds H.L., Hooper D.U., Lavorel S., Sala O.E., Hobbie S.E., Mack M.C. & Diaz S. (2000) Consequences of changing biodiversity. Nature, 405, 234-242 Chappell J. (1995) Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 46, 521-547 Choi D., Bostock R.M., Avdiushko S. & Hildebrand D.F. (1994) Lipid- derived signals that discriminate wound-responsive and pathogen- responsive isoprenoid pathways in plants - methyl jasmonate and the fungal elicitor arachidonic acid induce different 3-hydroxy-3- methylglutaryl-coenzyme A reductase genes and antimicrobial isoprenoids in Solanum tuberosum L. Proceedings of the National Academy of Sciences of the United States of America, 91, 2329-2333 Cipollini D.F. & Bergelson J. (2001) Plant density and nutrient availability constrain constitutive and wound-induced expression of trypsin inhibitors in Brassica napus. Journal of Chemical Ecology, 27, 593-610
80 MS3 – Biodiversity and defense responses of S. nigrum
Cipollini D.F. (2004) Stretching the limits of plasticity: Can a plant defend against both competitors and herbivores? Ecology, 85, 28-37 Edmonds J.M. & Chweya J.A. (1997) Black nightshades, Solanum nigrum L. and related species, Gatersleben, Rome. Halitschke R., Gase K., Hui D., Schmidt D.D. & Baldwin I.T. (2003) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VI. Microarray analysis reveals that most herbivore-specific transcriptional changes are mediated by fatty acid-amino acid conjugates. Plant Physiology, 131, 1894-1902 Haruta M. & Constabel C.P. (2003) Rapid alkalinization factors in poplar cell cultures. Peptide isolation, cDNA cloning, and differential expression in leaves and methyl jasmonate-treated cells. Plant Physiology, 131, 814-823 Kessler A. & Baldwin I.T. (2001) Defensive function of herbivore-induced plant volatile emissions in nature. Science, 291, 2141-2144 Kessler A. & Baldwin I.T. (2002) Plant responses to insect herbivory: The emerging molecular analysis. Annual Review of Plant Biology, 53, 299- 328 Körner C. (1993) Scaling from species to vegetation: the usefulness of functional groups. In: Schulze E.-D., Mooney H.A. (eds.) Biodiversity and ecosystem function. Ecological Studies 99, Springer, Berlin, 117–140. Lam H.M., Coschigano K.T., Oliveira I.C., Melo-Oliveira R. & Coruzzi G.M. (1996) The molecular genetics of nitrogen assimilation into amino acids in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology, 47, 569-593 Loreau M., Naeem S., Inchausti P., Bengtsson J., Grime J.P., Hector A., Hooper D.U., Huston M.A., Raffaelli D., Schmid B., Tilman D. & Wardle D.A. (2001) Biodiversity and ecosystem functioning: Current knowledge and future challenges. Science, 294, 804-808 Loreau M., Naeem S. & Inchausti P. (2002) Biodiversity and Ecosystem Functioning. Oxford University Press, Oxford. Lou Y. & Baldwin I.T. (2004) Nitrogen supply influences herbivore-induced direct and indirect defenses and transcriptional responses in Nicotiana attenuata. Plant Physiology, 135, 496-506 Matt P., Krapp A., Haake V., Mock H.P. & Stitt M. (2002) Decreased Rubisco activity leads to dramatic changes of nitrate metabolism, amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants. Plant Journal, 30, 663-677
81 MS3 – Biodiversity and defense responses of S. nigrum
Mulder C.P.H., Jumpponen A., Hogberg P. & Huss-Danell K. (2002) How plant diversity and legumes affect nitrogen dynamics in experimental grassland communities. Oecologia, 133, 412-421 Pearce G., Moura D.S., Stratmann J. & Ryan C.A. (2001) RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development. Proceedings of the National Academy of Sciences of the United States of America, 98, 12843-12847 Portis A.R. (1995) The regulation of rubisco by rubisco activase. Journal of Experimental Botany, 46, 1285-1291 Rajaniemi T.K., Allison V.J. & Goldberg D.E. (2003) Root competition can cause a decline in diversity with increased productivity. Journal of Ecology, 91, 407-416 Roscher C., Schumacher J., Baade J., Wilcke W., Gleixner G., Weisser W.W., Schmid B. & Schulze E.-D. (2004) The role of biodiversity for element cycling and trophic interactions: an experimental approach in a grassland community. Basic and Applied Ecology, 5, 107-121 Schmelz E.A., Alborn H.T., Engelberth J. & Tumlinson J.H. (2003) Nitrogen deficiency increases volicitin-induced volatile emission, jasmonic acid accumulation, and ethylene sensitivity in maize. Plant Physiology, 133, 295-306 Schmidt D.D., Kessler A., Kessler D., Schmidt S., Lim M., Gase K. & Baldwin I.T. (2004) Solanum nigrum: A model ecological expression system. Molecular Ecology, 13, 981-995 Schulze E.-D. & Mooney H. (1993) Biodiversity and ecosystem function. Ecological Studies 99. Springer Verlag, Berlin. Stitt M., Muller C., Matt P., Gibon Y., Carillo P., Morcuende R., Scheible W.R. & Krapp A. (2002) Steps towards an integrated view of nitrogen metabolism. Journal of Experimental Botany, 53, 959-970 Strong D.R., Lawton J.H. & Southwood R. (1984) Insects on Plants. Blackwell Scientific Publications, Oxford. Thaler J.S., Stout M.J., Karban R. & Duffey S.S. (1996) Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field. Journal of Chemical Ecology, 22, 1767-1781 Voelckel C. & Baldwin I.T. (2003) Detecting herbivore-specific transcriptional responses in plants with multiple DDRT-PCR and subtractive library procedures. Physiologia Plantarum, 118, 240-252 Yang Z.B., Park H.S., Lacy G.H. & Cramer C.L. (1991) Differential activation of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge. Plant Cell, 3, 397-405
82 MS 4 – Solanaceous responses to herbivory
Manuscript 4
Submitted to Plant Physiology
Specificity in ecological interactions: Attack from the same Lepidopteran herbivore results in species-specific transcriptional responses in two Solanaceous hostplants
Dominik D. Schmidt§#, Claudia Voelckel# and Ian T. Baldwin*
Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745 Jena, Germany
Key words: Nicotiana attenuata, Solanum nigrum, Manduca sexta herbivory, microarray analysis
§Present address: John Innes Centre, Department Disease and Stress Biology, Colney, Norwich NR7 4UH, UK
#These authors contributed equally to the manuscript.
*Correspondence: Ian T. Baldwin, Department Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans- Knöll-Straße. 8, D-07745 Jena, Germany, Fax: 03641 571102, E- mail: [email protected]
Running title: Solanaceous responses to herbivory
6666 words 4 figures 1 supplementary figure 1 supplementary table
83 MS 4 – Solanaceous responses to herbivory
Abstract Model systems have proven enormously useful in elucidating the biochemical function of plant genes. However their ecological function, having been sculpted by evolutionary forces specific to a species, may be less conserved across taxa. Responses to wounding and herbivore attack differ among plant families and are known to be mediated by oxylipin, ethylene and systemin signaling networks. We analyzed transcriptional responses of two native Solanaceous species to the attack of an herbivore whose elicitors are known not to be influenced by diet. With a 11,243 cDNA potato microarray, we compared the transcriptional responses of native tobacco (Nicotiana attenuata) with those of black nightshade (Solanum nigrum) when both were attacked by the Solanaceous generalist herbivore, Manduca sexta. Based on a ndhF (NADH dehydrogenase subunit F) phylogeny, S. nigrum is more closely related to potato than N. attenuata, but responded significantly less to M. sexta attack. Apart from transcriptional differences anticipated from their differences in secondary metabolism, such as the up-regulation of pyridine and glycoalkaloid alkaloid biosynthetic genes in N. attenuata and S. nigrum, respectively, both species showed distinct transcriptional patterns (with only 10% overlap in significantly regulated genes), which point to fundamental differences in the signaling cascades and downstream genes mediating herbivore resistance. Given that attack from the same herbivore elicits profoundly different response in two Solanaceaous taxa, “blueprints” for commonly regulated responses to plant-herbivore interactions appear unlikely.
84 MS 4 – Solanaceous responses to herbivory
Introduction Understanding the genetic basis of plant secondary metabolism will require multiple model systems (Kutchan 2001) because clades of plant species are biochemically specialized to produce particular classes of secondary metabolites (e.g. Brassicaceous taxa emphasize glucosinolates, while Solanaceous plants produce steroidal, tropane or pyridine alkaloids; Fraenkel 1959). Such metabolic specialization is thought to have evolved in response to selection pressures from the plant’s enemies (Ehrlich and Raven 1964, Fraenkel 1959, Jones and Firn 1991). For example, the model Brassicaceous and Solanaceous plants, Arabidopsis and tomato, respectively, have each evolved a different arsenal of biochemical weapons that can be activated upon attack from herbivores (Walling 2000). While it has long been clear that the downstream defense responses of a plant would differ among taxa, the mechanisms that activate the defense responses are thought to be more conserved, and it has been assumed that Arabidopsis would provide a “blueprint” for the mechanisms that elicit ecological responses in higher plants (Mitchell-Olds 2001). However evidence is emerging that this “blueprint” has undergone some revisions at the family level. Three plant hormones, jasmonic acid (JA), ethylene (ET), and salicylic acid (SA), mediate responses to wounding and attack from herbivores and pathogens in most taxa studied, but research on “crosstalk” among these signals has identified important differences at the family level. For example in Arabidopsis, wounding leads to the activation of two pathways: an oligosaccharide-dependent and a JA- dependent pathway in the damaged leaves (Walling 2000, Leon et al. 2001). The former produces ET, which antagonizes the elicitation of JA- responsive genes in the wounded leaves, but not in unwounded leaves from the same plant. In contrast, both ET and JA are required for the elicitation of wound-inducible genes in tomato (Walling 2000, Leon et al. 2001). In addition to differences in “crosstalk” among the same signal molecules, Brassicaceous and Solanaceous plants differ in the signal molecules that are recruited. Systemin, a polypeptide hormone, which
85 MS 4 – Solanaceous responses to herbivory mediates local and systemic responses to wounding, has only been found in Solanaceous species to date (Ryan 2000). In addition to these family- level differences, differences have also been found between taxa within the same family. Within-family differences have been observed in the crosstalk between JA and ET signaling in the elicitation of defense responses. While JA and ET interact synergistically to elicit proteinase inhibitor expression in tomato, attack from Manduca sexta larvae in Nicotiana attenuata elicits an ET burst, which antagonizes the JA-mediated increase in nicotine (Kahl et al. 2000, Winz and Baldwin 2001). Within-family differences in signals have also been found. Systemins of potato, bell pepper and black nightshade are structurally similar and differ each in three (of 18) amino acids from the tomato systemin and differ in their ability to elicit tomato proteinase inhibitors (Constabel et al. 1998). Two tobacco systemins that elicit the accumulation of proteinase inhibitors have been identified, but these are structurally dissimilar to the tomato systemin (Pearce et al. 2001). Tobacco does not produce peptide signals that activate the tomato systemin receptor and does not react to tomato systemin (Scheer et al. 2003) Given this evidence for differences in defense signaling among several Solanaceous taxa, we were interested in a more thorough characterization of the potential differences in responses to herbivore attack within a plant family. For this analysis, we compaired the herbivore-induced transcriptome of two native Solanaceous species – Nicotiana attenuata and Solanum nigrum - to attack from the same native herbivore, M. sexta larvae. Three aspects of this analysis make this a valuable comparison. First, we analyze the responses of two native plants, in which the responses observed to herbivore attack are not confounded by a history of artificial selection for yield-associated traits. Second, we measure the responses to attack by a shared native herbivore (Fraenkel 1959), which is particularly well-studied with regard to how it elicits responses in its host plants. M. sexta larvae produce a suite of 8 fatty acid-amino acid conjugates (FAC) which are thought to be introduced into wounds during feeding and are necessary and sufficient
86 MS 4 – Solanaceous responses to herbivory to account for all of the observed changes in the plant’s wound response - including defense metabolites, signals and transcriptional responses - that are elicited by larval feeding (Halitschke et al. 2001, 2003; Roda et al. 2004). Simply adding these FACs to plant wounds simulates the plant’s responses to herbivore attack. Moreover, M. sexta’s FAC profile is not substantially altered when it feeds on different hostplants (Alborn et al. 2003) and specifically does not quantitatively or qualitatively change when it feeds on N. attenuata or S. nigrum (D.D. Schmidt, A. Steppuhn and R. Halitschke, unpublished results). Third, the secondary metabolites produced by both species are well-studied and provide a valuable backdrop against which to compare their herbivore-elicited transcriptomes. N. attenuata and S. nigrum constitutively produce nicotine and glycoalkaloids (Baldwin 1999, Dopke et al. 1987, Ridout et al. 1989), respectively, and both species are capable of producing proteinase inhibitors in response to wounding and release monoterpenoid and sesquiterpenoid volatiles in response to herbivore feeding (Van Dam et al 2001, Kessler and Baldwin 2001, Schmidt et al. 2004). In summary, for this pair of hostplants, attack by M sexta larvae is likely to provide a standardized elicitation of the responses to herbivore attack, which, in turn, are likely sculpted by natural selection. In order to provide an unbiased comparison of the herbivore- regulated transcriptome in both species, we used a microarray with 11,243 potato cDNAs (representing approximately a third of the potato genome). We were particularly interested in the scope of the response (how many genes are involved?) and the specificity of the response (how many transcripts are commonly and specifically regulated?). The potato microarray was established through the NSF Potato Functional Genomics project (http://www.tigr.org/tdb/potato) and is available from The Institute of Genomic Research (TIGR, Rockville, Maryland, USA). The scope and specificity of the response could be influenced by the taxonomic similarity between the source of the genes on the array (S. tuberosum) the samples used in the hybridizations (S. nigrum and N. attenuata). To clarify the phylogentic relationship among the three species, we analyzed sequence similarities among the plastidial ndhF gene
87 MS 4 – Solanaceous responses to herbivory of several solanacaeous species. Based on this analysis, S. nigrum is more closely related to S. tuberosum than N. attenuata, which generated the expectation that the array would likely provide stronger signals in the analysis of S. nigrum’s responses than from that of N. attenuata.
Materials and methods Plant and insect growth and experimental setup Seeds of a Nicotiana attenuata inbred line were smoke-germinated on Phytagel as described by Krugel et al. (2002). Twelve days after germination seedlings were planted in soil into Teku pots (Waalwijk, the Netherlands), and after 12 additional days, transferred to 0.5 L pots with a peat based substrate (Klasmann Tonsubstrat, Geeste-Groß Hesepe, Germany). Seeds of a Solanum nigrum inbred line were germinated as described in (Schmidt et al. 2004) and ten day-old seedlings were transferred to 0.5 L pots with the same substrate as for N. attenuata. Both plant species were grown in the glasshouse of the Max Planck Institute for Chemical Ecology (Jena, Germany) at 24-26°C (16h light; supplemental lighting by Philips Sun-T Agro 400 and 600 W sodium lights; 65% humidity). One day prior to the herbivore treatment, 24 randomly selected plants of each species were placed in glass insect cages (30cm x 30cm x 60cm, each cage accommodating 4 plants). Twelve plants were used in both herbivore and control treatment; at harvest, four plants per cage were pooled to provide one biological replicate of the experiment. Eggs of Manduca sexta were obtained from a laboratory colony. On each plant of the herbivore treatment (n=12 plants for each species) ten freshly hatched caterpillars were placed on two different leaves and allowed to feed freely. After 24h of feeding, herbivores and their frass were removed and shoots and leaves of the herbivore-damaged and of non-attacked control plants were harvested, flash-frozen in liquid nitrogen, and stored at –80°C until microarray analysis.
88 MS 4 – Solanaceous responses to herbivory
Microarray analysis For gene expression analysis, we used the TIGR potato microarray that contains 11,243 annotated cDNA clones spotted as duplicates on the array. Detailed information about this microarray can be found under http://www.tigr.org/tdb/potato/microarray2.shtml. Gene expression data obtained from hybridizations of this potato microarray with a variety of Solanaceous species can be accessed through a database maintained at the TIGR website (http://www.tigr.org/tigr-scripts/sgedb/studies_SGED.pl) and thus compared and shared across different laboratories. Although Solanaceous species clearly differ in morphology, life cycle, secondary metabolism, tuber and fruit formation etc., they have similar genomes with respect to gene content and genome organization (see transcriptional analysis of several Solanaceous plants by Robin Buell, http://www.tigr.org/tigr-scripts/sgedb/search2_std.pl?study_id=35). Plant material was ground under liquid nitrogen and total RNA was extracted with TRI ReagentTM (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. All steps of microarray processing (cDNA production, cDNA labeling, microarray hybridization, data quantification, data normalization using LOWESS) were carried out by the TIGR Expression Profiling Service according to published methods (http://www.tigr.org/tdb/potato/microarray _SOPs.shtml). The cDNAs hybridized to an individual array was produced from RNA extracted individually from the four plants of a control cage (Cy5 labeled) and the four plants of the treatment cage (Cy3 labeled). The three biological replicates of the M. sexta-N. attenuata elicitation experiment are named NA1, NA2, and NA3. SN1, SN2, and SN3 designated the corresponding replicate microarrays of the M. sexta- S. nigrum elicitation experiment. For N. attenuata, one flip-dye microarray was hybridized in which the control sample was labeled with Cy3 and the treatment sample with Cy5. The raw data from the six hybridizations including all details of the experiment are available from the TIGR Solanaceae Gene Expression Database (SGED;
89 MS 4 – Solanaceous responses to herbivory http://www.tigr.org/tigr-scripts/sgedb/search2 _std.pl?study_id=53, experiment IDs 2450, 2455, 2460, 2465, 2470, 2475). We analyzed the normalized data of the six microarrays with GeneSpring 6.1 (Silicon Genetics, Redwood City, CA, USA) using Hierarchical Cluster Analysis. For further analysis we calculated the mean expression ratio (ER) from the biological replicates and defined a transcript as being differentially regulated when the following criteria were fullfilled: (1) The ER was significantly different from 1 as determined by a Student’s t-test (p<0.05); (2) the ER exceeded the thresholds of 0.67 and 1.5 for down- and up-regulation, respectively. These criteria had been previously tested and found to give reproducible results (Heidel and Baldwin 2004; Halitschke et al. 2003).
Figure 1. The single most parsimonious tree recovered using sequences of ndhF (length=372, CI=0.949, RI=0.835). The two species of interest (N. attenuata and S. nigrum) are in bold as is the source species for the cDNA clones spotted onto the TIGR potato array (S. tuberosum). Branch lengths correspond to parsimony steps and the numbers above branches indicate parsimony bootstrap support values for that clade.
90 MS 4 – Solanaceous responses to herbivory
Results Phylogenetic analysis To clarify the phylogenetic relationship between N. attenuata and S. nigrum we analyzed sequence similarities among the plastidial ndhF gene of several Solanaceous species. Based on this comparison, S. nigrum is more closely related to S. tuberosum than N. attenuata, as demonstrated by a 97% homology between S. nigrum and S. tuberosum and 94% between N. attenuata and S. tuberosum (Fig. 1).
Microarray analysis Three replicate TIGR chips were hybridized with RNA from thrice replicated M. sexta-infested N. attenuata plants and M. sexta-infested
Figure 2. Venn diagram of the numbers of overlapping and non-overlapping significantly upregulated (↑; ER>1.5) or downregulated (↓; ER<0.67) in N. attenuata and S. nigrum in response to M. sexta herbivory. In summary, 561 genes were found to be up-regulated and 203 to be down-regulated with an overlap of about 10% in both plant species. (S. nigrum sketch: http://www.rmc.sierraclub.org/outings/images/ weeds_blacknightshade_drawing.jpg)
91 MS 4 – Solanaceous responses to herbivory
S. nigrum plants. Of 11,243 cDNAs, a total of 754 were regulated (mean ratio >1.5 or <0.67 for up- and down-regulated genes, respectively) in either N. attenuata, S. nigrum or both species in response to M. sexta attack (Fig. 2). Interestingly, there were more genes up-regulated (561) than down-regulated (203) and only 75 cDNAs (10%) were equally regulated in both species (Fig. 2). When the expression ratios of the 754 responsive cDNAs were subjected to a cluster analysis, the three N. attenuata arrays were clearly separated from the three S. nigrum arrays and patterns of commonly and specifically regulated genes were discernable (Supplemental Fig.1). Based on the annotations of the 754 regulated cDNAs, we grouped the genes into functional categories (Supplemental Table 1) and in the following use this classification to discuss some striking differences in gene up-regulation that occur between these two Solanaceous species. For the details (expression ratios, annotations, categories) of gene down-regulation see Supplementary Table 1. Signal transduction. Additionally to a common up-regulation of lysophospholipase and lipoxygenase (oxylipin signaling), N. attenuata and S. nigrum activate different signal cascades in response to leaf-chewing M. sexta larvae. Increases in genes coding for a G-protein-coupled receptor, a GTP- binding protein, phospolipase C, diacylglycerol kinase, calmodulin, annexin, a Ca2+-activated kinase and a Ca2+-activated ion channel indicate calcium- and inositol phospholipid-based signaling, as well as, G-protein-mediated signaling in N. attenuata. Moreover, the generation of 2-hydroperoxides (α-dox) and glucosylated salicylate (UDP-glucose:SA glucosyltransferase) appears to be specific to N. attenuata, while a zeatin-glucosyltransferase and a 12-oxo-phytodienoate reductase seem to have in role in signaling in S. nigrum. An auxin-amino acid hydrolase and several kinases (calcium-dependent kinases, receptor kinases, and MAP kinases) are activated in both species. Proteolysis. Among the genes coding for proteolytic enzymes, a leucine aminopeptidase (LAP), which catalyzes the release of N-terminal residues from proteins and peptides, was up-regulated up to 20-fold in S. nigrum. In contrast, increases in ubiquitin-mediated proteolysis (polyubiquitin, ubiquitin-conjugating enzyme) are specific to N. attenuata.
92 MS 4 – Solanaceous responses to herbivory
Figure 3. Categories of up-regulated genes in response to M. sexta herbivory with exemplary genes for both plant species (Both, non-shaded boxes), for N. attenuata (NA, darkly shaded boxes), and for S. nigrum (SN, lightly shaded boxes).
93 MS 4 – Solanaceous responses to herbivory
An up-regulation of protein disulfide isomerase and peptidylprolyl isomerase was found in both species, but the response of the latter was stronger in S. nigrum. Secondary metabolism. Little overlap between N. attenuata and S. nigrum was found in the expression of genes involved in secondary metabolism, including up-regulation of hydroperoxide lyase, an important gene in the synthesis of C6 volatile organic compounds (VOCs), spermidine synthase, which is involved in polyamine synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR2), the key enzyme in the cytosolic route to the terpenoid precursor isopentenyl pyrophosphate (IPP). In S. nigrum, HMGR1 is also up-regulated and it has been shown for potato that HMGR2 is mainly involved in the biosynthesis of sesquiterpenes, whereas HMGR1 produces precursors for sterol biosynthesis (Choi et al. 1994). Moreover, enzymes involved in the biosynthesis of sterols, such as cholesterol (7-dehydrocholesterol reductase), were up-regulated in S. nigrum. Sterol contents in Solanum species are tightly linked to levels of glycoalkaloids and cholesterol is thought to be a precursor of steroidal alkaloids (Bergenstrahle et al. 1996), which in turn suggests increased production of glycoalkaloids in S. nigrum in response to M. sexta feeding. Further evidence for the deployment of alkaloidal defenses in S. nigrum is the up-regulation of a 2- oxoglutarate-dependent dioxygenase, which is homologous to hyoscyamine 6β-hydroxylase of Hyoscyamus niger (Lantin et al. 1999). Up- regulated genes of terpenoid biosynthesis include farnesyl pyrophosphate synthase and several sesquiterpene synthases, which are among the most strongly up-regulated genes in S. nigrum. It is known that S. nigrum produces a rich bouquet of VOCs in response to herbivory from flea beetles or the moth Acherontia atropos (Schmidt et al. 2004). In contrast to S. nigrum, N. attenuata induces transcripts involved in the plastid-localized glyceraldehydes/pyruvate pathway (DOX synthase, DOX reductoisomerase) of IPP production, suggesting that not only sesquiterpenes but also mono- and diterpenes, are elicited in N. attenuata upon M. sexta-herbivory. This is supported by the up-regulation of
94 MS 4 – Solanaceous responses to herbivory linalool synthase. IPP isomerase, which is recruited by both terpenoid pathways, is specifically up-regulated in N. attenuata. Also in contrast to the response in S. nigrum, N. attenuata plants elicit a strong transcriptional commitment to the production of phenol- based secondary compounds. Starting with the synthesis of shikimate (dehydroquinate synthase, shikimate kinase, 3-phosphoshikimate 1- carboxyvinyltransferase (EPSP synthase), chorismate synthase), preceding to the synthesis of prephenate (the committed step in phenylalanine and tyrosine synthesis catalyzed by chorismate mutase), continuing with the synthesis of cinnamic acid (PAL), p-coumaric acid (cinnamate-4-hydroxylase) and p-coumaroyl-CoA (coumaric acid-CoA ligase), the genes providing the precursors for flavonoid- and phenylpropanoid biosynthesis are induced. Transcripts related to flavonoid metabolism (UDP rhamnose-anthocyanidin-3-glucoside rhamnosyltransferase, dihydroflavonol reductase) and phenylpropanoid metabolism (p-coumaroyl shikimate 3'-hydroxylases, cinnamoyl-CoA reductase) were up-regulated as well. These results are consistent with the following earlier findings in the M. sexta - N. attenuata interaction: 1. the cloning of a UDP rhamnose-anthocyanidin-3-glucoside rhamnosyltransferase by DDRT-PCR from M. sexta-induced N. attenuata plants (Voelckel and Baldwin 2003), the production of phenylpropanoid- derived compounds (caffeoyl-putrescine, chlorogenic acid) in M. sexta- attacked N. attenuata plants (Kessler and Baldwin 2004) and the production of flavonoids (quercetin, rutin) in N. attenuata leaf trichomes (Roda et al. 2004). Several transcripts for polyphenyl oxidases, which catalyzes the production of o-quinones, which, in turn react with insect dietary proteins and impair their digestion (e.g. Constable et al. 2000), increase in abundance specifically in M. sexta-attacked N. attenuata. Proteinase inhibitor induction, an induced defense response well characterized in Manduca attacked N. attenuata (Zavala et al. 2004, Zavala and Baldwin 2004) was seen only in S. nigrum, probably because of the lack of tobacco-specific probes on the array. Primary metabolism. Manduca attack up-regulated in both plant species genes involved in cell wall biosynthesis, such as pectin methyl
95 MS 4 – Solanaceous responses to herbivory esterase (PME) and extensin; genes related to lipid metabolism (acyl- CoA synthetase); and genes that play a central role in carbohydrate metabolism (e.g. glyceraldehyd 3-phosphate dehydrogenase). Interestingly, transcripts for acetyl-CoA carboxylase which catalyzes the formation of malonyl-CoA, an essential precursor of flavonoid biosynthesis, increased specifically in N. attenuata. Other precursors of secondary metabolism whose production is elicited in N. attenuata include acetyl-CoA, S-adenosylmethionine, and putrescine. Putrescine is required for nicotine and polyamine synthesis and S-adenosylmethionine may be used in ethylene and polyamine synthesis. The up-regulation of two different isoforms of S-adenosylmethionine decarboxylase (secondary metabolism) suggests that a supply of decarboxlated S-adenosylmethionine is required to complement the supply of putrescine for higher polyamine synthesis. Additional evidence of tobacco-specific regulation in primary metabolism includes increases in xyloglucan endotransglycosylase, starch synthase, ATP citrate lyase, adenine phosphoribosyl transferase and nucleotide sugar epimerase. S. nigrum up-regulates genes involved in cell wall biosynthesis (cellulose synthase), amino acid metabolism and lipid synthesis. The most striking difference with the responses of primary metabolism in N. attenuata was the up-regulation of photosynthesis- related genes (e.g. Rubisco, light-harvesting complex b6) in caterpillar- infested S. nigrum plants. In general, herbivory is thought to down- regulate photosynthesis genes and, thereby, divert resources to secondary metabolism (e.g. Hermsmeier et al. 2001). Stress responses and selected genes. The stressed metabolic state of herbivore-attacked plants is reflected in the up-regulation of chaperones (HSP90, luminal binding protein, endoplasmin, etc.), dehydration induced proteins (with distinct genes elicited in both species, e.g. dehydrin in S. nigrum) and several cytochrome P450 monooxygenases. Among the most strongly up-regulated genes in both species were a putative acid phosphatase and the EEF53 gene. N. attenuata induces a vegetative storage protein reported to be herbivory-induced in Arabidopsis (Berger et al. 2002), a salt stress-related phospholipid-hydroperoxide glutathione peroxidase (Mittova et al. 2002), a TMV response related
96 MS 4 – Solanaceous responses to herbivory
Figure 4. Description see next page.
97 MS 4 – Solanaceous responses to herbivory
Figure 4 (p. 97). Simplified overview of major pathways of secondary- metabolite biosynthesis and their interrelationships with primary metabolism (modified from Gershenzon 2002). Genes found to be up-regulated are italicized and examples of Solanaceous secondary compounds are listed for some classes of secondary metabolites. Genes up-regulated in N. attenuata, S. nigrum, or both (#,*) are indicated. Abbreviations: G3P glycerol-3-phosphate; DHQS 3-dehydroquinate synthase; SHKK shikimate kinase; CS chorismate synthase; CM chorismate mutase; ADC arginine decarboxylase; ODC ornithine decarboxylase; SAMS S-adenosyl methionine synthase; SAMDC S-adenosyl methionine decarboxylase; SPDSYS spermidine synthase; PAL phenylalanine ammonia lyase; 4CL 4-coumarate coenzyme A ligase; C4H cinnamate-4-hydrogenase; DHFR dihydroflavonol reductase; LDOX leucanthocyanidin dioxygenase; PLC phospholipase C; LOX lipoxygenase; OPR oxophytodienoate reductase; HPL hydroperoxide lyase; VOCs volatile organic compounds; HMGR 3-hydroxy-3-methylglutaryl coenzyme A reductase; SabSYN sabinene synthase; DXR 1-deoxy-D-xylulose-5- phosphate reductoisomerase; DXS 1-deoxy-D-xylulose-5-phosphate synthase; Lis linalool synthase; 7DCR 7-dehydrocholesterol reductase. gene, an iron-stress related protein, a superoxid dismutase and a metal- transporting ATPase. A remarkably large number of chaperones are induced only in S. nigrum. Further stress-related responses in S. nigrum include the up-regulation of enzymes involved in oxidative stress, such as an herbivory-induced NADPH oxidase subunit (whitefly-induced gp91- phox), a peroxidase, and a glutathione transferase.
Discussion Small scale microarrays are often criticized for their bias in gene selection, which makes them suitable for answering the questions for which they were designed but unsuitable for other research questions. Here we used the potato 11,243 cDNA clone microarray (TIGR) to provide an unbiased comparison of the transcriptional responses of two Solanaceous species, N. attenuata and S. nigrum, to herbivory from the Solanaceous generalist, M. sexta. Based on the diversity of secondary metabolites produced by Solanaceous plants (e.g. Frohne and Jensen 19??) and the suite of signals that mediate plant responses to herbivory in
98 MS 4 – Solanaceous responses to herbivory this family (Walling 2000, Leon et al. 2001), we hypothesized that the analysis of M. sexta-elicited transcriptomes would yield two major results: (1) attack would elicit increases in secondary metabolism in both species, but the elicited pathways would differ, reflecting the different secondary metabolites constitutively produced by the two species and (2) signal transduction would be similar. We discuss our findings in the light of these two predictions and relate the results to what is known from other plant species. First, we evaluate what we learned about the M. sexta- responsive transcriptome in N. attenuata with a customized oligonucleotide microarray (Voelckel and Baldwin 2004) as compared to the large scale TIGR array. The TIGR array analysis confirmed previously measured increases in the expression of jasmonate cascade genes (13-lipoxygenase, α-dioxygenase), a green leaf volatile synthesis gene (hydroperoxide lyase), a polyamine synthesis gene (SAM-decarboxylase), phenylpropanoid synthesis genes (PAL, C4H, 4CL), polyphenoloxidase, xyloglucan- endotransglycosylase and UDP rhamnose:anthocyanidine-3-glucoside rhamnosyltransferase. Moreover, genes representing the above mentioned branches of metabolism, including p-coumaroyl shikimate 3'- hydroxylase and cinnamoyl-CoA reductase (phenylpropanoids and their conjugates, Gang et al. 2002), SAM synthetase and spermidine synthase (polyamines), arginine- and ornithine decarboxylase (precursors for polyamines and nicotine) and several polyphenol oxidases were found to be induced. Additionally, the TIGR array analysis of N. attenuata responses revealed the activation of 1) almost every gene in the shikimate pathway, 2) the plastidic route to terpenoid synthesis, and 3) additional cell wall related genes (pectin methyl esterase, extensin), to name just a few of the genes not monitored with the oligonucleotide array. Disagreements between the two analyses were mainly due to four reasons: 1) Some genes were not present on the TIGR array (e.g. thionins); 2) differential regulation was observed in less than three biological replicates (e.g. proteinase inhibitors) and hence the gene was automatically excluded from further analysis; 3) some genes were not regulated in the TIGR array analysis (e.g. Rubisco). 4) The raw signals
99 MS 4 – Solanaceous responses to herbivory of the hybridized TIGR arrays were not interpretable due to technical problems with the array (e.g. PR genes, ACC oxidase, threonine deaminase). In general, the large scale transcriptional analysis with the TIGR array has substantially extended our understanding of the plastic responses of N. attenuata to M. sexta attack, and additionally allowed for a detailed comparison of these responses with those elicited in S. nigrum by this Solanaceous generalist. Contrary to our assumption, N. attenuata and S. nigrum activated different sets of signaling genes. While jasmonic acid signaling was regulated in both species, calcium-based signaling, inositol-phospholipid signaling, and G-protein-mediated signaling were found to be specific to N. attenuata and cytokinin signaling was only detected in S. nigrum. While calcium-signaling has been implicated in systemin-mediated activation of phospholipase A2 and subsequent jasmonic acid synthesis (Ryan 2000), the role of phospholipase C and heterotrimeric G proteins in defense signaling in Solanaceous plants is largely unexplored. The only prosystemin gene on the array was up-regulated in two of three replicates of S. nigrum but did not yield interpretable results in N. attenuata, consistent with the structural dissimilarities of potato and tobacco systemins (Scheer et al 2003; Ryan 2000). Apart from the anticipated differences in alkaloid (steroidal alkaloids in S. nigrum) and alkaloid precursor (putrescine in N. attenuata) formation, our analysis revealed the production of different defense metabolites in both species. N. attenuata predominantly elicited genes for the production of antinutritive polyphenol oxidases, phenylpropanoids and their precursors, and plastidic isopentenyl pyrophosphate, which is primarily channeled into mono- and diterpene synthesis (Lichtenthaler 1999). In S. nigrum, the transcriptional emphasis was on sesquiterpene synthesis. Both species likely increase flavonoids, polyamines and green leaf volatiles in response to attack. Analyses of the metabolome are needed to test the predictions emerging from this transcriptional analysis. Our analysis also provided a deeper insight into the changes that accompany alterations in signaling and secondary metabolism,
100 MS 4 – Solanaceous responses to herbivory namely changes in carbohydrate and amino acid metabolism, nucleic acid and protein metabolism or changes associated with other biotic or abiotic stressors. For example, both plants increased similar and dissimilar transcripts for chaperones, dehydration-related genes and oxidative stress genes. Transcript levels of enzymes involved in protein degradation / processing were increased in both species but with distinct differences. Ubiquitin-related proteolysis, as suggested by the increased production of chaperones, plays an important role in eliminating misfolded or abnormal proteins that are probably accumulate in stressed plants and is activated only in N. attenuata. Furthermore, ubiquitin- dependent protein turnover influences many cellular processes by modulating levels of regulatory proteins (Hare et al. 2003) and there is increasing evidence that ubiquitin-dependent proteolysis is essential in regulating oxilipin-mediated plant responses. An important element in JA signal transduction, COI1 (CORONATINE INSENSITIVE 1), is part of an E3-type ubiquitin ligase complex and tomato mutants expressing non-functional COI1 are compromised in their resistance to two-spotted spider mites (Xie et al. 1998, Devoto et al. 2002, Li et al. 2004). Hence, ubiquitin-dependent proteolysis may play an important role in regulatory networks that mediate defense responses of N. attenuata. In S. nigrum other genes involved in protein metabolism are induced by M. sexta herbivory, with the most dramatic example being a LAP gene whose expression was up-regulated 20 fold. LAPs are present in pro- and eukaryotes and plant LAPs have been intensively studied in tomato, where they are elicited by JA, wounding and pathogens and are present in floral tissues (Tu et al. 2003 and references therein). Although tomato LAPs are biochemically and physiologically well characterized, their function in plant defense responses remains elusive. Animal aminopeptidases are involved in modulation of peptide and protein activities (Barr 1991) and therefore it is tempting to speculate that a LAP cleaves precursors of polypeptide hormones that are known especially among Solanaceous plants (Ryan et al. 2002). Alternatively, wound- induced LAPs may have a more general role in protein turnover that is an essential component of plant defense responses (Walling et al. 1995).
101 MS 4 – Solanaceous responses to herbivory
In addition to genes annotated with a putative function, the transcriptional analysis of M. sexta-infested plants revealed xx and xx genes whose functions are unknown, (Supplementary Table 1). These unknown genes providing interesting candidate genes for functional studies with plants in which their expression is silenced. The transcriptional comparison of M. sexta attacked N. attenuata and S. nigrum plants did not support the existence of a Solanaceous “blueprint” for herbivore defense. Most strikingly, the differences extended beyond the activation of different alkaloid pathways and included a profound divergence in signaling pathways mediating the elicited responses. Furthermore, the weaker response of S. nigrum plants could be due to S. nigrum’s general unresponsiveness or because N. attenuata has evolved a strong response to M. sexta attack, while S. nigrum hasn’t to this herbivore, but may have to others. Only by comparing responses to other herbivores (e.g. a flea beetle), which may elicit more common than specific responses in the two plant species, can this be resolved. Based on our analysis, we propose that only by analyzing a number of different Solanaceous taxa can the real wealth of responses to insect herbivores in this family be appreciated.
Acknowledgements We thank TIGR for generously providing the microarrays and the data acquisition; Dr. Ryan Oyama for invaluable support with the phylogenetic analysis; the Deutsche Forschungsgemeinschaft (BA 2132/1-1) and the Max Planck Gesellschaft for financial support.
Literature cited
Alborn HT, Brennan MM, Tumlinson JH (2003) Differential activity and degradation of plant volatile elicitors in regurgitant of tobacco hornworm (Manduca sexta) larvae. Journal of Chemical Ecology 29: 1357-1372
102 MS 4 – Solanaceous responses to herbivory
Baldwin IT (1999) Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. Journal of Chemical Ecology 25: 3-30 Bergenstrahle A, Borga P, Jonsson MV (1996) Sterol composition and synthesis in potato tuber discs in relation to glycoalkaloid synthesis. Phytochemistry 41: 155-161 Berger S, Mitchell-Olds T, Stotz HU (2002) Local and differential control of vegetative storage protein expression in response to herbivore damage in Arabidopsis thaliana. Physiologia Plantarum 114: 85-91 Constabel CP, Yip L, Patton JJ, Christopher ME (2000) Polyphenol oxidase from hybrid poplar. Cloning and expression in response to wounding and herbivory. Plant Physiology 124: 285-295 Constabel CP, Yip L, Ryan CA (1998) Prosystemin from potato, black nightshade, and bell pepper: primary structure and biological activity of predicted systemin polypeptides. Plant Molecular Biology 36: 55-62 Dopke W, Duday S, Matos N (1987) The isolation and identification of alkaloids and steroids from Solanum nigrum. Zeitschrift für Chemie 27: 64-64 Ehrlich PR, Raven PH (1964) Butterflies and plants - a study in coevolution. Evolution 18: 586-608 Fraenkel GS (1959) Raison d'etre of secondary plant substances. Science 129: 1466-1470 Gang DR, Beuerle T, Ullmann P, Werck-Reichhart D, Pichersky E (2002) Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases. Plant Physiology 130: 1536- 1544 Gershenzon J (2002) Secondary Metabolism and Plant Defense. In L Taiz, E Zeiger, eds, Plant Physiology, Ed Third Edition. Sinauer Associates, Inc., Sunderland, pp 283-308 Halitschke R, Gase K, Hui D, Schmidt DD, Baldwin IT (2003) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VI. Microarray analysis reveals that most herbivore- specific transcriptional changes are mediated by fatty acid-amino acid conjugates. Plant Physiol. 131: 1894-1902
103 MS 4 – Solanaceous responses to herbivory
Heidel A, Baldwin IT (2004) Microarray analysis of SA- and JA- signaling in Nicotiana attenuata's responses to attack by insects from multiple feeding guilds. Plant, Cell and Environment in press Hermsmeier D, Schittko U, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. I. Large-scale changes in the accumulation of growth- and defense-related plant mRNAs. Plant Physiology 125: 683-700 Jones CG, Firn RD (1991) On the evolution of plant secondary chemical diversity. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 333: 273-280 Kahl J, Siemens DH, Aerts RJ, Gabler R, Kuhnemann F, Preston CA, Baldwin IT (2000) Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore. Planta 210: 336-342 Kessler A, Baldwin IT (2001) Defensive function of herbivore- induced plant volatile emissions in nature. Science 291: 2141- 2144 Kessler A, Baldwin IT (2004) Herbivore-induced plant vaccination. Part I. The orchestration of plant defenses in nature and their fitness consequences in the wild tobacco Nicotiana attenuata. Plant Journal 38: 639-649 Krugel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002) Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology 12: 177-183 Kutchan TM (2001) Ecological arsenal and developmental dispatcher. The paradigm of secondary metabolism. Plant Physiology 125: 58-60 Lantin S, O'Brien M, Matton DP (1999) Pollination, wounding and jasmonate treatments induce the expression of a developmentally regulated pistil dioxygenase at a distance, in the ovary, in the wild potato Solanum chacoense Bitt. Plant Molecular Biology 41: 371- 386 Leon J, Rojo E, Sanchez-Serrano JJ (2001) Wound signalling in plants. Journal of Experimental Botany 52: 1-9 Lichtenthaler HK (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annual Review of Plant Physiology and Plant Molecular Biology 50: 47-65 Mitchell-Olds T (2001) Arabidopsis thaliana and its wild relatives: a model system for ecology and evolution. Trends in Ecology and Evolution 16: 693-700
104 MS 4 – Solanaceous responses to herbivory
Mittova V, Tal M, Volokita M, Guy M (2002) Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species. Physiologia Plantarum 115: 393-400 Pearce G, Moura DS, Stratmann J, Ryan CA (2001) Production of multiple plant hormones from a single polyprotein precursor. Nature 411: 817-820 Ridout CL, Price KR, Coxon DT, Fenwick GR (1989) Glycoalkaloids from Solanum nigrum L., alpha-solamargine and alpha-solasonine. Pharmazie 44: 732-733 Roda AL, Oldham NJ, Svatos A, Baldwin IT (2003) Allometric analysis of the induced flavonols on the leaf surface of wild tobacco (Nicotiana attenuata). Phytochemistry 62: 527-536 Ryan CA (2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology 1477: 112-121 Scheer JM, Pearce G, Ryan CA (2003) Generation of systemin signaling in tobacco by transformation with the tomato systemin receptor kinase gene. Proc Natl Acad Sci USA. 100: 10114– 10117 Schmidt DD, Kessler A, Kessler D, Schmidt S, Lim M, Gase K, Baldwin IT (2004) Solanum nigrum: A model ecological expression system. Molecular Ecology 13: 981-995 Tu CJ, Park SY, Walling LL (2003) Isolation and characterization of the neutral leucine aminopeptidase (LapN) of tomato. Plant Physiology 132: 243-255 Van Dam NM, Horn M, Mares M, Baldwin IT (2001) Ontogeny constrains systemic protease inhibitor response in Nicotiana attenuata. Journal of Chemical Ecology 27: 547-568 Voelckel C, Baldwin IT (2003) Detecting herbivore-specific transcriptional responses in plants with multiple DDRT-PCR and subtractive library procedures. Physiologia Plantarum 118: 240- 252 Voelckel C, Baldwin IT (2004) Herbivore-induced plant vaccination. Part II. Array-studies reveal the transience of herbivore-specific transcriptional imprints and a distinct imprint from stress combinations. Plant Journal 38: 650-663 Walling LL (2000) The myriad plant responses to herbivores. Journal of Plant Growth Regulation 19: 195-216. Walling LL, Pautot V, Gu YQ, Chao WS, Holzer FM (1995) Leucine aminopeptidase - a complex array of proteins induced
105 MS 4 – Solanaceous responses to herbivory
during the plant defense response. Journal of Cellular Biochemistry: 137-137 Winz RA, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-induced ethylene reduces jasmonate-induced nicotine accumulation by regulating putrescine N-methyltransferase transcripts. Plant Physiology 125: 2189-2202
106 MS 4 – Solanaceous responses to herbivory
Supplementary figure 1. Hierarchical cluster ana- lysis of the replicate microarrays (NA1-3 = N. attenuata; SN1-3 = S. nigrum) based on the expression ratios of the significantly regulated genes (Supplementary Table 1). The pattern of up- and down-regulated genes contained sets of similarly regulated genes, but clearly distinguished the transcriptional re- sponses of both species to attack from the same Lepidopteran herbivore.
107
108 6. General discussion
6. General discussion The central question was to study how intra- and interspecific competition influences inducible defense traits of S. nigrum. Here I discuss the main results of the presented manuscripts: (1) The establishment of molecular and ecological tools for the native species, S. nigrum; (2) The general response of S. nigrum to herbivory from different insects; (3) Responses of S. nigrum to competition in general and to specific competitors; (4) Lastly, the integrated response of S. nigrum to competition and herbivory.
The toolbox for S. nigrum A common way of studying plant-insect interactions is to manipulate the interacting partners for example by controlling the number of herbivores on a plant (MS4; Fig. 1-4). If it is known which signals mediate the response to a certain herbivore, these can be elegantly used as chemical elicitors to trigger a similar suite of responses with the great advantage of easier standardization of the treatment (Thaler et al. 1996; Baldwin 1998). S. nigrum was shown to activate the oxylipin signaling cascade in response to herbivory (MS1, 4) and, hence, methyl jasmonate (MeJA) could be used as standardized ‘herbivore’ treatment that elicits a comparable suite of responses as herbivory does (MS1; Fig. 6-4). However, the use of chemical elicitors may cause pleiotropic effects that make the interpretation of the results difficult. The solution for this problem is a ‘bottom-up’ manipulation of interactions using transformation. Yet, transformation protocols developed for one model system can be difficult to transfer to other systems without substantial investment in technique development. Most techniques have been developed for agronomically and economically important organisms and cannot be readily applied to wild relatives. The establishment of an efficient Agrobacterium-based transformation system for S. nigrum (MS1) provides a powerful tool for stably manipulating the genetic basis of ecologically important traits. Additional techniques for transient transformation, such as virus-induced gene silencing (VIGS), are now also available for S. nigrum (Brignetti et al. 2004), but are
109 6. General discussion impossible to use for field-based ecological research due to legal restrictions in Germany. With the ability to transform the native species, S. nigrum, the next task was to decide which genes to manipulate and how to interpret the responses to the manipulations. Microarrays allow biologists to examine the expression of hundreds or thousands of genes simultaneously, and their use in combination with elicitation studies provides a powerful means of identifying ‘suspect’ genes relevant for ecological interactions (Korth 2003; Thomas & Klaper 2004). In the studies presented here I used three microarrays that were different regarding the number of spotted genes, namely 60 or 568 genes on the two small-scale oligonucleotide microarrays (MS1-3) and 11,243 clones on the TIGR potato cDNA microarray (MS4). Even though only few of the sequences on this ‘Solanaceous’ microarray were designed from S. nigrum specific- sequences, it is clear from the verifications of selected array responses by RNA gel blot analysis and real-time qPCR (MS1; MS2) that the microarrays provided valuable information about differential gene expression in S. nigrum. Remarkably, the expression patterns of field-grown plants largely reflected the responses of glasshouse-grown plants (MS2). Field conditions led to the up-regulation of few stress-responsive genes, but the characteristic transcriptional patterns that were found in glasshous experiments were largely retained and not confounded by environmental effects.. Hence, array techniques can be successfully used for field-based ecological experiments. Ecologists frequently manipulate complicated biotic interactions in the field, but rarely consider using the established array technology to characterize the transcriptional responses to these interactions. Microarray platforms that have been publicly funded (e.g. http://www.tigr.org/tdb/potato/microarray2.shtml) offer ecologists a means of rapidly identifying molecular targets that could be manipulated in complex interactions. The successful establishment of molecular techniques for S. nigrum facilitated the analysis and manipulation of genes relevant for biotic interactions.
110 6. General discussion
S. nigrum’s responses to herbivory For the study of plant-herbivore interactions it is essential to know which insects attack or visit the studied plant species and how the plant responds to its attackers. S. nigrum is attacked by a variety of herbivorous insects that leave distinct phenotypic and transcriptional imprints (Fig. 6- 1, MS1,4). Flea beetles (Epitrix pubescens) that are specialized on Solanaceous species were among the often-observed insect herbivores that cause considerable damage on S. nigrum in the field sites around Jena (Fig. 6-1B,C). The response of S. nigrum to E. pubescens is characterized by the strong up-regulation of defense-related genes that is correlated to the increased production of direct (TPIs) and indirect defenses (VOCs; MS1; Fig. 6-2A, 6-4C). Interestingly, flea beetle-induced VOC emissions quantitatively and qualitatively differed from those induced by caterpillars, such as Acherontia atropos or Manduca sexta. Despite the phenotypic differences that are caused by special herbivores, the signal processes mediating these responses appeared to be similar. Consistent with a regulatory role of JA signaling in insect-induced plant responses (Blee 2002; Farmer et al. 2003), genes coding for oxylipin biosynthetic enzymes are up-regulated in flea beetle- and caterpillar- infested S. nigrum plants. However, if plants that are directly elicited with the signal compound, namely the methyl ester of JA, MeJA, the gene expression analysis explained most of the herbivore-induced transcriptional changes with some exceptions(MS1,2; Fig. 6-4A,C). It is known that compounds from oral regurgitants of herbivores play an important role in eliciting an adapted response of a plant (Halitschke et al. 2001) and induction using MeJA omits those specific elicitors. Flea beetles additionally induced up-regulation of several pathogenesis- related (PR) genes and of genes involved in ethylene (ET) synthesis and perception. Hence, from the microarray analysis it is clear that S. nigrum does not rely on a linear signal pathway in response to flea beetle herbivory, but activates additional signal cascades, such as ethylene signaling and putatively salicylic acid (SA) signaling; the latter could explain the increased transcript levels of PR genes. This is consistent with the increasing evidence that JA and SA not necessarily act
111 6. General discussion
Figure 6-1. Feeding patterns of different insect herbivores that attack S. nigrum. (A) Intact leaf. (B-F) Leaves that are infested by (B,C) Flea beetles (Epitrix pubescens). (D) Colorado potato beetle (Leptinotarsa decemlineata). (E) Tomato hornworm (Manduca sexta). (F) Aphids.
antagonistically. Schenk et al. (2001) found 25% of 2,375 genes co- regulated when they treated Arabidopsis thaliana with JA or SA. In N. attenuata, SA levels uniformly increased in response to different herbivores and the SA mimic, BTH (benzo[1,2,3]thiadiazole-7- carbothioic acid-S-methyl ester), induced SA- and JA-elicited genes (Heidel & Baldwin 2004). Hence, the commonly invoked tradeoff between SA-mediated resistance of plants to microorganisms and JA- mediated resistance to insect herbivores (Felton et al. 1999; Thaler et al. 2002) needs to be refined because of the complex interrelationships between these signaling pathways. Transcription factors may play an important role in coordinating these many signal cascades. In S. nigrum attacked by flea beetles, a transcription factor of the WRKY family (WRKY3) was strongly up- regulated, as it was shown to be in N. attenuata attacked by M. sexta larvae (Hui et al. 2003). WRKY transcription factors occur in large gene families and are known to regulate numerous stress-related genes, including those responsive to pathogens and wounding (Eulgem et al. 2000). Recently, the WRKY70 transcription factor has been identified as common compound of JA- and SA signaling (Li et al. 2004). The large-
112 6. General discussion scale transcriptional analysis of M. sexta-infested S. nigrum plants revealed more transcription factors, such as WRKY65, SCARECROW, KNAT3, etc., that are important candidates for central regulatory elements in plants attacked by herbivores (MS4). Notably, the suite of signals and regulatory elements that S. nigrum activated in response to M. sexta attack significantly differed from the response profile of another Solanaceous species, N. attenuata, that was attacked by the same herbivore in an identically designed experiment (MS4). Responses to wounding and herbivore attack are known to differ among plant families, but both N. attenuata and S. nigrum belong to the family of Solanaceae, for which a more common response might have been expected (Walling 2000; Leon et al. 2001). However, only 10% of almost 800 repressed or induced genes were co-regulated in both species (MS4). Apart from expected differences, such as induction of nicotine biosynthesis in N. attenuata and activation of genes leading to glycoalkaloids in S. nigrum, mono-, sesqui-, and diterpenoid metabolism showed distinct regulation and genes involved in the biosynthesis of phenolic compounds were up-regulated solely in N. attenuata. In addition to the mentioned differential expression of transcriptional regulators, furthermore, the activation of calcium/calmodulin- and inositol/phospholipid-based signaling as well as G-protein-mediated signaling was restricted to N. attenuata attacked by M. sexta. In general, S. nigrum activated significantly less genes than N. attenuata in response to M.sexta, which naturally feeds on both species (Fraenkel 1959). It remains to be elucidated, whether this lack of response in S. nigrum is due to its adaptation to M. sexta or vice versa. Despite the clear differences of N. attenuata and S. nigrum with respect to signal transduction and secondary metabolism, both species share similarities in their transcriptional responses to herbivory that extended beyond the up-regulation of stress-responsive transcripts to include a coordinated down-regulation of growth-related transcripts, which is most clearly seen in the negative correlation between the expression of photosynthetic genes and that of defense genes (Hermsmeier et al. 2001; Schittko et al. 2001; MS1-3; Fig. 6-2B). It is
113 6. General discussion important to note that the transcriptional changes in MeJA-elicited S. nigrum plants provided a reliable prediction for the fitness consequences; MeJA-elicited plants produced significantly less fruits than non-elicited control plants (MS1; Fig. 6-2B). However, MeJA did not reduce plant
Figure 6-2. Summary of effects of herbivores and competitors (separately and in combination) on S. nigrum. Herbivores or MeJA elicitation induced defense responses, such as the production anti-digestive proteins (TPIs) and the emission of VOCs (A); concomitantly, herbivory or MeJA caused a down-regulation of genes that are related to growth, such as photosynthetic genes (B), and this negative effect was greater when plants competed (J). Competition alone also repressed photosynthesis-related genes (H) and led to up-regulation of stress-related genes, for example when S. nigrum competed with Plantago lanceolata (F); this effect was stronger, if plants that were competing with P. lanceolata were induced with MeJA. These negative effects of herbivory, MeJA or competition invoked fitness costs, such as decreased fruit numbers or decreased biomass (B, H, J). If S. nigrum was induced with MeJA at an early developmental stage, no fitness costs were detectable at the end of reproductive development (C). At an early stage of competition, S. nigrum activated signaling genes mediating growth responses (G), but defense- related traits were not influenced by competitors (D). However, in mature plant communities, defense traits of S. nigrum were negatively influenced in a competitor-dependent manner (E).
114 6. General discussion height (Fig. 6-2C), a conclusion Thaler et al. (1996) had reached with regard to JA treatment of tomato. The array of differential responses of Solanaceous plants emphasizes the importance of extending the range of model species to disentangle the complexity and diversity of plant responses to insect herbivores.
S. nigrum’s responses to competition Competition-mediated responses of S. nigrum were studied using two approaches; (1) S. nigrum was grown in presence of three intra- or interspecific competitors in field and glasshouse mesocosms. (2) S. nigrum was planted into plots of the ‘Jena biodiversity experiment’ (Roscher et al. 2004), in which the plant diversity was factorially manipulated to study the role of biodiversity for element cycling and trophic interactions. The analysis of plants grown in different competitive environments allowed to determine effects common to competition in general and, furthermore, facilitated the identification of effects that are specific for competition regimes of different functional diversity. Using S. nigrum as a focal plant in a range of competition experiments revealed effects that were common to competition in general (MS2). The transcriptional analysis using a 568-gene array facilitated the identification of signaling pathways, namely ET and gibberellin signaling, that are activated in S. nigrum at an early stage of competition, when the canopy space is not fully occupied by the competing plants. ET is known to have multiple and diverse effects in all aspects of plant life (Wang et al. 2002) and has recently been recognized as an important mediator of competitive interactions. ET-insensitive tobacco plants, which are unresponsive to blue light signals from a competitor’s canopy, perform poorly when competing against wildtype neighbors (Pierik et al. 2003, 2004). Plants use these blue light signals in addition to the alterations in red:far-red ratios (R:FR) to adjust their growth responses and increase competitive ability (Ballaré 1999). Biosynthesis and catabolism of GA, which profoundly alters stem growth and competitive ability, is also influenced by light signals (García- Martinez & Gil 2002). The molecular mechanisms by which a plant
115 6. General discussion responds to external signals that signify competitive environments, such as light and possibly ET, to trigger endogenous hormone cascades that finally alter the phenotype in an adaptive manner remain to be determined. Despite similar effects of different competitors at an early stage competitive interaction, the picture was modified, when the mesocosms had developed dense canopy structures. The species I used as competitors differed significantly in their life histories. S. nigrum is a fast- growing annual; these are usually superior competitors in high-light conditions (Callaway et al. 2003). P. lanceolata as well as L. perenne and T. pratense are perennials, which develop more slowly and therefore provide a different competitive environment in comparison to intraspecific competition. The phenotypic consequences (Fig. 6-2G,H) invoked by differential competitors were evident in field-and glasshouse-grown plants. Plant height and subsequent lifetime fruit production of S. nigrum varied according to species (MS3; Fig. 6-3). Intraspecific competition decreased fruit production most in comparison to interspecific competition. In the field the competing S. nigrum grew taller than non- competing plants (Fig. 6-2G) and, hence, showed a typical shade- avoidance response, which is a well-documented example of density- dependent plasticity (Dudley & Schmitt 1995). The differences between GH- and field-grown plants (Fig. 6-3) are most likely due to pot-bound effects, namely stronger nutrient depletion and less rooting space in pots compared to field plots; such effects have for example been demonstrated for N. sylvestris plants, which lost their ability to increase nicotine production in response to leaf damage when the plants were grown in pots (Baldwin 1988). After having found effects of different competitors on growth- related traits of S. nigrum the next task was to determine, whether this effect was related to the species diversity or to the functional diversity of competitors. Therefore we transplanted S. nigrum into plots of the ‘Jena Biodiversity Experiment’ (Roscher et al. 2004) in which 60 grassland species were factorially manipulated and found a correlation of growth- related traits (plant height, above-ground dry mass) to morphological
116 6. General discussion
traits of the competitors. Hence, life history differences between S. nigrum and its interspecific competitors drove the distribution of plant height, which is consistent with results of other studied. Size asymmetries are important in competitive interactions, as was found for the establishment of Solidago seedlings in different sized vegetation openings (Goldberg & Werner 1983). To test whether this effect is species-dependent, Tremmel & Bazzaz (1995) grew target plants in the presence of species having different morphologies; they found that the plant morphology resulting in different above-ground biomass allocation patterns was responsible for observed changes in the target plants, rather than the species identity of the neighbors. However, the transcriptional analysis had not only identified genes commonly activated in plants grown under different competition regimes, but it also pointed to responses that are likely to be specific to growth with particular competitors (MS2). The most strikingly different response was observed when S. nigrum competed with P. lanceolata. Under A B a 8000 GH plants 100 GH plants b field plants c 6000 field plants b b 4000 80 ab A A c AB C BC 60 2000 A
40 Fruits / plant 600 Plant height (cm) B B 400 C 20 200
0 0 no competition S. nigrum P. lanceolata 3 species no competition S. nigrum P. lanceolata 3 species
Competition Competition Figure 6-3. Fitness estimates for S. nigrum, growing under different levels of competition in glasshouse (GH) and field mesocosms (3 species: S. nigrum competed with Lolium perenne, P. lanceolata and Trifolium pratense; for detailed description of the experimental design see MS2). Significance levels are shown by different letters (capitals for the GH experiment; lower-case letters for the field experiment). (A) Plant height differed significantly against the different levels of competition (ANOVA; GH: F=11.59, p<0.0001; field: F=23.38, p<0.0001); (B) Fruit numbers were significantly lower in competing plants compared to control plants (ANOVA with Tukey-Kramer HSD post hoc-test; GH: F=19.42, p<0.0001; field: F=18.91, p<0.0001).
117 6. General discussion this competitive regime, an additional suite of genes was elicited, which are thought to be involved in responses to stresses, such as herbivory. It is tempting to speculate that there may be species-specific interactions, but they need to be verified. In summary, S. nigrum showed distinct responses to differences in the competitive regime
S. nigrum’s integrated response to herbivory and competition Studies on interactive effects of competition and herbivory have revealed that plants are less defended when they grow under competition (Cipollini & Bergelson 2001; Gurevitch et al. 2000). It is clear that conditional responses, such as induction of defenses, are strongly influenced by the plant’s physiological condition and resource-based trade-offs between growth- and defense-related traits are well- documented (Baldwin 1998; Lou and Baldwin 2004; Schmelz et al. 2003). However, the coordination of plant responses to competition and herbivory are also thought to be achieved by hormonal linkages (Cipollini 2004). In MS2 and 3, I present transcriptional evidence for both mechanisms and, lastly, ask whether diverse competitive environments cause differences in the expression of defense-related traits. Competition and herbivory (separately and in combination) negatively influenced growth-related traits in S. nigrum. That was most clearly seen in the down-regulation of photosynthesis- and glycolysis- related genes in response to flea beetle herbivory, to MeJA elicitation and to competition (MS1-3; Fig. 6-4; Fig. 6-2J). This effect was pronounced, if competition and MeJA elicitation were combined, which is consistent with the finding that competition reinforces the effects of herbivory (Müller-Schärer 1991; McEvoy et al. 1993; Gurevitch et al. 2000). Apart from overlapping responses of S. nigrum to these two biotic stresses, there were significant differences in the transcriptional patterns. Competing plants upregulated genes related to ET- and GA-signaling only when they were not elicited by MeJA. For herbivore resistance traits, ET is known to positively and negatively interact with oxylipin-
118 6. General discussion mediated responses. For example, JA and ET synergistically activate defense-related genes (O'Donnell et al. 1996; Penninckx et al. 1996) and many genes induced by ET are also elicited by MeJA in A. thaliana (Schenk et al. 2000). ET is an important regulator in competitive interactions (see above), suggesting that the responses mediated by oxylipins, ET and GA could account for changes in growth and defense processes in competing plants. Moreover, MeJA and competition had opposing effects on genes involved in nitrate metabolism and cell wall
Figure 6-4. Venn diagram of overlapping and non-overlapping transcriptional responses of S. nigrum to flea beetle herbivory, MeJA elicitation and the combination of competition and MeJA elicitation. Approximately 50% of the genes up-regulated by flea beetle herbivory were commonly up-regulated by MeJA, suggesting that oxylipin signaling is important for mediating defense responses in S. nigrum. If MeJA-elicited plants were grown under competition, several photosynthesis-related genes were down-regulated as they were in flea beetle-infested plants indicating that down-regulation of photosynthesis may be a general response of S. nigrum to biotic stresses. S. nigrum up-regulated a nitrate transporter gene in responses to MeJA elicitation and flea beetle herbivory only when plants were not competing; this indicates a resource-based interaction of competition and herbivory. The analysis integrates data of MS1 and MS2; annotated gene lists are in the corresponding MSs or available on the supplementary CD-ROM.
119 6. General discussion biosynthesis indicating resource-based interactions of competition and MeJA. In conclusion, the transcriptional analysis of S. nigrum revealed insights into the molecular basis of the often-invoked trade-off between growth- and defense-related traits. After the discovery of interactive effects of MeJA elicitation and competition on a transcriptional level, the next task was to determine whether induced defense responses are influenced by the biodiversity of competitors (MS3). Microarray analysis combined with multivariate statistics proved as extremely useful tool for detecting effects of different competitive regimes. Principal component analysis (PCA) identified a pattern in the MeJA-induced transcriptome when the microarrays were classified by the canopy height classes of the competing plant species. Photosynthesis- and defense-related genes were differentially regulated in plants grown with short canopy competitors as compared to those grown with medium or tall canopy competitors. The concentration of defense proteins trypsin proteinase inhibitors (TPIs) were correlated with the expression of TPI transcripts and increased significantly in response to MeJA elicitation; however, the increase in TPI levels was attenuated in plants grown with short or tall competitors. Reduced TPI levels are most likely the effect of resource competition as has been demonstrated for Brassica napus and Nicotiana attenuata. The decrease of TPIs in B. napus in response to intraspecific competition could be buffered if plants were grown under conditions of high nutrient availability (Cipollini and Bergelson 2001). In N. attenuata lower N availability was correlated to lower TPI production in response to MeJA or herbivory (Lou and Baldwin 2004). The attenuation of this nitrogen-intensive defense was associated with increases in the expression of genes involved in the biosynthesis of C-based defense metabolites (sesquiterpenes). The morphological characteristics of interspecific competitors had a stronger effect on S. nigrum’s transcriptional and TPI responses to MeJA elicitation than the number of competing species, suggesting that functional categorizations are useful in characterizing the influence of biodiversity on plant-herbivore interactions.
120 7. Summary
7. Summary Plants evolved different strategies to cope with competition and herbivory, which are two of the most important selective pressures for plants. While the traits responsible for herbivore resistance have long been studied and are known to include complex adaptations involving direct and indirect defenses, little is known about the molecular basis of competitive ability. In this study, S. nigrum has been established as an ecological model system to determine effects of competition on inducible defense traits. For this purpose, a number of molecular and ecological tools were developed and their usefulness for studying ecological interactions was verified under glasshouse and field conditions. Gene expression profiling using microarrays identified many target genes for manipulative studies of ecological interactions. In order to study the interactive effect of competition and herbivory on S. nigrum, it first was necessary to characterize the responses to either stress alone. S. nigrum induced a suite of direct (glycoalkaloids, TPIs) and indirect (VOCs) defenses in response to different herbivores that triggered complex signaling networks involving oxylipins and ethylene. This response could be largely mimicked by applying MeJA to the plants. Ethylene is also involved in mediating competitive interactions of S. nigrum and it could be demonstrated that MeJA inhibits transcription of genes involved in ethylene and gibberellin signaling. The interaction on the signaling level translated into opposite regulation of genes related to nitrogen metabolism in competing- and MeJA-elicited plants indicating the involvement of resource-based mechanisms in the defense-growth tradeoff. The transcriptional effects reliably predicted phenotypic consequences (fitness costs) of competition and herbivory. After the discovery of common effects of competition, it could be demonstrated that the expression of jasmonate-inducible traits is influenced by morphological traits of the competitors rather than by their species diversity. The regulated genes, especially the unknowns, provide targets for future manipulative studies of plants in natural environments as to fully integrate the power of ecology and molecular biology. 121
8. Zusammenfassung
8. Zusammenfassung Pflanzen sind in ihrer natürlichen Umwelt umgeben von Konkurrenten und Herbivoren. Dies hat dazu geführt, dass Pflanzen ausgeklügelte Abwehr- und Konkurrenzstrategien entwickelt haben. Während die Grundlagen der Resistenz gegen Herbivorie zunehmend aufgeklärt werden, liegen die molekularen Grundlagen der pflanzlichen Konkurrenzkraft weitgehend im Verborgenen. Da Pflanzen am natürlichen Standort oft gleichzeitig von Schädingen befallen und Konkurrenten beeinträchtigt werden, ergeben sich interaktive Effekte von Konkurrenz und Herbivorie. Diese werden durch die den Pflanzen zur Verfügung stehenden Resourcen bestimmt oder durch direkte Verbindungen der involvierten Hormonkaskaden vermittelt. Ziel dieser Dissertation war es, Effekte von Konkurrenz auf induzierbare Abwehrmechanismen von Pflanzen zu untersuchen und dazu wurde das Modellsystem S. nigrum entwickelt. S. nigrum, ein wilder Verwandter wichtiger Kulturpflanzen wie z.B. Kartoffel oder Tomate, wächst in Pflanzengemeinschaften, in denen wechselnde Konkurrenzbedingungen herrschen, und wird dort durch einer Vielzahl von Insektenarten befallen. Um die molekularen Grundlagen der Herbivoren-Resistenz und des Konkurrenzverhaltens von S. nigrum untersuchen zu können, war es zuerst notwendig, molekularbiologische Techniken für S. nigrum zu entwickeln und ihre Aussagekraft im Zusammenhang ökologischer Studien zu überprüfen. Im nächsten Abschnitt der Arbeit wurden die Reaktionen von S. nigrum auf Herbivorie und die Effekte von Konkurrenz auf S. nigurm getrennt charakterisiert. Auf diesen Grundlagen aufbauend wurde schliesslich untersucht, wie Konkurrenz und Herbivorie interaktiv auf S. nigrum wirken und welche Mechanismen zu der postulierten Interaktion führen. Eine zentrale Frage war hierbei, ob die Biodiversität der konkurrierenden Pflanzen einen Effekt auf S. nigrum hat. Moderne molekularbiologische Methoden sind anwendbar für ökologische Feldforschung. Microarrays stellen eine sensitive Technik zur Expressionsanalyse dar mit dem Vorteil, viele 122
8. Zusammenfassung
Gene gleichzeitig untersuchen zu können. Diese Technik wurde mit großem Erfolg in einer Vielzahl von Laborstudien bei der Identifzierung von Genen eingesetzt, die essentielle Rollen in der Resistenz von Pflanzen gegen Schädlinge spielen. Ausserdem konnten dadurch transkriptionelle Verknüpfungen zu anderen für die Pflanze wichtigen Prozessen aufgezeigt werden. Da die Bedingungen in Feldexperimenten im Vergleich zu klimatistierten Gewächshäusern “außer Kontrolle” sind, bestand die Frage, ob die transkriptionelle Analyse von Pflanzen im Feld die Ergebnisse von Gewächshausstudien widerspiegelt. Beim Vergleich von elizitor-induzierten Pflanzen konnte gezeigt werden, dass trotz der Aktivierung einiger stressinduzierbarer Gene Feld-Pflanzen weitestgehend das gleiche Transkriptionsmuster wie Gewächshauspflanzen aufwiesen. Zudem können durch die Etablierung einer Agrobacterium-basierten Transformation die regulierten Gene in S. nigrum manipuliert werden, um ihre Signifikanz in ökologischen Interaktionen zu untersuchen. S. nigrum aktiviert als Reaktion auf Herbivorenbefall die Produktion von direkten und indirekten Abwehrstoffen, wobei letztere von der Art des Herbivors abhängen. Pflanzen haben inzuzierbare Abwehrstrategien entwickelt, um Resourcen in Gemeinschaften mit geringem Herbivorendruck zu sparen. Bei Befall durch Flohkäfer (Epitrix pubecens) oder Tabakschwärmer (Manduca sexta) schaltet S. nigrum Gene der Steroidalkaloid-Biosynthese an und produziert erhöhte Mengen an Trypsin-Proteinaseinhibitoren (TPIs), die verdauungshemmend im Darm von Insekten wirken können. Da jedoch angepasste Herbivoren wie z.B. E. pubescens nicht durch TPIs beeiflußt werden, schaltet S. nigrum zusätzlich die Emission von Duftstoffen (flüchtige Alkohole und Terpene) an, die als Anlockung von Prädatoren oder Parasitoiden der Herbivoren wirken. Die Spektren an emittierten Stoffen hängen vom Herbivoren ab, der S. nigrum befällt. Die Aktivierung der Abwehr gegen Herbivorie in S. nigrum erfolgt über die Oxylipin-Kaskade und weitere Signalwege. Die transkriptionelle Analyse von durch Flohkäfer
123
8. Zusammenfassung befallenen und mit Methyljasmonat (MeJA) behandelten Pflanzen hat ergeben, dass die Oxylipin-Signalkaskade eine zentrale Rolle in der Vermittlung der Reaktion von S. nigrum auf Herbivore darstellt. Deshalb wurde bei weiteren Experimenten MeJA als standardisierter und leicht zu dosierender “Herbivor” eingesetzt. Zusätzlich zu Oxylipin-Signalen spielen auch noch andere Signale wie Ethylen und wahrscheinlich Salicylsäure bei der Herbivoren-Resistenz von S. nigrum eine Rolle. Transkriptionsfaktoren wie z.B. WRKY, die ebenfalls durch Herbivorie aktiviert werden, sind in der übergeordneten Regulation der Signalnetzwerke von Bedeutung. Die Reaktion von S. nigrum auf Herbivorie unterscheidet sich deutlich von der anderer Vertreter der Solanaceae. Der Vergleich der Expression von über 11.000 Genen in S. nigrum und Nicotiana attenuata, die beide von Manduca sexta befallen waren, hat gezeigt, dass nur 10% der Herbivor-induzierten Gene in beiden Pflanzenarten koreguliert waren. Deutliche Unterschiede waren vor allem in Signaltranduktions-Genen und Genen des Sekundärmetabolismus zu finden. S. nigrum aktivierte generell weniger Gene als N. attenuata. Konkurrenz im allgemeinen induziert die Expression von Ethylen-bezogenen Genen. Pflanzen bemerken Konkurrenten, lange bevor der Kampf um die Nährstoffe beginnt. Die Lichtverhältnisse und Ethylen spielen hierbei als Signale eine wichtige Rolle. Letzteres konnte durch die Transkriptionsanalyse von S. nigrum, der in Gegenwart verschiedener Konkurrenten wuchs, belegt werden. Gene, die involviert sind in die Biosynthese und Wahrnehmung von Ethylen, werden durch Konkurrenz aktiviert. Ausserdem induzierte Konkurrenz in S. nigrum einen in die Regulation von Gibberellinsäure involvierten Transkriptionsfaktor, der auf die Aktivierung von Wachstumsreaktionen hinweist. Phänotypische Effekte von Konkurrenz auf S. nigrum ist von der Art der Konkurrenten abhängig. Das Wachstum und nachfolgend die Fruchtproduktion als wichtiges Indiz für die Fitness von
124
8. Zusammenfassung
S. nigrum war abhängig von Typ der Konkurrenz. Als wichtig erwies sich hier der funktionelle Typ an Konkurrenten und nicht die Artzugehörigkeit. Am grössten war der Fitnessverlust bei Pflanzen, die in Gegenwart von hochwüchsigen Pflanzen wuchsen. Das Oxylipin Methyl-Jasmonat hemmt konkurrenz- induzierte Signale in S. nigrum. Die Interaktion von Konkurrenz und Herbivorie findet bereits auf der Ebene der Signaltransduktion statt. Konkurrierende Pflanzen konnten Ethylen- und Gibberellinsäure- Signale nur aktivieren, wenn sie nicht mit MeJA behandelt wurden. Die durch MeJA induzierte Oxylipin-Signalkaskade scheint daher in S. nigrum antagonistisch zu durch Konkurrenz angeschalteten Ethylen- und Gibberellinsäure-Signalen zu wirken. Die Ethylenbiosynthese wurde auch durch Herbivorie von E. pubescens angeregt und verdeutlicht somit, daß die Feinregulation der Reaktion auf Herbivorie und Konkurrenz in S. nigurm hauptsächlich durch Zusammenspiel von Oxylipin-, Ethylen- und Gibberellinsäure-Signalen erreicht wird. Konkurrenz beeinflusst induzierbare Abwehr- reaktionen von S. nigrum in Abhängigkeit von der Art der Konkurrenten. Die durch MeJA induzierte Genexpression und Produktion von TPIs in S. nigrum wurde signifikant durch verschiedene Konkurrenz-Regimes beeinflusst. Jedoch war dieser Effekt nicht von der Artzugehörigkeit bzw. der reinen Artzahl (Biodiversität) der Konkurrenten abhängig, sondern stand in Bezug zu morphologischen Eigenschaften. Hierbei waren die Gehalte an TPIs in S. nigrum in Gemeinschaften mit kleinen und hohen Pflanzen verringert. Die korrelierte Genexpression lieferte Indize, daß die Reaktion von S. nigrum durch verringerte Stickstoff-Verfügbarkeit beeinflusst wurde.
125
9. References
9. References
Agrawal AA (2001) Phenotypic plasticity in the interactions and evolution of species. Science 294, 321-326. Aslanov SM (1971) Glycoalkaloids of Solanum nigrum. Khimiya Prirodnykh Soedinenii, 674. Bais HP, Park SW, Weir TL, Callaway RM, Vivanco JM (2004) How plants communicate using the underground information superhighway. Trends in Plant Science 9, 26-32. Baldwin IT (1988) Damage-induced alkaloids in tobacco - pot-bound plants are not inducible. Journal of Chemical Ecology 14, 1113-1120. Baldwin IT (1998) Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences of the United States of America 95, 8113-8118. Ballare CL (1999) Keeping up with the neighbours: phytochrome sensing and other signalling mechanisms. Trends in Plant Science 4, 97-102. Beg MZ, Khan AH (1988) The mechanism of speciation and evolutionary trend in Solanum nigrum complex. Cytologia 53, 297-306. Begon M, Harper JL, Townsend CR (1996) Ecology: Individuals, Populations and Communities Blackwell Science, Oxford. Benidze MM (1994) Steroid glycosides of Solanum nigrum. Khimiya Prirodnykh Soedinenii, 683-684. Blee E (2002) Impact of phyto-oxylipins in plant defense. Trends in Plant Science 7, 315-321. Bonser SP, Reader RJ (1995) Plant competition and herbivory in relation to vegetation biomass. Ecology 76, 2176-2183. Brigneti G, Martin-Hernandez AM, Jin H, et al. (2004) Virus-induced gene silencing in Solanum species. Plant Journal 39, 264-272. Buchmann SL (1983) Buzz pollination in angiosperms. In: Handbook of Experimental Pollination Biology (ed. Jones CE), pp. 73-113. Van Nostrand Reinhold, New York. Callaway RM, Pennings SC, Richards CL (2003) Phenotypic plasticity and interactions among plants. Ecology 84, 1115-1128. Cipollini ML, Levey DJ (1997) Secondary metabolites of fleshy vertebrate- dispersed fruits: Adaptive hypotheses and implications for seed dispersal. American Naturalist 150, 346-372.
126
9. References
Cipollini DF, Bergelson J (2001) Plant density and nutrient availability constrain constitutive and wound-induced expression of trypsin inhibitors in Brassica napus. Journal of Chemical Ecology 27, 593-610. Cipollini DF (2002) Does competition magnify the fitness costs of induced responses in Arabidopsis thaliana? A manipulative approach. Oecologia 131, 514-520. Cipollini DF, Bergelson J (2002) Interspecific competition affects growth and herbivore damage of Brassica napus in the field. Plant Ecology 162, 227-231. Cipollini ML, Paulk E, Cipollini DF (2002) Effect of nitrogen and water treatment on leaf chemistry in horsenettle (Solanum carolinense), and relationship to herbivory by flea beetles (Epitrix spp.) and tobacco hornworm (Manduca sexta). Journal of Chemical Ecology 28, 2377-2398. Cipollini DF (2004) Stretching the limits of plasticity: Can a plant defend against both competitors and herbivores. Ecology 85, 28-37. Creelman RA, Mullet JE (1997) Biosynthesis and action of jasmonates in plants. Annual Review of Plant Physiology and Plant Molecular Biology 48, 355-381. Dehmer KJ, Hammer K (2004) Taxonomic status and geographic provenance of germplasm accessions in the Solanum nigrum L. complex: AFLP data. Genetic Resources and Crop Evolution 51, 551-558. del Campo ML, Miles CI, Schroeder FC, et al. (2001) Host recognition by the tobacco hornworm is mediated by a host plant compound. Nature 411, 186-189. Delaney TP, Uknes S, Vernooij B, et al. (1994) A central role of salicylic acid in plant disease resistance. Science 266, 1247-1250. Dicke M, Bruin J (2001) Chemical information transfer between plants: back to the future. Biochemical Systematics and Ecology 29, 981-994. Dopke W, Duday S, Matos N (1987) The isolation and identification of alkaloids and steroids from Solanum nigrum. Zeitschrift für Chemie 27, 64- 64. Dudley SA, Schmitt J (1995) Genetic differentiation in morphological responses to simulated foliage shade between populations of Impatiens capensis from open and woodland Sites. Functional Ecology 9, 655-666. Edmonds JM, Chweya JA (1997) Black nightshades, Solanum nigrum L. and related species, Gatersleben, Rome. Eltayeb EA, Al Ansari AS, Roddick JG (1997) Changes in the steroidal alkaloid solasodine during development of Solanum nigrum and Solanum incanum. Phytochemistry 46, 489-494. 127
9. References
Heil M, Baldwin IT (2002) Fitness costs of induced resistance: emerging experimental support for a slippery concept. Trends in Plant Science 7, 61-67. Herms DA, Mattson WJ (1992) The dilemma of plants - to grow or defend. Quarterly Review of Biology 67, 283-335. Hermsmeier D, Schittko U, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. I. Large-scale changes in the accumulation of growth- and defense-related plant mRNAs. Plant Physiology 125, 683-700. Hui D, Iqbal J, Lehmann K, et al. (2003) Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata: V. Microarray Analysis and Further Characterization of Large-Scale Changes in Herbivore- Induced mRNAs. Plant Physiology 131, 1877-1893. Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: The emerging molecular analysis. Annual Review of Plant Biology 53, 299- 328. Korth KL (2003) Profiling the response of plants to herbivorous insects. Genome Biology 4, 221. Leon J, Rojo E, Sanchez-Serrano JJ (2001) Wound signalling in plants. Journal of Experimental Botany 52, 1-9. Li L, Zhao YF, McCaig BC, et al. (2004) The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development. Plant Cell 16, 126-143. Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: A node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16, 319-331. Lou Y, Baldwin IT (2003) Manduca sexta recognition and resistance among allopolyploid Nicotiana host plants. Proceedings of the National Academy of Sciences of the United States of America 100, 14581-14586. Lou Y, Baldwin IT (2004) N supply influences herbivore-induced defenses and transcriptional responses in Nicotiana attenuata. Plant Physiology 135, 496-506. Mathe I, Vanmai H, Sen M (1979) Variation in the solasodine production in stands of various stages of development of Solanum nigrum L. during the vegetation period. Planta Medica 36, 237-238.
129
9. References
McEvoy PB, Rudd NT, Cox CS, Huso M (1993) Disturbance, competition, and herbivory effects on ragwort Senecio jacobaea populations. Ecological Monographs 63, 55-75. Muller C, Renwick JAA (2001) Different phagostimulants in potato foliage for Manduca sexta and Leptinotarsa decemlineata. Chemoecology 11, 37-41. O'Donnell PJ, Calvert C, Atzorn R, et al. (1996) Ethylene as a signal mediating the wound response of tomato plants. Science 274, 1914- 1917. Pierik R, Visser EJW, De Kroon H, Voesenek L (2003) Ethylene is required in tobacco to successfully compete with neighbours. Plant Cell and Environment 26, 1229-1234. Pierik R, Voesenek L, de Kroon H, Visser EJW (2004a) Density-induced plant size reduction and size inequalities in ethylene-sensing and ethylene-insensitive tobacco. Plant Biology 6, 201-205. Pierik R, Whitelam GC, Voesenek L, de Kroon H, Visser EJW (2004b) Canopy studies on ethylene-insensitive tobacco identify ethylene as a novel element in blue light and plant-plant signalling. Plant Journal 38, 310-319. Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene expression. Current Opinion in Plant Biology 1, 404-411. Ridout CL, Price KR, Coxon DT, Fenwick GR (1989) Glycoalkaloids from Solanum nigrum L., alpha-solamargine and alpha-solasonine. Pharmazie 44, 732-733. Roscher C, Schumacher J, Baade J, et al. (2004) The role of biodiversity for element cycling and trophic interactions: an experimental approach in a grassland community. Basic and Applied Ecology 5, 107-121. Ryan CA (2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology 1477, 112-121. Ryan CA, Pearce G (2003) Systemins: A functionally defined family of peptide signal that regulate defensive genes in Solanaceae species. Proceedings of the National Academy of Sciences of the United States of America 100, 14577-14580. Sanford LL, Domek JM, Cantelo WW, Kobayashi RS, Sinden SL (1996) Mortality of potato leafhopper adults on synthetic diets containing seven glycoalkaloids synthesized in the foliage of various Solanum species. American Potato Journal 73, 79-88. Schenk PM, Kazan K, Wilson I, et al. (2000) Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proceedings of
130
9. References
Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends in Plant Science 5, 199-206. Farmer EE, Almeras E, Krishnamurthy V (2003) Jasmonates and related oxylipins in plant responses to pathogenesis and herbivory. Current Opinion in Plant Biology 6, 372-378. Felton GW, Korth KL, Bi JL, et al. (1999) Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory. Current Biology 9, 317-320. Fowler NL, Rausher MD (1985) Joint effects of competitors and herbivores on growth and reproduction in Aristolochia reticulata. Ecology 66, 1580- 1587. Fraenkel GS (1959) Raison d'être of secondary plant substances. Science 129, 1466-1470. Garcia-Martinez JL, Gil J (2001) Light regulation of gibberellin biosynthesis and mode of action. Journal of Plant Growth Regulation 20, 354-368. Genoud T, Santa Cruz MBT, Metraux JP (2001) Numeric simulation of plant signaling networks. Plant Physiology 126, 1430-1437. Glazebrook J (2001) Genes controlling expression of defense responses in Arabidopsis - 2001 status. Current Opinion in Plant Biology 4, 301-308. Goldberg DE, Werner PA (1983) The effects of size of opening in vegetation and litter cover on seedling establishment of goldenrods (Solidago spp.). Oecologia 60, 149-155. Goldberg DE (1990) Components of resource competition in plant communities. In: Perspectives on plant competition (eds. Grace JB, Tilman D), pp. 27-49. Academic Press, San Diego. Gurevitch J, Morrison JA, Hedges LV (2000) The interaction between competition and predation: A meta-analysis of field experiments. American Naturalist 155, 435-453. Halitschke R, Schittko U, Pohnert G, Boland W, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore- specific plant responses. Plant Physiology 125, 711-717. Heidel A, Baldwin IT (2004) Microarray analysis of SA- and JA-signaling in Nicotiana attenuata's responses to attack by insects from multiple feeding guilds. Plant, Cell and Environment in press.
128
9. References
the National Academy of Sciences of the United States of America 97, 11655- 11660. Schittko U, Hermsmeier D, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. II. Accumulation of plant mRNAs in response to insect-derived cues. Plant Physiology 125, 701-710. Schmelz EA, Alborn HT, Engelberth J, Tumlinson JH (2003) Nitrogen deficiency increases volicitin-induced volatile emission, jasmonic acid accumulation, and ethylene sensitivity in maize. Plant Physiology 133, 295-306. Sharma SC, Chand R (1979) Steroidal sapogenins from different parts of Solanum nigrum L. Pharmazie 34, 850-851. Sinden SL, Sanford LL, Osman SF (1980) Glycoalkaloids and resistance to the Colorado Potato Beetle in Solanum chacoense. American Potato Journal 57, 331-343. Singh SP, Raghavendra K, Singh RK, Subbarao SK (2001) Studies on larvicidal properties of leaf extract of Solanum nigrum L.. Current Science 81, 1529-1530. Strong DR, Lawton JH, Southwood R (1984) Insects on Plants Blackwell Scientific Publications, Oxford. Sultan SE (2000) Phenotypic plasticity for plant development, function and life history. Trends in Plant Science 5, 537-542. Thaler JS, Stout MJ, Karban R, Duffey SS (1996) Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field. Journal of Chemical Ecology 22, 1767-1781. Thaler JS, Karban R, Ullman DE, Boege K, Bostock RM (2002) Cross-talk between jasmonate and salicylate plant defense pathways: effects on several plant parasites. Oecologia 131, 227-235. Thomas MA, Klaper R (2004) Genomics for the ecological toolbox. Trends in Ecology & Evolution 19, 439-445. Tremmel DC, Bazzaz FA (1995) Plant architecture and allocation in different neighborhoods - Implications for competitive success. Ecology 76, 262-271. Walling LL (2000) The myriad plant responses to herbivores. Journal of Plant Growth Regulation 19, 195-216. Wang KLC, Li H, Ecker JR (2002) Ethylene biosynthesis and signaling networks. Plant Cell 14, S131-S151. Winz RA, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural 131
9. References
host Nicotiana attenuata. IV. Insect-induced ethylene reduces jasmonate- induced nicotine accumulation by regulating putrescine N- methyltransferase transcripts. Plant Physiology 125, 2189-2202.
132
10. Appendix
10. Appendix 10.1 Supplementary Data Supplementary data for MS1, MS3, MS4 and the General Discussion can be found as electronic copies on the attached CD-ROM.
133
10. Appendix
10.2 Curriculum vitae Name: Dominik Daniel Schmidt Date of birth: March 1st 1974 in Hamm (Germany) Nationality: German
School education 09/1980 – 07/1984 Elementary school in Augsburg (Germany) 09/1984 – 07/1993 Secondary school (Gymnasium St. Stephan) in Augsburg (Grade: Abitur) University education 10/1995 – 09/1997 Undergraduate degree of Biology at the University of Hohenheim (Stuttgart, Germany) 09/1998 – 11/2000 Diploma of Biology at the University of Hohenheim Thesis of diploma: “Isolation and characterization of compounds from sunflower pollen“
Practical experiences and positions held 10/1993 – 12/1994 Social Service in the Association for the Protection of the Environment and Nature (BUND), Radolfzell-Möggingen (Germany) 02/1995 – 07/1995 Internship at the Alabor Horticulture Co. Binningen (Switzerland) 10/1997 – 02/1998 Practical training at the Estación Científica San Francisco, Loja (Ecuador) in the program “Ecosystem characteristics of tropical montane rain forest“ 04/1998 – 09/1998 Practical training at the Group for Landscape Planning Detzel & Matthäus (GÖG), Stuttgart (Germany) 10/1998 – 12/2000 Work for the Institute of Botany (University of Hohenheim) as assistant in courses and in the lab of Prof. O. Spring 01/2001 – present PhD in the Department of Molecular Ecology (Prof. I.T. Baldwin) at the Max Planck Institute for Chemical Ecology, Jena (Germany); Topic: „Transcriptional and phenotypic plasticity of Solanum nigrum: Can it defend against both competitors and herbivores?
134
10. Appendix
10.3 Publication list
Müller N, Schmidt DD (1995) Zur Florendynamik in den Lechauen bei Augsburg, Berichte des Naturwissenschaftlichen Vereins für Schwaben 99: 3-7.
Halitschke R, Gase K, Hui D, Schmidt DD, Baldwin IT (2003) Molecular interactions between the specialist herbivore Manduca sexta and its natural host Nicotiana attenuata. VI. Microarray analysis reveals that most herbivore-specific transcriptional changes are mediated by fatty acid-amino acid conjugates. Plant Physiology 131, 1894-1902.
Schmidt DD, Kessler A, Kessler D, Schmidt S, Lim M Gase K, Baldwin IT (2004) Solanum nigrum: A model ecological expression system and its tools. Molecular Ecology 13: 981-995.
Schmidt DD, Baldwin IT: The effect of competition on transcriptional responses elicited by methyl jasmonate in Solanum nigrum—a glasshouse and field comparison. Submitted to Molecular Ecology.
Schmidt DD, Baldwin IT: Canopy height of interspecific competitors influences jasmonate-induced transcriptional and phenotypic responses of Solanum nigrum. Submitted to Ecology Letters.
Schmidt DD, Voelckel C, Baldwin IT: Attack from the same Lepidopteran herbivore results in species-specific transcriptional responses in two Solanaceous species. Submitted to Plant Physiology.
135
10. Appendix
10.4 Oral and poster presentations
Deutsche Botanikertagung; Freiburg/Germany; September 2002; Solanum nigrum: A model ecological expression system
Kurth-Mothes-Doktoranden-Workshop Sekundärstoffwechsel, Jena/ Germany; October 2002; Solanum nigrum: A model ecological expression system
MPI for Molecular Plant Physiology Symposium; Golm/Germany; January 2003; The influence of plant biodiversity on fitness- and defense- related traits in Solanum nigrum – first results from the field
Functional Genomics: theory and hands-on data analysis; Utrecht/The Netherlands; August 2003; Solanum nigrum: genomic tools for ecological questions
MPI for Chemical Ecology Institute Symposium; Jena/Germany; January 2004; Competition influences defense traits of Solanum nigrum
136
10. Appendix
10.5 Danksagung
Ich möchte mich bei allen, die zum Gelingen dieser Arbeit beigetragen haben, bedanken, inbesondere bei: Prof. Ian Baldwin für intensive und fundierte Betreuung, die mir den weiten Horizont der chemischen Ökologie eröffnet hat. Danny Kessler für seine aufopfernde Hilfe bei der Feld- und Laborarbeit. Dr. André Kessler und Rayko Halitschke für inspirierende Gespräche und praktische Hilfe. Susan Kutschbach, Wibke Kröber, Thomas Hahn und Dr. Klaus Gase für wertvolle Hilfe bei der Microarray-Arbeit. Michelle Lim für die Transformation von S. nigrum. Dr. Tamara Krügel für Hilfe in botanischen und anderen Fragen und Ihrem Gärtnerteam für die Kultivierung zahlreicher Pflanzen Der “Solanum-Gruppe”, Silvia Schmidt und Markus Hartl, für gute Zusammenarbeit. Antje Hermsdorf und Celia Diezel für Probenaufbereitung im Feld und im Labor. Der Forschergruppe “Biodiversität” für gute Zusammenarbeit. Meinen Eltern für die jederzeitige und grosszügige Unterstützung. Meiner Freundin, Dajana Lobbes, für die Energie, die Doktorarbeit abzuschliessen.
137
10. Appendix
10.6 Eigenständigkeitserklärung
Entsprechend der Promotionsordnung der Biologisch- Pharmazeutischen Fakultät der Friedrich-Schiller-Universität erkläre ich, dass ich die vorliegende Arbeit selbständig und nur unter Verwendung der angegebenen Hilfsmittel und Literatur angefertigt habe. Personen, die an der Durchführung und Auswertung des Materials und bei der Herstellung der Manuskripte beteiligt waren sind am Beginn der Arbeit („Manuscript Overview“) und jedes Manuskriptes angegeben. Die Hilfe eines Promotionsberaters wurde nicht in Anspruch genommen. Die vorgelegte Arbeit wurde weder an der Friedrich-Schiller- Universität Jena, noch an einer anderen Hochschule als Dissertation eingereicht.
Dominik D. Schmidt
138