Massimo Maffei, Plant Physiology Unit, Department of Plant Biology, University of Turin, Innovation Centre, Via Quarello 11/A 10135 Turin, Italy

August, 2010

Introduction

Plant defenses against herbivory are mediated by a comprehensive network of interacting signal transduction pathways downstream of sensors/receptors that recognize the attacking herbivore (see textbook pp. 705–715 on induced plant defenses against insect herbivory). Many aspects of plant-insect interaction resemble the initial key events occurring in the response of plants to microbes. Recognition can occur through physical interaction—through adhesins, fimbriae, flagella, or signaling by small molecules. Early events during pathogen attack (before gene expression) involve the release of cell wall oligosaccharides (so called elicitors), which can be recognized by specific receptors able to trigger signaling cascades involving ion fluxes and activation of reactive oxygen species (ROS) forming enzymes. Two of the earliest occurrences following recognition are a Ca²⁺ flux across the plasmalemma and the generation of the superoxide anion and H₂O₂, the so called “oxidative burst,” with these two events appearing to be mutually regulated. The generation of the plant oxidative burst has been linked to the initiation of electron flow across the plasmalemma via a NADPH oxidase complex, analogous to that found in mammalian neutrophils.

The ablity of plants to withstand biotic stresses depends on their ability to quickly recognize and adequately respond to a wide array of attacking biotrophs. Current research in plant-insect interaction is focusing mainly on genomics and proteomics which are late events induced by biotic stress. In contrast, events within the first seconds to minutes, which are responsible for recognition and triggering of signal transduction pathways, are still poorly understood. Early events in plant-insect interactions start from damage-induced ion imbalances, causing variations in membrane potentials, Ca²⁺-signaling, production of reactive oxygen species, kinase activities, phytohormones, and their cross-talk up to processes that precede gene expression (Maffei et al. 2007a).

Plants are continuously interacting with the external world. The coordination of internal processes and their balance with the environment are connected with the excitability of plant cells. The primary candidate for intercellular signaling in higher plants is the stimulus-induced change in plasma membrane potential (Maffei et al. 2007a). The plasma membrane of plant cells is the only compartment in direct contact with the environment and represents a sensing element able to recognize changes and to initiate cascades of events eventually leading to specific responses. Changes in plasma transmembrane potential (V_m), or modulation of ion fluxes at the plasma membrane level, are amongst the earliest cellular responses to biotic and abiotic stresses. By using this sophisticated sensing system, plants are able to discriminate mechanical wounding (MW) from herbivore wounding (HW) (or more generally, biotic wounding). It has been recently demonstrated that V_m variations occurring after herbivore feeding on lima bean (Phaseolus lunatus L.) leaves are significantly higher than those occurring after a single or a repeated mechanical wounding (Maffei et al. 2004) and the same is true for other biotic interactions. Thus, the direct interference of the biotic agent adds something very important to the tissue damage, starting a chemical interaction with plant cells.

In plants, a variety of electrical phenomena have been described: local voltage transients vanish after a distance of a few millimeters, action potentials (APs) and so-called variation potentials (VPs) may carry information over long distances from organ to organ. Herbivory-induced V_m changes are preceded by a fast electrical signal (APs) that travels through the entire plant from the point of origin of the perceived input.

While the action potential is a momentary change in electrical potential on the surface of a cell which can propagate up to 40 m sec^-1 (which is too fast for the movement of a chemical signal along with either phloem or xylem), V_m changes are much slower: for a distance of 6–7 cm (dimensions of lima bean leaves), the overall process takes about 5-6 min and involves signaling molecules traveling with the same speed (approximately 1 cm min^-1). Moreover, as recently pointed out by Zimmermann and co-workers (2009), apoplastic ion flux analysis reveals that, in contrast to action or variation potentials, ion movements occur after the voltage change begins, suggesting that these wound-induced “system potentials” might represent a possible way of electrical long-distance signaling in higher plants.

Of particular importance in early recognition between the host and pathogen/herbivore is the role of signal molecules which can affect V_m either directly or via receptors. In the case of lima bean, herbivore attack is associated with: 1) a strong V_m depolarization at the bite zone causing a wave of V_m depolarization spreading throughout the entire attacked leaf; 2) a consistent influx of Ca²⁺ (at the very edge of the bite), which is halved by application of the Ca²⁺ channel blocker Verapamil; and a ROS burst. Larvae regurgitants (R) and N-acyl-amino acid conjugates interact with the plasma membrane and alter V_m. R from lima bean-reared larvae altered V_m in a concentration-independent fashion and its effect is clearly different from that observed in V_m studies with the individual compounds (Maffei et al., 2004). The time-course and distance-dependence spreading of the V_m depolarization upon herbivore attack in intact leaves is probably associated with a molecule able to disperse within tissues at a relatively high speed. Studies done perfusing leaves with H₂O₂, Ethephon and ABA indicate a V_m depolarizing effect of these molecules. Another interesting target is the analysis of the early events in the interaction of volatiles (including VOCs, ethylene, hydrogen peroxide and NO) emitted from wounded plants and/or perceived by neighbored healthy plants (see also Web Essay 23.5: The Plant Volatilome).

In this essay we will explore how electrophysiology can be applied to evaluate early responses of a legume, lima bean (Phaseolus lunatus L.), to a generalist herbivore, Spodoptera littoralis.

Materials and Methods

The system we developed to measure V_m in leaves after insect attack is the result of many technical tests which gave at the end a useful set of both electrical, electronic and hydraulic instrumentations, with the aim of on-line (or real-time) recording of electrical variations through the plant plasma membrane (Maffei and Bossi 2006). This system is shown in Figure 1.

Figure 1   Schematic representation of the system used for the evaluation of V_m in leaf segments. (Modified from Maffei and Bossi 2006).

V_m can also be recorded in whole tissues. To this end we developed a chamber where molecules diffused by air can interact with leaves. Figure 2 shows how a cutting bearing entire leaves can be analyzed. The membrane potential is captured with a glass electrode directly on an entire leaf fixed on a stative.

Figure 2   Scheme of the new system used for the detection of V_m in intact leaves. (Modified from Maffei and Bossi 2006).

Results

The results of the measurement of membrane potential after MW and HW indicate a specific response of the leaf tissue. In lima bean, leaf V_m varies according to the cell type: epidermal cells have an average V_m of -50 mV (± 5.7 mV), guard cells have an average V_m of –200 mV (± 12.2 mV), palisade cells have an average V_m of -140 mV (± 9.8 mV), and spongy parenchyma cells have an average V_m of -100 mV (± 10.5 mV). Lima bean palisade cells are the most responsive cells when leaf tissues are attacked by herbivores. When V_m is evaluated at increasing distances from the site of damage, the response is a strong V_m depolarization in the bite zone, followed by a transient V_m hyperpolarization and, finally, a constant V_m depolarization throughout the rest of the attacked leaf (Figure 3). The V_m of the MW leaf (control, represented by the green bars) does not significantly change. Exponential interpolation of V_m data shows a strong V_m depolarization up to about 1.5 mm from the bite zone, whereas a V_m hyperpolarization is found at about 2.5-3 mm from the bite zone, immediately followed by a second strong V_m depolarization. V_m differences from control in the zone from 3.5 to about 6 mm from the bite zone are not significant, but V_m displayed depolarized values from 6 mm throughout all the HW leaf (see Figure 3).

Figure 3   Lima bean (Phaseolus lunatus) leaf V_m values as a function of distance from the bite zone 15 min after herbivore damage. The histogram superimposed on lima bean leaf wounded by a larva of Spodoptera littoralis represents V_m values (and standard deviations) measured at increasing distances from the bite zone. The green bars represents the average V_m value from a mechanically wounded lima bean leaf. In the close vicinity of the bite zone (up to 1.5 mm) there is a strong drop in the V_m (depolarization), whereas at about 2.5-3 mm from the bite zone an increase of V_m is observed (hyperpolarization). About 6 mm from the bite zone throughout all leaf there is a constant V_m depolarization. (Modified from Maffei et al. 2004).

To probe whether the feeding activity of the herbivore is perceived as a V_m variation even at considerable distances from the bite zone in the same leaf, an intact leaf from a potted plant was fixed to the V_m apparatus and the V_m determined. When V_m reached a constant value, S. littoralis was allowed to start its feeding activity. Figure 4 depicts V_m variations as a function of time and distance from MW lima bean leaf tissue. Feeding activity starts a series of V_m variations eventually leading to V_m depolarization within the first 15 min after the onset of the feeding activity. The recognition of the bite activity of S. littoralis is quickly perceived in the same leaf at increasing distances from the bite area. However, the attempt to find variations in neighboring opposite leaves (OL) resulted in no obvious variations as did MW on the same leaf.

Figure 4   Time-course of the V_m variations in palisade cells distant 5, 30, and 60 mm from the bite activity of the herbivore Spodoptera littoralis. The feeding larva induces a series of V_m variations leading to V_m depolarization after about 15 min from the onset of the feeding activity. (Modified from Maffei et al. 2004).

In response to herbivory, lima bean leaves are able to produced H₂O₂, in concentrations which were higher when compared to MW leaves. Cellular and subcellular localization analyses revealed that H₂O₂ is mainly localized in MW and HW zones and spreads throughout the veins and tissues. Furthermore, increasing amounts of added H₂O₂ correlated with a higher cytosolic calcium ([Ca²⁺]_cyt) concentration (Maffei et al. 2006).

Since Ca²⁺ can be present and stored in several cell compartments in different forms as well as in the buffering solution, one way to assess Ca²⁺ involvement is perfusing MW and HW tissues with Ca²⁺ chelating agents or transport inhibitors.

Chelation of extracellular Ca²⁺ by EGTA has been found to completely abolish the increase in [Ca²⁺]_cyt and the activation of downstream responses in several plant cells. However, when 250 μM EGTA was applied to MD (Figure 5A) and HW (Figure 5B) leaves, no significant differences were found in comparison to control tissues (i.e., tissues perfused with the sole 15 mM H₂O₂).

Figure 5   Effect of increasing H₂O₂ concentrations on V_m of mechanically damaged (A) and herbivore wounded (B) lima bean leaves. A. Increasing H₂O₂ prompts increased transient V_m depolarization up to 600 ppm. Washing tissues with fresh buffer caused V_m hyperpolarization. B. Herbivore-derived wounding caused a significant V_m depolarization (compare starting V_m values in A and B). Increasing H₂O₂ concentrations caused a transient V_m depolarization which is followed by a V_m hyperpolarization. Bars indicate standard deviation. (Modified from Maffei et al. 2006.)

In lima bean the use of Verapamil, a voltage-gated Ca²⁺ channel antagonist, reduced the Ca²⁺ influx after MD and HW (Maffei et al. 2004). Preincubation of lima bean leaves with 100 μM Verapamil completely suppresses H₂O₂-dependent V_m depolarization in MD leaves (see Figure 5A). Increased values of [Ca²⁺]_cyt may also depend on release of Ca²⁺ from internal stores. Another important inhibitor is ruthenium red, which has been successfully used as an inhibitor of Ca²⁺ release from internal stores. After incubation of MD and HW leaves with ruthenium red, application of 15 mM H₂O₂ showed the same effects. In both, MD and HW leaves, ruthenium red completely suppressed V_m depolarization (Figure 6). Mechanically induced oxidative burst in soybean cells was prevented by gadolinium (Gd), an inhibitor of stretch-activated channels, therefore suggesting the involvement of these channels in oxidase activation. Gd was also efficient to prevent the activation of the hypoosmotically induced oxidative burst in tobacco cell suspensions. When 100 μM Gd was applied to MD leaves, the same suppression of H₂O₂-dependent V_m depolarization caused by Verapamil could be observed (see Figure 6A). Even in this case HW leaves did not show any difference when Gd was applied (see Figure 6B).

Figure 6   Effect of chelation of extracellular Ca²⁺ by EGTA and inhibition of Ca²⁺ uptake/release by specific inhibitors on H₂O₂-dependent V_m depolarization in mechanically damaged (A) and herbivore wounded (B) lima bean leaves. The effect of 600 ppm H₂O₂ without the use of inhibitors is indicated; metric bars represent standard deviation. (Modified from Maffei et al. 2006).

Thus, in MW leaves, verapamil, Gd and ruthenium red, the latter a potent inhibitor of the release of Ca²⁺ from internal stores, completely suppressed H₂O₂-induced V_m depolarization, whereas the use of the Ca²⁺ chelator EGTA did not exert any effect on both MW and HW leaves. Evidently, the removal of free Ca²⁺ from the extracellular space (and buffer solution) was not sufficient to abolish V_m depolarization. On the other hand, the blockage of voltage-gated channels and internal store release of Ca²⁺ indicated that these two sources of Ca²⁺ could be associated with V_m depolarization. However, it cannot be excluded that Verapamil, Gd and Ruthenium red may target the same calcium pool in this system.

One of the many questions arising from these results was: How fast is V_m depolarization in leaf tissues? What is the nature of the depolarizing agent?

In order to evaluate the speed of depolarization in an intact leaf we grounded the basal part of the lima bean stem and impaled a palisade cell with a micropipette (see Figure 2). When the V_m was stable (around -140 mV) mechanical damage was done with a small scissor at different distances from the impaled cell on the same leaf. An immediate action potential was recorded, and when V_m was again stable a drop of a strong oxidant was applied at the wounded zone. After the application of the oxidant a small action potential was also recorded. After a few seconds a significant V_m depolarization was recorded and the original resting potential was not re-established, indicating that the voltage change was not an action potential. The V_m was then allowed to reach a stable value. The new value was a V_m hyperpolarized value with respect the beginning of the experiment. A second damage was then performed in another part of the leaf and an action potential was recorded, once again a drop of a strong oxidant was applied and a V_m depolarization was observed after a longer period (Figure 7). By using the data of the latter experiment we can try to calculate the theoretical speed of V_m depolarization. In lima bean, the speed of the fastest V_m depolarization after a strong oxidant application was found to be about 1 mm sec^-1, and the speed of the slowest V_m depolarization was 4 mm sec^-1, that is 0.1 cm sec^-1 and 0.4 cm sec^-1_, respectively.

Figure 7   Action potentials and membrane potentials (V_m) in lima bean intact leaves in response to mechanical wounding and application of a strong oxidant. (Modified from Maffei and Bossi 2006.)

Concluding Remarks

The few examples given in this essay show that depolarization of the V_m is one of the first responses of the plasma membrane, which is mainly depending on ion fluxes, including the calcium release from internal stores or influx from the apoplast. Thus electrophysiology is indeed a valuable tool to study and understand what is going on at the very beginning of plant interaction with other organisms (including other plants) and V_m evaluation, more that the single patch analysis, gives a tissue image of cooperative interplay among wounded and unwounded cells.

In our specific case-study, understanding insect–plant interactions is of interest not only from an ecological and evolutionary perspective but also for the development of novel crop protection strategies. Owing to the massive damage that herbivores cause to valuable crops, the deciphering of early signals from plants represent one of the most exciting fields of research in the first line defense. Recent studies have advanced our understanding of the mechanisms by which plants recognize herbivores and subsequently activate direct and indirect defense responses. Three areas where future efforts might result in major breakthroughs are related to the identification of herbivore-specific signal molecules, their recognition, and further signal transduction. The challenge for further research in this area is to determine their mode of action, whether these signals are transduced by receptor-mediated processes or simply interact with the plant membranes thereby initiating signal transduction pathways. One approach to achieve this goal might be the use of plant mutants that are not responsive to a particular herbivory-related signal. Characterization and the use of such mutants could result in the identification of both the genes encoding proteins involved in signal perception or proteins acting downstream in signal transduction and regulation of the defense response. Such an approach might yield important insight into the nature, the organization, and the integration of signal perception/transduction. There is a general need to identify putative components of signal transduction pathways by using biochemical and genetic methods, to study their possible interactions, and to analyze causal relationships (Maffei et al. 2007a, Maffei et al. 2007b).

Not only can such studies uncover individual signaling pathways, but they can also establish links in a network of alternative routes regulating the multitude of inducible plant defenses. Much more has to be done in this field, but the promising results obtained in intact rooted plants following biotic and abiotic stress may lead to interesting new discoveries.

References

Maffei, M., and Bossi, S. (2006) Electrophysiology and Plant Responses to Biotic Stress. In Plant Electrophysiology – Theory and Methods. Edited by Volkov, A. pp. 461-481. Springer-Verlag, Berlin.

Maffei, M., Bossi, S., Spiteller, D., Mithofer, A. and Boland, W. (2004) Effects of feeding Spodoptera littoralis on lima bean leaves. I. Membrane potentials, intracellular calcium variations, oral secretions, and regurgitate components. Plant Physiology. 134: 1752-1762.

Maffei, M.E., Mithofer, A., Arimura, G.I., Uchtenhagen, H., Bossi, S., Bertea, C.M., Cucuzza, L.S., Novero, M., Volpe, V., Quadro, S. and Boland, W. (2006) Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide. Plant Physiology. 140: 1022-1035.

Maffei, M.E., Mithofer, A. and Boland, W. (2007a) Before gene expression: early events in plant-insect interaction. Trends in Plant Science. 12: 310-316.

Maffei, M.E., Mithofer, A. and Boland, W. (2007b) Insects feeding on plants: Rapid signals and responses preceding the induction of phytochemical release. Phytochemistry. 68: 2946-2959.

Zimmermann, M.R., Maischak, H., Mithofer, A., Boland, W. and Felle, H.H. (2009) System Potentials, a Novel Electrical Long-Distance Apoplastic Signal in Plants, Induced by Wounding. Plant Physiology. 149: 1593-1600.