In: Action Potential ISBN 978-1-61668-833-2 Editor: Marc L. DuBois, pp. 1-26 © 2010 Nova Science Publishers, Inc.

Chapter 1

WHAT DO PLANTS NEED ACTION POTENTIALS FOR?

Elżbieta Król, Halina Dziubińska, and Kazimierz Trębacz Department of Biophysics, Institute of Biology, Maria Curie-Skłodowska University, Akademicka 19, 20–033 Lublin, Poland

ABSTRACT

For many years the physiological significance of electrical signalling in plants has been neglected, even though the very first action potentials (APs) were recorded in insectivorous plants in 1873 (1). Still many aspects of plant excitability are not sufficiently well elaborated. However, nowadays it is common knowledge that in animals as well as in plants: (i) ion fluxes through plasma membrane provide AP biophysical bases; (ii) AP transmission is electrotonic, without a decrement and is followed by a refractory period; (iii) there is an ―all-or-nothing‖ principle fulfilled, with an exponential dependency of threshold stimulus strength on stimulus duration; (iv) APs are initiated and propagated by excitable tissues to control a plethora of responses indispensable for growth, nutrient winning, reproduction, and defence against biotic and abiotic challenges. AP can be viewed as a burst of electrical activity that is dependent on a depolarizing current. In plants the depolarization phase of AP consists of Cl-- and Ca2+-fluxes. The following phase—a repolarization—relies in turn on K+ fluxes and active H+ flows that both drive membrane potential back to more negative values. Thus, the AP mechanism is electrochemically governed by the selective properties of the plasma membrane with ion selective conduits as key players. A more detailed understanding of how these membrane proteins work hand in hand during excitation and signal transduction is eagerly awaited. The existence of ion channels was first hypothesized by Alan Hodgkin and Andrew Huxley (2-8), and next confirmed with a patch-clamp technique by Erwin Neher and Bert Sakmann (9). These experiments though conducted on neurons and muscles, respectively, prompted plant electrophysiology as well. Since then substantial evidence for APs in a wide array of plants has been emerging and consequently growing in number.

2 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz

INTRODUCTION

Electrical sensitivity of living organisms originates from selective membranes that surround each cell. Thanks to active transport of ions by pumps and transporters (mainly K+, Na+, H+ and Ca2+ but also Cl-) and selective properties of the channels embedded in membranes, a transmembrane potential difference is generated (10-13). This membrane voltage (= membrane potential, transmembrane potential) is the difference between the inside and the outside (by convention set to 0) potential. The magnitude of the membrane potential directly depends on the membrane selective properties and hence on concentration of ions facing both sides of the membrane (12). There is a negative membrane resting potential (difference at rest) in most living cells. At rest, the net flow of ions through a selective membrane equals zero, which means that outflows and inflows of ions transported are counterbalanced. Any unbalanced movement of ions results in changes in the resting potential. Such imbalances can be triggered by stimuli as different as: electric current, light, pressure (mechanical or osmotic) and chemical substances of various derivation. The above- listed stimuli are either directly or indirectly responsible for ion channel, transporter or pump activation/inhibition, which transiently changes membrane permeability for corresponding ions and thus make the resting potential change (14). Evoked membrane potential changes hold (i) various shapes, (ii) kinetics, (iii) duration, (iv) properties and (v) functions and accordingly can be classified as:

i. hyperpolarization / depolarization (if it drives the potential to more negative / less negative values); ii. graded / of constant amplitude (with an amplitude depending on / independent of stimulus strength); iii. transient / long-lasting; iv. propagable / non-propagable v. systemic / local (spreading within the whole organ or organism / appearing locally).

Among them the best studied and characterized are action potentials (APs), which are a transient membrane depolarization with all-or-nothing characteristics (14-17), propagating systemically (18-20) with a cell-specific velocity (14,21) and without a decrement (an amplitude decrease). As for the AP amplitude that is cell-specific, too, it cannot be increased by an increase in stimulus strength. The physical depiction of the latter statement is reflected in the above-specified ―all-or-nothing characteristics‖. In addition, the relation between threshold stimulus charge (strength) and stimulus duration can be represented by Weiss's experimental formula—the exponential dependence of threshold stimulus strength on its duration (22). The AP transmission along excitable membranes is achieved through electrotonic transmission. A local current flows between the just activated part and the adjacent yet unexcited part. After the passage of each single AP there is a refractory period—the period of transient unexcitability or, in other words, time needed for a cell to restore its excitability. Finally, one must keep in mind that excitability in electrophysiology nomenclature means the ability to generate and transmit APs. The cells on whose membranes the other potential changes but not APs occur are not considered excitable (23). What Do Plants Need Action Potentials for? 3

A PINCH OF HISTORY

When in 1786 Luigi Galvani dissected a frog, touched its leg with a charged scalpel and saw the frog‘s leg kicking after the charge had jumped from the scalpel to the muscle tissue, he had no idea that the charge flow induced an AP and that the muscles contracted as a result of AP (excitation) spreading. However, his observation made Galvani the first investigator to appreciate the relationship between electricity and movement in living organisms. His associate and intellectual adversary Alessandro Volta went deeper into the nature of electrochemical processes (which allowed him invent the first battery—a galvanic cell). His intuition that ―animal electricity‖ has the same underpinnings as electrochemical reactions proved correct and widely contributed to our understanding of ion-pulling forces in living systems (24). Starting from 1830 till his death in 1865 another Italian scientist, the physician and neurophysiologist Carlo Matteucci pursued experiments on frog muscles, using them as a kind of electricity-detector (25). His work influenced directly the German physician Emil du Bois-Reymond, who, trying to duplicate Matteucci‘s results, ended up with the discovery of APs. At that time he termed them ―negative variations‖. The results of du Bois-Reymond‘s inquiries were being compiled systematically in his life-work Researches on Animal Electricity, the first part of which appeared in 1848, and the last in 1884. Though the story of bioelectricity began with a frog, its impact on plant biology was equally impressive. The very first APs recorded in plants were reported by the English physician-physiologist Sir John Scott Burdon-Sanderson, who—encouraged by Charles Darwin—was the first to recognize the electric phenomena in carnivorous plants (1). For his pioneering work in the field of electrophysiology and plant physiology, the Royal Society awarded Burdon-Sanderson a Royal Medal in 1872. Inspired by Burdon-Sanderson‘s work, another British scientist Walter Gardiner (a botanist) devoted himself to carnivorous spp. trying to find a link between excitation and histological changes in secretory glands (26). Next, the Austrian botanists and father of a plant cell totipotentiality theory, Gottlieb Johann Friedrich Haberlandt found non- carnivorous plants to move after electrical stimulation (27). At the same time in Bengal, Haberlandt‘s peer Sir Jagadish Chandra Bose (a polymath: physicist, biologist, archaeologist and writer) studied the correlation between plant development and environmental stimulants (wounds, chemical agents, light and temperature changes) with the help of his self-invented devices (a crescograph to measure plant growth; microamperemeter for current assessments; an electric probe for voltage recordings). On intact plants he measured electrical conduction and corresponding changes in the cell membrane potential in response to chemical and physical stimulation (28-29). Having generalized that all strong stimuli produced a transient diminution of growth rate, a negative mechanical response (cell shrinking) and an electric response of ―galvanometric negativity‖ (= AP), he was very close to discerning excitation as an endogenous form of cell signalling for stress/danger-sensing (29-30). He also worked on isolated vascular bundles to conclude that plants contain organs which are analogous to muscle and nerves in animals (30), just as Burdon-Sanderson had suspected (31). However, because of prevailing prejudices and general acceptance that plants should not be compared to animals, Bose‘s observations were not taken into serious consideration and neglected for over 70 years. Letting higher plants fall into oblivion, the first intracellular recordings (with a cell- inserted microelectrode) of APs were registered in lower plants (32-34). Varied 4 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz responsiveness of higher plants according to season, vigour, water status, temperature, age and previous history of stimulation (all of which Sir Jagadish Chandra Bose himself had struggled with) stumped the researchers effectively. Another reason to work on lower plants was the fact that higher species have just selected cells/tissues that are excitable while the entire body of a lower plant is so. Thus the advantage was taken from elongated alga internodes that were both excitable and accessible—big enough for a measuring electrode to be inserted into (35). That kind of recordings was almost simultaneously adopted for giant cells of plants (33) and animals (36). The former were soon considered more complicated than the latter, because of the existence of some structures missing from animal cells, namely a cell wall and a large central vacuole. Moreover, a tonoplast - a membrane embracing the vacuole - turned out to be excitable in some spp., so double-peaked APs were recorded, when the measuring electrode was placed into the vacuole (37). Because of this structure unique approaches developed (e.g. open vacuole method) to proceed electophysiological studies on algae (38); in spite of these structures Characean cells became a model tool for understanding membrane function (39). The giant neurons of squids were scrutinized simultaneously and independently by Howard James Curtis and Kenneth Stewart Cole at Woods Hole (U.S.A.) and by Sir Alan Lloyd Hodgkin and Sir Andrew Fielding Huxley at the laboratory of the Marine Biological Association in Plymouth (Great Britain). Among the scientists mentioned, a pioneering role in unifying plant and animal membrane responses was played by the biophysicist Kenneth Stewart Cole (40). He was the first to show that all principles of excitable membranes are equally applicable to plants (41-42) and animals (43-44). In his model of excitability Cole depicted an excitable cell as an electrical circuit with resistive and capacitive properties (45), which lent substance to the future ―sodium theory‖, which – in turn - validated depolarizing currents during nerve excitation. His demonstration of a large increase in membrane conductance during excitation with a parallel invariability of capacitance was a major landmark and fitted perfectly into the prevailing membrane theory of Bernstein (46). Julius Bernstein was a German physician (a student of Emil du Bois-Reymond) and neurophysiologist, who developed a differential rheotome—an instrument for resolving the time course of APs. Bernstein‘s membrane theory provided the first physico-chemical model of bioelectricity valid hitherto (47). Bernstein correctly assumed that the membrane of a cell is selectively permeable to K+ at rest and that the membrane permeability to some other ions increases during excitation. Accordingly, his theory gave reasons for the negative resting potential as a consequence of the tendency of positively charged potassium ions to diffuse from their high concentration inside a cell (cytoplasm) to their low concentration in the extracellular solution (apoplast) while the counter ions (anions) are held back (48). In Bernstein‘s theory two pivotal postulations were applied: (i) Walther Nerst’s equation describing electrical potentials as a result of concentration gradients separated by a biological membrane; (ii) Wilhelm Ostwald‘s calculation of the electrical potential at artificial semi- permeable membranes (ion sieves). On the basis of Bernstein‘s and Cole‘s assumptions (―potassium theory‖ and ―sodium theory‖, respectively), a correct model of the ion mechanism of neuronal AP was elaborated. It can be summarized as a transient increase in Na+ permeability followed by K+ outflow. Thanks to Cole‘s devotion (awarded in 1967 with the National Medal of Science) the intracellular technique designed to directly measure APs and the membrane potential immediately came to be widely employed and applicable (49). During next years the intracellular technique became successively complemented with high- What Do Plants Need Action Potentials for? 5 gain amplifiers and voltage-clamping circuits so that current assessments could commence instead of voltage measurements. Two electrodes for current passing (to set the voltage at command value) and another two independent electrodes for voltage measurements were initially used, until a time-sharing system made single-microelectrode voltage-clamping possible (50). In a voltage-clamp mode the current necessary to set the command voltage is measured. For an isolated single cell it is also possible to apply a current-clamp mode, in which the membrane current is held at zero by the feedback circuit while measuring voltage necessary to nullify the flow of charges (51). The earliest measurements of ion currents known as voltage-clamp were conducted by the two above-mentioned Nobel Prize winners (Nobel Prize in Physiology or Medicine in 1963), Sir Alan Lloyd Hodgkin and Sir Andrew Fielding Huxley (36). Thanks to the voltage- clamp technique, they published a mathematical formula - the Hodgkin-Huxley model (1952) - describing currents flowing through the hypothetical ion channels and giving rise to APs in excitable neurons of the Atlantic squid Loligo pealei (3). Their model largely stemmed from Cole‘s theory (52). 24 years later the existence of ion conduits was elegantly confirmed with a patch-clamp technique – a sophisticated version of voltage-clamping – by Erwin Neher and Bert Sakmann (9), a German physicist and physician, respectively, awarded for that with the Nobel Prize in 1991. From then on succeeding characterisation of various ion channels takes place (52). Now it is a common knowledge that APs in nervous cells involve the transient opening of Na+-channels and Na+ influx, in cardiac muscles the main depolarizing current flows through the Ca2+-channels, while in plant this is accomplished by a release of negative chloride ions. The subsequent release of positive potassium ions is common to plants and animals and is responsible for a repolarization – a return to the resting potential. In addition, more detailed studies on plants revealed that: (i) apart from chloride (14,53-59) also calcium is involved in the depolarization phase of AP (60-69); (ii) the Ca2+ ions may have external (70-73) and/or internal origin (64,74-76); their function is to activate calcium-dependent Cl-- channels (56,77-79) and to inactivate plasma membrane H+-ATPase (80-81); (iii) along with potassium ions (55,59,82) H+-ATPase plays an important role in the repolarization (66,83); (iv) AP-delimited ion fluxes additionally serve signalling functions, such as turgor regulation, gene expression or Ca2+-dependent kinase activation (84-86); (v) in contrast to animals, plant AP-associated channels seem to be additionally regulated by cytoplasmic messengers (Ca2+, H+, ATP) and/or regulatory enzymes (kinases, phosphatases); (vi) many aspects of the plant AP-mechanism which include second messenger-activated channel and calcium ion liberation from internal stores still await more careful consideration (87). Let us say then that during the last 50 years enormous progress has been made in electrophysiological techniques, which brought about our better (but not complete yet) understanding of physico-chemical processes occurring on membranes at rest and during excitation. Action potentials are triggered when the stimulus causes transient opening of selective ion channels so that ions can start to flow down their electrochemical gradients. The various AP-mechanisms are electrochemically governed by the selective properties of the plasma membrane with different ion selective conduits as the key players. APs are initiated and propagated by excitable tissues to control a plethora of responses which in plants include growth synchronization, nutrient winning, reproduction (fertilization), defence against abiotic and biotic assaults together with an increase in pathogen-related gene expression. The role of the AP in plant movements, wound signalling, and turgor regulation is now well documented (87). Nevertheless, how exactly membrane excitation influences the nucleus (genes) and/or 6 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz other organelles is still obscure and a more detailed understanding of how membrane proteins work hand in hand during signal transduction and to what extent APs are involved in intracellular signalling is eagerly awaited. Likewise, AP involvement in invasion by pathogens, chilling injury, light, and gravity sensing needs further investigation (87). The following chapter is focused on the documented aspects of excitability in the plant kingdom.

AP SIGNIFICANCE

Trap Closure and Enzyme Secretion

Electrical signals are one of the fastest means of information transmission within a plant (88). For the first time recognized in the Venus flytrap Dionaea muscipula (1), next also found in its closest relative - the waterwheel plant Aldrovanda vesciculosa, they were linked with a trap closure right away. In the waterwheel plant they were shown to propagate at the rate of 80 mm/s (89), while different AP velocities (depending on the course along which they move) were noted for Dionaea. Accordingly, AP reached up to 250 mm/s in midrib direction (the highest value reported in plants hitherto), and ―only‖ 60 – 170 mm/s if running towards the trap margins. Moreover, while the first AP propagated to the sister-lobe with the average velocity of 100 mm/s, the succeeding one did it twice as fast (90). Considering the differences in propagation rates of succeeding APs in Dionaea, it is postulated that the first excitation facilitates the spread of the successive one (90). Likewise the propagation rate, also AP duration of 1s and 2 s in A. vesciculosa (91) and D. muscipula (92), respectively, are outstanding among plants. For comparison one should realize that APs in closely related Drosera rotundifolia last on average from 10 to 20 s (93), and in lower plants a single AP can even last up to dozens of minutes (94), propagation, in turn, hardly exceeds a few cm/s. The reported AP amplitudes of Dionaea muscipula and Aldrovanda vesciculosa exceed 100 mV (63,91,95-96) and are independent of a kind of the stimulus (mechanical stimulation, 2+ electrical stimulation, cold, light) (67). They depend, however, on [Ca ]ext in such a way that 2+ the amplitude of AP follows [Ca ]ext increases (92,97). Accordingly, Ca-ionophores or chemicals disturbing Ca-homeostasis hamper AP amplitudes (96) and slow down trap closure (98). Apart from Ca2+ also Cl- ions participate in the depolarization phase, since Cl-channel blocker A9C (anthraceno-9-carboxylic acid) lowers AP amplitude (67). K+ efflux is responsible for AP repolarization (99), which altogether perfectly matches ion mechanisms of excitation in plants (100). To make the Dionaea trap snap within 100 ms (101) at least two APs are needed, and the interval between the first and the second AP cannot exceed 10 s. The longer the breaks between succeeding APs, the more APs are necessary for a trap to snap (85,102). However, the trap is not completely closed yet. For a hermetical closure consecutive APs are needed; if not stimulated again, the trap re-opens relatively fast. The same refers to Aldrovanda vesciculosa which also needs more APs than one to keep the trap closed and to trigger corresponding turgor changes indispensible for the hermetical closure (85,103). Since all the cells of traps in both species are electrically coupled and all are equally excitable, they participate in fast AP transmission evenly (90-92,104). However, not all of them respond to What Do Plants Need Action Potentials for? 7

AP equally - effector responses differ. To close the trap completely, the loss of water takes place preferentially in the upper epidermis and adjacent mesophyll cells (85) while the lower epidermis extends (104). An answer to the question why the same AP makes only some cells shrink remains obscure. It may be speculated that the corresponding channels in the upper and lower sides of the trap are differently regulated by the same stimulus. Alternatively, a number of channels (channel density) differs in both sides, hence the discrepancy in the extent of water loss. In digestive glands, in turn, APs control the enzymatic activities (26), with successive APs sufficient to induce enzyme secretion (unpublished results). Moreover, excitation and secretion seem to be mutually linked (APs induce digestion - digestion products trigger APs), as many chemical substances is able to trigger both processes (105). It seems reasonable that excitation from one digestive gland spreads to the others to fully prepare the whole trap to digest a prey effectively. As a prey break-up boosts up a mineral uptake in the roots of carnivores (106), there is also a possibility that APs might be involved in inter-organ signalling. Whether APs play a role in trap-root communication after all, is still an open issue, because APs ―outside‖ the traps have never been reported so far (107). Since at least two succeeding APs are needed for Dionaea to take action, it is postulated that the plants may possess a kind of memory, which allows them to respond only to the second AP. Because the membrane potential goes back to the resting value right after the passage of an AP, the resting potential cannot act as an ―accumulator‖ in the process of memory. There is also no indication that the memory is associated in any way with a receptor potential – stimulus-dependent depolarization which if large enough leads to AP generation (108). Instead, for analogy to animal nerve systems, stepwise accumulation of bioactive substances during successive stimulations of the trap was suggested. Irrespective of the biochemical basis, the process of two successive APs during trap closure surely serves to protect a plant against any accidental mechanical stimulation. It can also be seen as a kind of protection against light-stimulation. Keeping in mind that light transiently depolarizes the membrane and that an excitable cell fires AP whenever membrane depolarization reaches the threshold value, it is not surprising that APs are noted after trap illumination (95). Another analogy to animals can be postulated, if one considers the AP-trigger trap closure as excitation-contraction coupling in muscles (1,109-110), especially that APs lead to a production of lysophosphatidic acid that increases membrane permeability to water and makes a cell shrink (111). A fast movement of the trapping organ under the control of an electric signal (AP) prompted Darwin to name Dionaea muscipula ―the most wonderful plant in the world‖ (112). However, other carnivorous spp. are very fast, too. Beside Dionaea and Aldrovanda also Drosera burmanni and D. glanduligera are able to execute snapping movements; they can bend a tentacle within 5 s and 0.15 s, respectively. As a matter of fact, the traps of all spp. of Drosera (sundews) and some of Pinguicula (butterworts) are mobile and ―use‖ APs to control the movement. Two layers of cells surrounding the conductive bundles constitute the excitable tissue of Drosera‘s tentacle and are responsible for a rapid electrotonic transmission of APs (93). These cells are electrically coupled by numerous plasmodesmata, which suits them for the fast propagation of APs (113). Moving down the stalk, APs travel with the velocity of 5 mm/s, while propagation upwards is twice as fast (114b). In addition to tentacle movements, most species of Drosera are also able to bend the whole leaf, which usually takes a couple of hours, and requires consecutive APs and subsequent turgor changes (85). The 8 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz successive APs are very probably indispensible for induction of enzyme secretion in these plants, too.

Fertilization

The suggestive paper of Sinyukhin and Britikov published on Incarvillea grandiflora and Incarvillea delavayi (gloxinia) has reported that: (i) an AP is triggered when a pollen sets on the stigma of the pistil; the AP appears in response to mechanical irritation, too – it can be triggered with a soft brush; damaging stimuli do not evoke the AP; (ii) the extracellularly recorded AP of 30-40 mV spreads to the base of the stigma with the velocity of 18 mm/s in order to make it close; the stigma closes in 6-10 s; (iii) another AP of 80 - 90 mV appears if the pollen has turned out to be respective; if the mechanical irritation is not followed by the corresponding chemical stimulation, the stigma re-opens in 17 - 22 min; (iv) the second AP courses down the style of the pistil at the rate of 29 mm/s and enhances respiration in the ovary; the second AP has been postulated to make the ovary ready for pollination (115). In the same paper the ability to control ovary metabolism by pollen-triggered APs has also been suggested for Lilium martagon, and Zea mays. Similar experiments conducted on Hibiscus rosa-sinensis has shown that either self- or cross-pollination results in a series of 10 to 15 APs propagating down the vascular tissue of the style with a velocity of 13 - 35 mm/s (116). AP-induction is proceeded by a hyperpolarization that takes place 50 – 100 s before AP firing. Only with the passage of AP series is an increase in ovary respiration correlated; neither cold nor wounding are coupled with CO2 increases though they also produce membrane potential changes and moreover cold stimulation is associated with a single AP (116). By analogy to Sinyukhin and Britikov‘s results, it can be concluded that electrical signalling of AP is informative only if accompanied by additional – most probably pollen derived - stimulants. It can be speculated that there must be a signalling cascade leading to pollen recognition, which triggers APs. In case of Hibiscus rosa-sinensis cation efflux and thus membrane hyperpolarization must be involved, while in Incarvillea spp. membrane stretch was suggested to be of crutial importance (115). Since both, negative membrane potential (hyperpolarization) and positive pressure (stretch), are known to activate respective Ca2+-channels (117), they might serve AP initiation. However, the exact sequence of events leading from pollen germination to AP initiation has yet to be deciphered. It is very likely, for example, that receptor like kinases (RLK) known to be involved in pollen-pistil communication (118) might mediate in channel activation, too. An ovary response only to the second AP (chemically-induced AP) greatly resembles a protection system of carnivorous plants against accidental stimulations, and points to the interesting fact that a single electrical change itself may hardly be satisfactory if not backed up by supportive information – a recurrent issue, recently raised by Pyatygin et al. (119).

Mechanical Stimulation and Thigmonastic Movements

The movements of plant parts (e.g.: leaves, stamens, stigma, stems, tendrils) caused by touch are referred to as thigmonastic if independent of the stimulus direction and tigmotropic when they follow the stimulus course. Described in the insectivorous plants first (1,112), the What Do Plants Need Action Potentials for? 9 thigmonastic movements soon turned out to be a characteristic phenomenon for a few other species: Mimosa pudica (27,30), Biophytum sensitivum (120) and Incarvillea spp. (115). Leaf folding by Mimosa is the best elaborated thigmonastic response that has long been linked with AP propagation (90). It is enough to touch a single pinnate leaflet to trigger an AP and let the thigmonastic movement start, when AP propagation along the entire leaf make the leaflets fold up consecutively. The pathway of AP transmission comprises the elongated cells of phloem and parenchymal cells surrounding both the xylem and the phloem (90,121). The transmission velocity varies enormously from 4 to 40 mm/sec, depending on leaf age and general condition as well as on ambient temperature and humidity (30,90,122). If the AP reaches the pulvinus, another type of AP (pulvinar AP) appears with a latency of 0.2 – 0.4 s (102). The pulvinar AP with the amplitude of 100 – 140 mV arises with a rate of 0.5 – 2 V/s and endures on average 10 s (90,102). Within 0.3 s after generation it propagates throughout the whole pulvinus (102). As a consequence the abaxial (lower) cells of the pulvinus lose turgor vigorously, which causes the leaf drop (85). Time needed to lose water amounts to 0.1 – 0.2 s (122). From the pulvinus the signal (AP) occasionally enters the stem and next ingresses the other pulvini so that the other leaves drop and fold (now the AP moves from the pulvinus up to the pinnate leaflets; thusly, APs have a nature of propagating waves in both basipetal and acropetal direction). The transmission rates in the petiole and the pina-rachis only slightly depend on the direction (basipetal vs acropetal), but they increase with an increase in the number of excitable cells involved or - in other words - with the width of the petiole (90). This means that the extent of excitation transmission and velocity depends on the co-operation of many cells, which manifests itself in such a way that thicker organs transmit the signal wider and faster (30). Accordingly, the transmission along the stems of Mimosa takes place only as a result of the co-operation of a number of cells. The transmission is electrotonic and occurs longwise excitable cells as well as transversely - then ―jumping over‖ an unexcitable tissue separating excitable bundles (90). In the pulvinus, the so called collocytes (adhesive cells) occupying the phloem/cortex interface are responsible for lateral transduction of APs toward motor cells (123). Since folding is under the control of AP, this reaction runs either completely or not at all. With the passage of excitation wave Cl- and K+ ions are released first; they ―drag‖ water out of cells next. When water leaks out, leaf movement begins. The coincidence of Cl- / K+ release to the apoplast of a pulvinus and ―tissue contraction‖ (leaf drop) has been proven with the use of Cl-selective electrodes (102) and radioactive potassium ions (124). However, not - all excitable cells ―expel‖ ions to the same extent. Accordingly, the increase in [Cl ]ext is observed only in the lower half of the pulvinus (102,125-126), while the APs are detected in both halves (90,102,127-128). The situation resembles the conditions from Dionaea‘s trap where a loss of turgor is coupled with AP passage but do not embrace all cells excited (but upper epidermis only). Therefore, turgor-losing cells can be viewed as effectors (motor cells), while the other excitable cells as both efferent fibres connecting sensors with effectors and the sensors themselves, as every part of Mimosa‘s leaf is receptive to touch. The slumped leaves return to their starting position after 15 to 30 minutes of recovery (129). This time is needed for restoring ionic gradient and turgoid water status in each pulvini autonomously (130). Leaf folding brings some consequences for Mimosa, among of which an impediment of photosynthesis seems quite obvious (131). Moreover, an AP controlling the leaf movements also triggers phloem unloading of sucrose (132). It appears that with the transmission of excitation through the phloem the flow of assimilates stops, sucrose enters the apoplast and 10 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz the excited cells shrink (129,132-133). As a matter of fact, the temporal loss of photoassimilates seems to control all movements of Mimosa (nyctinasty, thigmonasty, gravitropism), since they all depend on turgor changes. Should excitation be evoked by phloem injury, then phloem shrinking could serve as a kind of protection against photoassimilate (energy) loss, as well. The latter hypothesis can be partly substantiated by showing that the sugar unloading is a more general response of the excited phloem (134-135). Mechanically triggered APs propagating throughout the length of a pinna-rachis or a peduncle have also been reported in Biophytum sp. (120). The AP of 60 to 100 mV (extracellular recordings) is followed by the absolute refractory period of 20 - 50 s and the relative one of 30 - 70 s. The AP transmission is restricted to the base of the leaf or peduncle; its velocity of is about 2 mm/s; and there is no difference in the velocity between the acropetal and basipetal directions. The mechanism of the transmission is electrotonic and similar to that in Mimosa pudica. Other plants in which mechanical-APs are registered include not only the above- mentioned sensitive plants or carnivorous spp. (Dionaea (63), Aldrovanda (91), Drosera (114,136)) but also Pinus (137), Ipomoea, Xanthium, Pisum (138) and algae (139-140). Mechano-stimulation of carnivorous plants is connected with bending of trigger hairs, the organs responsible for prey sensing (sensors). Deviation of the trigger-hair of Dionaea and Aldrovanda or bending of the head of Drosera‘s tentacle results in activation of the stretch- activated channels located in the bending zones. The channels allow Ca2+ entry and hence membrane depolarization (63). In Chara APs can be stimulated by touching (dropping a glass rod on) the node (140) or by pressure changes (139); they appear as a consequence of membrane stretching of the node cells and propagate along intermodal cells, proving that there is an electrical coupling between nodes and internodes. Characean internodal cells can be mechanically stimulated either by direct decompression of the plasma membrane or thanks to osmotic changes of a bath solution. Exchanges from hypertonic to hypotonic media or their accompanying membrane stretching, always induce large membrane depolarization (141) that is accompanied by APs (142). By contrast, APs have been never observed during exchanges from hypotonic to hypertonic solutions (=membrane compression). A link from membrane stretch to AP generation in Chara can be quite straightforward, if stretch-activated Cl-- channels are engaged (143). Alternatively, likewise for carnivores, the activation of the mechano-sensitive Ca2+-channels triggered by membrane decompression has been proposed (142-144). Additionally, stretch-activated Ca2+-channels in the chloroplast have also been shown to participate in plasmalemma excitation (145). Because in Acetabularia mediterranea APs accompany pressure regulations in the critical range and their frequency is increasing with turgor raises (146), it seems convincing that APs may constitute a ―valve‖ releasing osmotically active ions (Cl-, K+) and thus lowering turgor, as it is the case in bacteria (147). It has already been suggested that the original function of electrical excitability of biological membranes is related to osmoregulation which has persisted through evolution in plants, whereas the osmotically neutral action potentials in animals have evolved later towards the novel function of rapid transmission of information over long distances (148). As for higher plants, the osmoregulation-hypothesis might be well substantiated by APs induced by wetting dry roots (hypo-osmotic shock). Additionally, such APs initiated in the roots and registered in the stem are suggested to coordinate physiological responses with water availability in the soil (149); the results were further supported in maize (150). In the plumular hook of pea epicotyls, in turn, mechanically evoked APs are proposed to mediate an What Do Plants Need Action Potentials for? 11 increase in mechanical durability during stem growth (151). Their involvement in induction of an ethylene release (a hormone that among others inhibits the opening of the pulmular hook and in this way enables the plumule to penetrate soil) has been suggested (151). Growth-associated spontaneous fluctuations of the membrane potential occurring individually or in series have been also recorded in shoots of Ipomoea, Xanthium and Pisum (138). With the use of intracellular recordings they were noted as the putative action potentials of 1-4 s duration, however, their function was never deciphered. The same holds true for spontaneous APs reported in cucumbers and sunflowers (152). More often than not, local membrane potential changes instead of APs appear in the place of growth (apical tips, elongation zones and pollen tubes). Corresponding transmembrane currents seem to control such plant reactions as gravitropism (roots and shoots), thigmotropism (tendrils), chemotropism (pollens), (circum)nutations (roots and shoots), which all together is of great interest for electrophysiology but is not an issue for the present review. Up to this day, for example, the suggestion of AP involvement in geotropic responses (84) has not been experimentally confirmed (87,153). On the other hand, circumnutation-associated APs were shown to appear in sunflowers with 24-h-rhythmicity (154). The same results were independently obtained by Stahlberg et al. who postulated a casual relationship between stem growth and stem spontaneous excitability (152), but the question of the exact role of APs in growth progression has not been answered yet.

Light/dark - Guided Signalling

The existence of the above mentioned circumnutation-associated APs is rather linked with darkness, as those APs predominately occur at nights (152,154), when the membrane resting potential is known to be relatively depolarized (38,94,150,155-161). The boosting effect of light on the plasmalemma polarization can be connected with the stimulating action of photosynthesis on plasma membrane H+-ATPase, which was experimentally shown by the use of PSII inhibitors (156,162). Therefore, the enquiry of whether those APs are light/dark- message transmitting signals to synchronize dark-induced growth of a plant (152) or just a consequence of membrane depolarization (163) is really hard to be answered, especially that both solutions do not have to be mutually exclusive. The very first response of a plasmalemma to light-on is a short and transient membrane depolarization (if strong enough, leading to AP in excitable cells) followed by a long-running hyperpolarization (light boosting effects as mentioned above). Both responses are associated with photosynthesis, because they are absent in cells deprived of chloroplasts (158), inhibited by DCMU (an electron transport inhibitor) (69,94-95,164-165) and stimulated by CCCP (a proton gradient uncoupler) (164). These data also suggest that electron flow in chloroplasts (either cyclic or non cyclic) is somehow sensed by a plasmalemma (38). The mechanisms of light induced potential changes in excitable cells has been elaborated on lower plants predominately (38,53,73,165). Since approximately a half of alga‘s resting potential is fuelled by ATP (another half by K+-diffusion) and both photosynthesis and respiration are ATP-yielding processes, therefore any transition from darkness to illumination (and vice versa) may be connected with the very temporal and local ―loss‖ of ATP, sufficient enough to be sensed by adjacent plasmalemma H+-ATPase, thusly leading to H+-ATPase inhibition and hence membrane depolarization (14,157,166). Light-induced inhibition of the 12 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz electrogenic proton pump during the onset of AP has been assessed at 50% - 80% of the resting value (167-168). Another possible explanation of AP initiation by light-on is put forward by Mimura and Tazawa, who have suggested that light-induced chloroplast surface charge is able to influence plasmalemma (164). This is very much consistent with inhibitory effects of DCMU (38). This is also in accordance with previous Tazawa‘s papers which reported that not the stoppage of the pump but membrane depolarization is a necessary condition for the generation of light-induced rapid potential changes (169). Moreover, light- induced potential changes on thylakoid membranes are long known to precede those occurring on the plasmalemma (165). Like ―thylakoid-voltage‖ influences plasma membrane potential, so electrical excitation of the plasmalemma can modulate events in the thylakoid membrane (170-172). The plasmalemma-chloroplast coupling factors might be again ATP/ADP/Pi, Ca2+ and membrane depolarization itself. Additionally, AP-associated pHcyt changes have been postulated to influence photosynthesis directly (173). Since electrical signals interfere with photosynthesis (107,131,172-178) and photosynthetically active light triggers different membrane potential changes, therefore multifunctional and bilateral communication between plasmalemma and chloroplast must exists, where chloroplast-plasmalemma vicinity enables their mutual interactions (38). Chloroplastic Ca2+ release could be a plausible explanation of membrane excitation under dark (179-180). This Ca2+ flux does not occur immediately after the light-to-dark transition but begins circa 5 min after light off and slowly increases to a peak at 20 to 30 min after the onset of darkness, affecting cytosolic Ca2+ concentration as well (179). Ca2+ influence on membrane proteins (H+-pumps, transporters, channels and various membrane-bound enzymes) is difficult to be summarized in a few sentences, as its aftermaths depend on Ca- concentration itself as well as on numerous Ca-binding proteins (kinases, phosphatases, CaM, CBL). However Ca2+-activated Cl--channels or Ca2+-inhibited H+-ATPase seem to suffice to justify induction of APs. Light-induced APs have been so far reported in the moss Physcomitrella patens (94), the liverwort Conocephalum conicum (16,73,181), the bean Phaseolus vulgaris (182) and Dionaea muscipula (96), whereas dark-induced ones in Helianthus annuus (152), Physcomitrella patens (94), the hornwort Anthoceros punctatus (165), the green alga Eremosphaera viridis (183) and Acetabularia spp. (14,53). Recently Shabala et al. have demonstrated in maize seedlings that light exposure in the shots can have a strong impact on root ion transport, visible within a range of seconds to minutes (184). Such fast shoot-root communication must be accounted for with transmittable membrane potential changes. Since light-induced potential changes may be of AP character, therefore APs involvement in the control of root uptake machinery is not excluded, though it needs experimental confirmation. Finally, not only chlorophyll but also other receptors (phytochromes, cryptochromes, phototropins) can mediate the light-induced membrane potential changes (185). As those receptors are cytosolic - membrane bound proteins, their signalling cascade leading to membrane potential changes seems at first glance quite simple (via e.g. light-activated channels (186-187)). Such an attitude, however, may be in most cases misleading, as molecular studies have recently acknowledged the complicated and multilevel nature of light- perception systems in plants (188). In general, they do not cause AP generation (189), hence they are out of interest of this presentation. An exception is the UV-C perception complex in algae known to interfere with visible light to evoke APs (190). Additionally, for a few What Do Plants Need Action Potentials for? 13 reasons, two papers of Ermolayeva reporting on phytochrome-mediated membrane depolarization of the moss Physcomitrella patens are worth mentioning as well, although in those papers the light-induced membrane changes have never been named APs (191-192). First of all, the rapid and transient membrane depolarization of 100 mV has shown graded response below and all-or-nothing characteristics above the threshold value. Secondly, the depolarization has been followed by a transient 30 mV hyperpolarization and the refractory period of 12 - 15 min, which reflects AP characteristics. Thirdly, the ionic mechanism of the red-light induced depolarization resembles AP evolution. At last but not least important is the fact that the moss is excitable, thus able to generate APs in response to different stimuli (light, cold, current). The phytochrome evoked potential changes (putative APs) have been shown to initiate the development of primary side branches on caulonemal filaments of Physcomitrella (191). In accordance with this is the further report of Mishra et al. who have demonstrated that an electrical stimulus can probably overcome the requirement of photo-exposure to induce primary leaf formation in etiolated seedlings of Sorghum bicolor (193). Therefore it can be postulated that light-induced APs could be competent signals controlling photomorphogenesis. Still AP-controlled light sensing needs deeper consideration.

Temperature Sensing

Although as early as in 1837 Dutrochet observed that rapid cooling leads to an abrupt cessation of protoplasmic streaming in Chara (23), it took almost 100 years to realize that a sudden temperature drops evoke APs in this alga (35). Rapid cooling (unlike gradual cooling) acts as a stimulus upon nearly all plant cells. As a result of temperature drops membrane depolarization takes place (23). In excitable cells, the depolarization develops into an AP, as it is a case of Mimosa pudica (90,129), Biophytum sensitivum (120), Dionaea muscipula (67), Hibiscus rosa-sinensis (116), Zea mays (134), Cucurbita pepo (23,80,194-195), Cucumis sativus and Triticum aestivum (196), Luffa cylindrica (197), Populus trichocarpa (176), Conocephalum conicum (68), Physcomitrella patens [unpublished results] and numerous algae (14,35,197-198), and very likely for Arabidopsis hypocotyls (186). There have been differences in AP duration recorded after cold and other types of stimulation, with cold-AP lasting significantly longer, and no differences seen in AP amplitudes (68,116). Moreover, the lower the temperature, the slower the repolarization, which may simply reflect a dependency of an enzymatic activity of pumps on the temperature (= Q10 coefficient) (194). In unexcitable cell the cold-induced depolarization shapes after a stimulus strength and duration (199), and even if it surpasses the amplitude of 100 mV, it does not develop into an AP (23). Such magnitude originates form an influx of Ca2+ that is driven by a huge cell-interior directed electrochemical gradient (the negative transmembrane potential + the equilibrium potential ECa of circa +100 mV). More detailed experiments have demonstrated that these Ca-increases are adjusted by the rate of cooling irrespective of the absolute value and are ―sensitive‖ to previous stimulation, showing large desensitization (attenuation) (199). Consequently, not each consecutive cold treatment leads to APs in excitable plants (23,195,200) as well as Ca- influxes in unexcitable cells are hardly comparable when successively repeated (201). Since cold-induced Ca2+-flows were of interest of a host of researchers, their existence has been proved with the use of a plethora of experiments, e.g.: radioactive ions (202), voltage measurements (68), current measurements (199), luminescence measurements (186,203-204). 14 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz

As cold-induced calcium increases are the very first measurable cell responses, the cold- activated Ca-channels have been hypothesized to be temperature sensors in plants (205). Moreover, fast accommodation is one of the characteristics of a receptor system whose thresholds depends on the steepness of stimulus rise (195), which perfectly matches cold- activated Ca-currents. However, molecular entities and corresponding genes of Ca-channels have not been found yet, thus the channel-sensor hypothesis awaits verification. Another plausible explanation for cold-induced depolarization is an inhibition of plasmalemma H+-APTase (194-195,206-207). As mentioned above, the same may refer to light action linked with transient membrane depolarization. Both temperature and light are the so called physiological stimuli. As ambient environmental factors they control the whole plant metabolism shaping [ATP]cyt availability. Thus, it is not surprising that under certain circumstances they are able to evoke APs through [ATP]cyt-disturbances, and hence such APs can be named ―metabolic‖ (14). Since light and temperature act on the whole plant at a time, describing all the functions of ―metabolic APs‖ may be very problematic. In the case of cold stimulation, however, APs can be considered as ―hardening signals‖ (119), especially that AP-induced pre-adaptation has already been successfully provided for some plants, namely maize (208), wheat and cucumber (196).

Stress or Damage-associated APs

Not only physiological (pressure, light, temperature) but also notorious stimuli (burning, freezing, mashing, cutting) can make Mimosa and Biophytum fold down rapidly (90,120, respectively). Obviously, the sensitive plants can ―feel‖ pain, as Bose postulated already in 1926 (30). 50 years later after Bose‘s publication the view of plants sensing environmental danger became prevailing (149). After all, in many ―not-sensitive‖ species APs have been noted after harsh stimulation, e.g.: pine seedling (137), poplar (176-177), lupine (209), pea (210), broad bean (211), cucumber (212), tomato (213-215), beggartick (216-217), hibiscus (116), sunflower (160), Arabidopsis (218), barley (219), maize (173), liverworts (220), and various species of algae (14,139,221-222). In fact four types of electric signals have been noted in Chara (222) and two in higher plants after severe wounding (160,223-225); in the latter – ―fast‖ APs and ―slow‖ VPs, variation potentials. VPs are generated only if a xylem continuity is disrupted and a subsequent increase in xylem pressure takes place (212). Consequently, at saturating humidity, when xylem tension is negligible, VPs do not appear (225). Accordingly, the speed of conduction is related to the velocity at which water moves in the xylem and amounts from 0 to 7 mm/s (160,214). VPs cannot be evoked by electrical stimulation and are able to pass through the zone of killed plant tissue, which strongly differs them from APs (160). They simply are a manifestation of a pressure wave that makes stretch- activated channels open in living cells adjacent to the xylem (160). They also are responsible + 2+ for a transient shutdown of plasmalemma H -ATPase there, probably through [Ca ]cyt increases (225). As a consequence, two types of responses are recorded at a time after severe wounding – on the shoulder of VP, APs occur (160,223). Since VPs may ―interfere‖ with APs and because they never appear after electrical stimulation (160), thus a depolarizing current (DC) is often a stimulus of choice, when APs are to be explored. With the use of DC the view that excitable tissues act as ―neuroid‖ system was elaborated for: the lupine Lupinus angustifolius (17,19,226-227), the sunflower What Do Plants Need Action Potentials for? 15

Helianthus annuus (18,160,224), the cress Arabidopsis thaliana (228), the flytrap Dionaea muscipula (96), the waterwheel Aldrovanda vesiculosa (97), the sensitive plants Mimosa pudica (128) and Biophytum sensitivum (120), the potato spp. Solanum (229), the tomato Lycopersicon esculentum (229-231), the pumpkin Cucurbita pepo (232), the bean spp. Phaseolus (233), the buckwheat Fagopyrum sagittaeum (after (149)), the sorgo Sorghum bicolor (193), the willow Salix viminalis (234), the liverwort Conocephalum conicum (235), and numerous algae (reviewed by (38)). The careful reader must have already noticed, that every time APs are numbered in response to either non-damaging or severe stimulation the same plants are quoted, which simply reflects the fact that in an excitable cell/tissue/organ APs occur with a threshold stimulus but irrespective of the stimulus kind. Thus it is not surprising that DC is a means of evoking APs, that simplifies the experimental procedures, still allowing to deal with AP purposes and functions. The most splendid example is PIN (proteinase inhibitor) expression occurring systemically after wounding (213) as well as after electrical stimulation (229-231), thus proving that APs may control such a process as gene transcription and are meaningful for defence processes (236-237). In general, damage- or DC-evoked APs are linked with growth arrest (197,217), photosynthesis drops (107,131), enhancement of respiration (238-239), induction of ethylene emission (211), JA biosynthesis (86) and ROS generation (after (119)), which altogether resembles responses associated with danger perception (229). It is likely that under unfavourable circumstances plants stop growing and start self-defending, and that APs may synchronize both processes (86,240). Such a scenario perfectly corresponds to damage- 2+ triggered APs and repair-associated accomplishments in algae (241). In these taxa, [Ca ]cyt increases during the passage of an action potential; next, Ca2+ activates the protein kinase that phosphorylates myosin; this inhibits myosin interaction with actin and finally terminates cytoplasmic streaming (241-242). Cessation of streaming, in turn, grants the cell time for controlling damage. Moreover, cessation serves to protect the cell from leakage, while 2+ increased [Ca ]cyt participates in wound-clotting mechanism (241). It is tempting to speculate 2+ that phloem clotting, for which increased [Ca ]cyt is indispensable as well (243), also takes place after damage-induced AP passage and that such a succession of events has preserved in higher plants since algae acquired it. One of the consequences of tissue damage is a loss of turgor pressure that is sensed by adjacent intact cells (221-222), another - a release of molecules which being associated with a cell interior, when released, can serve signalling functions, e.g.: systemin, hydroxyproline- systemin, PEP, ATP/ADP, acetylocholine, GABA, free amino acids or even KCl (at high concentration). Both stimulations (pressure or chemicals) are known to bring about profound membrane potential changes, but only for wound-associated membrane stretching (221-222) and for a few cytoplasmic compounds (KCl, Gly, Glu and GABA) was an induction of APs reported (172,218-219,244-246). With the discovery of ionotropic glutamtate/glycine receptor genes in Arabidopsis, the quest for their role in plant physiology has begun (247). Though their functioning as Ca-permeable channels has only been indirectly proven, such a scenario fits perfectly to their putative role in AP generation (244). Their high expression in roots (248) may explain why these cells treated with amino acids generate APs that propagate to the leaves, where such APs induce changes in the rates of transpiration and photosynthesis (172). Pathogen attack, which is a quite common insult affecting plant development, is associated with tissue damage, too (249). However, pathogen recognition is most frequently 16 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz linked to a depolarization of insular characteristics (250). Therefore, in spite of tremendous amplitude (up to 150 mV), duration (over 1 h), and significance for plan survival (251), most of the pathogen-associated membrane potential changes are beyond the scope of this review. Nonetheless, it must be stressed here that AP function has already been suggested to be coupled with local/insular changes in ion concentration (Ca2+, H+, K+, Cl-), which lead to modified activities of enzymes in the cell wall (e.g. pectinase), the plasma membrane (e.g. cellulose synthetase, callose synthetase), and the cytoplasm (e.g. protein kinase), and may be indispensable for protection against injury and pathogen invasion (84,244). Accordingly, for AP-induced PIN expression only action potentials with a complete Ca2+ signature are required (86). Therefore, the notion has been put forward that AP propagation is a nonspecific component of signalling pathway which needs additional messengers to become specific (119). Such an additional role of ions (Ca2+, H+), sugars, aminoacids, nucleotides and phytochormones has long been known; more and more often VP and not-propagable potential changes are being included in the signalling network, too. It can be concluded that transmittable APs are the fastest systemic signal but represent just a part of the effector response (219,252). Quite obviously, there is an urgent call for further examination of AP- concurrent effector-sensor ―intermediators‖ and their dependencies on electrical membrane changes in plants (87). So prevailing are APs associated with stress or damage that they should be viewed as a kind of arms. The will to decipher the exact purposes of APs and AP-coupled sensor-effector links forces us to look at plants more carefully. A recent concept of viewing excitable plant cells as neurons is only partly justified (109). It seems like, for example, that plant APs carry no frequency-coded information (119,246). A series of AP occurring after severe wounding (14,218,220) should rather be connected with the leakage of excitatory compounds than with stimulus strength. Alternatively, AP series might fulfil a requirement for additional information, since a single AP means nothing unless followed by supplementary ―instructions‖ (86,105,115). Thus, apart from following AP-associated end responses (e.g. gene expression), dissecting the pathways of AP transmission and generation is equally important, as all these cells (sensors, conductors and effectors) seem to constitute AP-specific ―instructions‖ concurrently.

PATHWAYS OF TRANSMISSION

Bundles of phloem with companion cells and living cells of xylem (protoxylem, metaxylem); or the entire organ such as an active trap of Dionaea; or even the whole organism as it is a case of lower taxa (algae, mosses, liverworts); are pathways of AP transmission (88). As for higher plants, the living vascular bundles displaying highly negative membrane potential of circa -200 mV, having numerous plasmodesmata that guarantee good electrical conductivity over long distances, keeping low longitudinal resistance and being relatively insulated from the surrounding cortex (assuring minimal loss of excitation current) are the best suited for systemic and electrotonic transmission (177). Simultaneous acro- and basipetal direction results from the absence of ―rectifying‖ synapses. Instead, architecture of vascular system determines AP reach. Thus, the restricted areas exist, e.g. a base of peduncles of Biophytum sensitivum (120) or leaves of Helianthus annuus, whose vascular architecture What Do Plants Need Action Potentials for? 17 hampers APs from entering the petiole (224). In contrast, in Mimosa pudica (90), Vicia faba (238) or Arabidopsis thaliana (228) AP can ingress/leave leaves easily. The transmission along the vascular bundles takes place as a result of the co-operation of a number of cells, hence being preferred in stems rather than in leaves, after all. Moreover, most cells in leaves except conductive bundles are unexcitable in vascular plants but carnivores. Accordingly, no AP has ever been registered from mesophyll cells (except in carnivores), though local changes of a different character appear as a result of adjacent bundle excitation (219,244). Apart from excitation spreading excitable tissues in plants fulfil many other function (e.g. metabolite distribution, metabolite loading/unloading, photosynthesis, secretion, absorption). Since they have stop short of differentiating into nerve-like exclusively, the rate of AP transmission in plant (from 0.5 up to 300 mm/s) lags behind nerve impulse velocities (0.03 – 120 m/s). Still it is enough to shut up an organ within 100 ms or ―excite‖ the whole plant within a few minutes.

HOW TO RECORD AND MEASURE ACTION POTENTIALS

Electrode Techniques

Electrophysiology - a study of living objects, which deals with voltage, current, capacity, resistance or conductivity measurements and covers an ample variety of scales, beginning from entire organisms through excitable organs and cells to finish at a single channel activity level. Its goal is to describe the electrical properties of the living world. Classical electrophysiology make use of (micro)electrodes either placed outside or inside a living cell (100). The latter allows for the accurate measurement of a resting potential value and AP amplitude, a membrane capacitance, conductance and resistance; the former – for monitoring of AP occurrence, transmission and coincidence with physiological responses (177). With a multi-electrode installation an exact assessment of the transmission rate is possible. Extracellular recordings offer also such an advantage that the measurement can be conducted over several days (246). One must keep in mind, however, that during extracellular recordings electrode arrangement is of great importance for a few reasons: (i) the electrodes must be localized nearby excitable cells; (ii) there must be a link of sufficient resistance between the measuring and reference electrode to record potential drops; (iii) the electrodes must be localized on the way of excitation spread when physiological interdependence is to be worked out (224). With intracellular impalements, the difficulties may also begin when the exact positioning of a measuring electrode is important while working on an intact plant. This problem was solved by Wright and Fisher who used aphid‘s stylets to penetrate the phloem exclusively (253); the procedure was so suitable that it was used by the others, as well (132- 134). Equally prosperous is the method of placing the measuring electrode into substomatal cavities of the open stomata nearby an AP-conductive tissue (219,244,252). With the use of giant cells the step from intracellular recordings to voltage-clamp technique was taken quite smoothly (see Introduction). Even then, however, two severe problems remained: (i) spatially non-uniform voltage control; (ii) the lack of control over intracellular ionic composition (254). Solution to both problems came along with the patch- clamp technique which additionally allowed for a single channel measurements (9,255). AP- 18 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz clamp (76) and Self-clamp (256) techniques, in which an AP is recorded and repetitively replayed as the command voltage to the same cell under voltage control, proved to be reliable with excitable cells. Nowadays the sophisticated system of microstructured chips called a patchliner or planar patch-clamping facilitates an automatic and simultaneous patch-clamp analysis of many cells with high outputs (254). It is very useful for fast screening of channels; it is devoid of noises, very sensitive and designed to display an increased accessibility of the membrane for optical detection techniques (e.g. FRET - Fluorescence Resonance Energy Transfer) (254). Another sophisticated electrode technique is MIFE (Microelectrode Ion Fluxes Estimation) - a selective measurement of ion fluxes appearing near living cells (257), which originated form a vibrating probe (microelectrode) method (258). Both techniques are non- invasive and has a resolution of 2 - 20 micrometer in position and 10 seconds in time, which for membrane potential changes lasting a minute or longer is sufficient to be resolved (100). A typical MIFE measurement implements an ion-selective electrode and assesses the net flux of ions (nmol/m2s) on the basis of a change in ion concentrations (change in voltage of the ion-selective microelectrode) over a small known distance (184). An additional scanning function of a computer-controlled microelectrode position system offers two-dimension resolution via: SVET (Scanning Vibrating Electrode Technique) that can measure voltage gradients down to nV at a minimum speed of approximately 50 ms per scan point; or SIET (Scanning Ion-selective Electrode Technique) that can measure ion concentrations down to picomolar levels but at a slow speed of 500 - 1 000 ms per point so as not to disturb the measured ion gradient.

Optical Methods

Optical electrophysiological techniques were established to follow the one- or two- dimension distribution of electrical changes occurring in a living cell/tissue/organ. They are grounded in fluorescent techniques and make use of voltage sensitive dyes – the molecules capable of emitting light in response to applied voltage (259). After introduction of one or more such compounds into a cell via perfusion, injection or gene expression, the spatial and temporal patterns of electrical activity may be observed and recorded. Apart from voltage sensitive dyes, ion-selective fluorescent indicators can be engaged to monitor ion concentration changes during excitation. Commercially available Ca2+-, H+-, K+- and Cl--indicators [http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes- The-Handbook.html] can be introduced to excitable cells either through infiltration or by iontophoresis. The latter approach was successfully applied in algae to correlate membrane 2+ potential changes such as APs with [Ca ]cyt-increases (260). One can also imagine breeding of transgenic plants which would express apo-aequorin (a bioluminescent Ca2+-indicator) in excitable tissues exclusively; this is a futuristic idea, however.

Computational Studies

If a mathematical model of AP can be built up, its analysis may help to substantiate an experimental hypothesis, which indeed was the case for postulating osmotic changes during What Do Plants Need Action Potentials for? 19

APs or for proving intracellular Ca2+ involvement as well as H+-ATPase inhibition during a depolarization phase of APs or for reconstruction the main dynamical features of APs in plants (21,64,80,148,261-264). However, elaboration of such models is always restricted by the volume of experimental data. Nevertheless, modifications of the models could be a tool for theoretical investigation. Recently, an assemble of the number of AP models has been suggested as a means for the analysis of AP propagation (80).

Working on Mutants

Since the ion mechanism of APs elaborated on algae turned out to be consistent for all plants, the full knowledge of the channel proteins/genes involved in plant excitability has been on its way. The involvement of voltage-gated channels is unquestionable and surely comprises voltage-dependent Cl-- (56) and K+-channels (265). However, the voltage control over Ca2+-conduits is only assumed. Besides CNGC (Cyclic Nucleotide Gated Channels) and GLR (Glutamtate Receptor Like) nothing is known about the molecular entities of the plasmalemma calcium ―passive conductors‖ (266). Accordingly, stretch-, cold- or light- regulated Ca2+-channels remain as presumptions. No better situation appears with putative genes for intracellular Ca2+-conduits, as there is little direct evidence linking their products to intracellular calcium increases (267). The same holds true for Cl--channels, whose gene identification is in infancy (268). With the recently characterized S-type Cl-channels (SLAC and SLAH - (269-270)) a quest for voltage-gated Cl--conduits has just begun. Light- (186- 187,271) or stretch-activated anion channels (272) still need to be identified at the genetic level. As for genes and their products voltage-gated K+-channels are unique; they are Shaker- type inward (AKT1, AKT2-3, AKT5, AKT6, KAT1, KAT2, silent KC1) and outward (GORK, SKOR) rectifiers, which are very well described and genetically, molecularly and functionally characterized (273). Nevertheless, their involvement in membrane excitability has not been examined in detail. Neither has this been done for any of the mutants of the above mentioned channels. Thanks to enormous progress in genetics, a cornucopia of channel genes and channel mutants is ultimately expected. As most of these mutants are commercially available right away, working on such plants will open new perspectives for electrophysiology.

CONCLUSION

Most if not all of the plants possess excitable tissues. Action potentials in plants arose independently of those in metazoan excitable cells, nevertheless some analogies to animal APs can be found (195,274). For instance, they coincide with movement. They occur in mobile excitable organs such as traps/leaves or pistil to function in movement/turgor regulation. Moreover, they are also generated and transmitted in immobile parts of a plant to carry out intercellular and intracellular signalling indispensible for growth, photosynthesis and respiration adjustment, stress/danger perception and self-defence commencement (88). In spite of a lack of purely specialized cells devoted to AP transmission exclusively, plants are able to spread information systemically. This long distance communication is guaranteed by 20 Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz electrically coupled plasma membranes of excitable cells constituting conductive bundles. Apart from systemic transmission, AP-associated local signalling accomplished by changes in the subcellular localization of ions (Ca2+, H+, K+, Cl-) and perhaps membrane depolarization itself is equally important (252,275).

ACKNOWLEDGMENTS

This work was supported by the Ministry of Science and Higher Education Grant No. N N301 464534.

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