WHAT DO PLANTS NEED ACTION POTENTIALS FOR

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.

Elżbieta Król, Halina Dziubińska and Kazimierz Trębacz 2

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 Ca

2+ 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


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-


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


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,

electrical stimulation, cold, light) (67). They depend, however, on [Ca2+

]ext in such a way that

the amplitude of AP follows [Ca2+

]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


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


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


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


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


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


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 Ca

2+ concentration as well (179). Ca

2+ 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 Ca

2+-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


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).


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

for a transient shutdown of plasmalemma H+-ATPase there, probably through [Ca

2+]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


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-

triggered APs and repair-associated accomplishments in algae (241). In these taxa, [Ca2+

]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

increased [Ca2+

]cyt participates in wound-clotting mechanism (241). It is tempting to speculate

that phloem clotting, for which increased [Ca2+

]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


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


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-


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

potential changes such as APs with [Ca2+

]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


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


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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