Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. I. Membrane Potentials, Intracellular Calcium Variations, Oral Secretions, and Regurgitate Components1

Massimo Maffei, Simone Bossi, Dieter Spiteller, Axel Mitho¨fer, and Wilhelm Boland* Department of Plant Biology, University of Turin, Turin, Italy (M.M., S.B.); and Max Planck Institute for Chemical Ecology, D–07745 Jena, Germany (D.S., A.M., W.B.)

Membrane potentials (Vm) and intracellular calcium variations were studied in Lima bean (Phaseolus lunatus) leaves when the Mediterranean climbing cutworm (Spodoptera littoralis) was attacking the plants. In addition to the effect of the feeding insect the impact of several N-acyl Glns (volicitin, N-palmitoyl-Gln, N-linolenoyl-Gln) from the larval oral secretion was studied. The results showed that the early events upon herbivore attack were: a) a strong Vm depolarization at the bite zone and an isotropic wave of Vm depolarization spreading throughout the entire attacked leaf; b) a Vm depolarization observed for the regurgitant but not with volicitin {N-(17-hydroxy-linolenoyl)-Gln} alone; c) an enhanced influx of Ca21 at the very edge of the bite, which is halved, if the Ca21 channel blocker Verapamil is used. Furthermore, the dose-dependence effects of N-acyl Gln conjugates- triggered influx of Ca21 studied in transgenic aequorin-expressing soybean (Glycine max) cells, showed: a) a concentration- dependent influx of Ca21; b) a configuration-independent effect concerning the stereochemistry of the amino acid moiety; c) a slightly reduced influx of Ca21 after modification of the fatty acid backbone by functionalization with oxygen and; d) a comparable effect with the detergent SDS. Finally, the herbivore wounding causes a response in the plant cells that cannot be mimicked by mechanical wounding. The involvement of Ca21 in signaling after herbivore wounding is discussed.

Several plant species, including Lima bean (Phase- 2000a). Expression of these genes very often requires olus lunatus), when attacked by herbivores emit some of the early events in the signal transduction volatiles that attract natural predators of the damag- cascade such as calcium influx and protein phosphor- ing insects. This signaling by the plant to higher tro- ylation/dephosphorylation, jasmonate, ethylene, and phic levels has been interpreted as the plant’s cry for salicylates (Blumwald et al., 1998; Poppy, 1999; help (Dicke and Sabelis, 1992; Turlings et al., 1995; Arimura et al., 2000a, 2002; Winz and Baldwin, 2001). DeMoraes et al., 1998) and involves at least three The first events following leaf wounding and in- different levels of trophic interaction (Agrawal, 2000). troduction of the herbivore elicitors are not well un- Thus, volatile plant compounds released in response derstood, but in several cases the activation of the to insect feeding are directly associated with the octadecanoid-signaling cascade has been demon- feeding herbivore, which allows the plant to differen- strated (Koch et al., 1999, and references cited therein). tiate between mechanical wounding and wounding However, damaging leaves also generates a first-line caused by the chewing insect (Pare´ et al., 1998). In fact, of cell reactions involving both electrical signals and upon herbivore attack, plants activate a series of genes production of reactive oxygen species. indicating that feeding insects are able to elicit and up- Plants are continuously interacting with the external regulate defense in plants (Thaler, 1999; Baldwin et al., world. The coordination of internal processes and 2001; Schittko et al., 2001; Hui et al., 2003). Further- their balance with the environment are connected with more, uninfested Lima bean leaves activate defense the excitability of plant cells. The primary candidate genes when exposed to volatiles from conspecific for intercellular signaling in higher plants is the leaves infested with herbivores, e.g. spider mites stimulus-induced change in plasma membrane poten- (Tetranychus urticae), but not when exposed to volatiles tial (Vm; Labady et al., 2002). Vm are the result of an from artificially wounded leaves (Arimura et al., imbalance in the quantity of cations and anions across biological membranes. However, the binding of many plant natural products to membranes causes confor-

1 mational changes in the ion channels and membrane- This work was supported in part by the Department of Plant bound proteins as well as formation of new ion Biology (Turin) and by the MIUR-ITALY (grant ex 60%). Financial channels or pores, increasing or decreasing ion flow support by the Fonds der Chemischen Industrie, Frankfurt a.M., is gratefully acknowledged. (Warber, 1998; Engelberth et al., 2001; Maffei et al., * Corresponding author; e-mail [email protected]; fax 49–3641– 2001). Recent electrophysiological studies allowed to 571202. identify the involvement of a rapid electrical signal in Article, publication date, and citation information can be found at root to shoot communications in Sorghum bicolor www.plantphysiol.org/cgi/doi/10.1104/pp.103.034165. (Mishra et al., 2001), whereas in soybean (Glycine

1752 Plant Physiology, April 2004, Vol. 134, pp. 1752–1762, www.plantphysiol.org Ó 2004 American Society of Plant Biologists Effects of Feeding Spodoptera littoralis on Lima Bean Leaves

Figure 1. Lima bean leaf Vm 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 S. littoralis represents Vm values (and SD) measured at increasing distances from the bite zone. The dotted line (and its SD) represents the average Vm 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 Vm (depolarization), whereas at about 2.5 to 3 mm from the bite zone an increase of Vm is observed (hyperpolarization). About 6 mm from the bite zone throughout all leaf there is a constant Vm depolarization. max) phloem appears to participate in the transmission Recently, it has been demonstrated that when plants of fast root-to-shoot action potentials upon stress are wounded, jasmonate is synthesized and employed (Shvetsova et al., 2001). as a long-distance signal that activates the wound The Vm of the plasma membrane, which lies in the response program in unwounded leaves (Stratmann, range of 120 to 200 mV in plant cells, may be shifted 2003). Also the floral scent methyl jasmonate has been either to more negative (hyperpolarization) or to more demonstrated to be involved in gene-activation con- positive values (depolarization) in response to various trol and systemic long-distance signaling (Cheong and biotic or abiotic stresses. In plant cells Ca21 plays a key Choi, 2003). Much less is known about the individual physiological role as intracellular second messenger. It components from the salivary secretions of the feeding is especially important for the maintenance of cellular insects that lead to the up-regulation of the jasmonate homeostasis and signal transduction pathways (Evans signaling. Volicitin, N-(17-hydroxylinolenoyl)-Gln, et al., 1991; Pin˜eros and Tester, 1997; Roh et al., 1998). a major component from the regurgitant of Spodoptera Among the Ca21-permeable channels characterized in littoralis Boisd. larvae, has been shown to induce the plasma membrane of plant cells, voltage-activated a systemic release of volatiles from maize (Zea mays) Ca21 channels may contribute to calcium signaling plants (Alborn et al., 1997) and N-acyl Glus, for (Thuleau et al., 1998). Interestingly, the plant plasma example N-linolenoyl-Glu, from the regurgitant of membrane contains at least three distinct classes of the tobacco hornworm (Manduca sexta) elicit nicotine voltage-activated Ca21 channels stimulated by hyper- biosynthesis in tobacco (Nicotiana tabacum) leaves. polarization (Gelli and Blumwald, 1997), depolariza- Both compound types are highly surface active tion (Pin˜eros and Tester, 1995) and voltage insensitive amphiphiles and were shown to act via the jasmonate channels (White, 2000), respectively. pathway (Halitschke et al., 2001; Schmelz et al., 2003).

Plant Physiol. Vol. 134, 2004 1753 Maffei et al.

Other molecules are also involved in wound responses variations superimposed on the wounded Lima bean and the most studied are H2O2 (Orozco-Ca´rdenas et al., leaf tissue. The ordinates represent Vm expressed in 2001; Pellinen et al., 2002), salicylic acid (Shah, 2003), mV, while in the abscissa the bands (and the cor- and ethylene (Winz and Baldwin, 2001). responding histogram bars) represent different dis- So far, most of the work on plant-insect interaction tances (and the corresponding Vm values) from the has been done on gene activation and evaluation of the bite zone. The Vm of the mechanically wounded leaf various elements of the signaling pathway in plant (control) is represented by the dashed line. Expo- cells. To our knowledge almost nothing is known nential interpolation shows the trend of Vm variation. about the early signals upon herbivore attack at the A strong Vm depolarization was found up to about membrane level and the connections between in- 1.5 mm from the bite zone, whereas a Vm hyperpo- dividual components of the salivary secretions of the larization was found at about 2.5 to 3 mm from the insects and the subsequent up-regulation of the bite zone, immediately followed by a second strong octadecanoid biosynthesis. In this work we present Vm depolarization. Vm differences from control in the data on the early events during feeding of S. littoralis zone from 3.5 to about 6 mm from the bite zone on Lima bean leaves and on the effect of individ- were not significant, but Vm displayed depolarized ual, especially the surface active spit components, on values from 6 mm throughout all the attacked leaf changes in the Vm and their correlation with intra- (Fig. 1). cellular calcium variations. The trend of the Vm variation prompted a series of experiments aimed to better understand the nature and the reasons for this effect. The first attempt was RESULTS to probe whether the feeding activity of the herbivore was perceived as a Vm variation even at Effect of Feeding S. littoralis on Lima Bean Leaf Vm considerable distances from the bite zone in the same leaf. An intact leaf from a potted plant was In plants unable to produce constitutive defenses fixed to the V apparatus and the V determined. such as glandular trichomes filled with irritating es- m m When V reached a constant value S. littoralis was sential oils or morphological structures such as thorns m allowed to start its feeding activity. Figure 2 depicts and stinging hairs, the attack of herbivores is a de- V variations as a function of time and distance vastating experience, leading to the destruction of fed m from mechanically wounded (MW) Lima bean leaf tissues. However, some plants react to this event by tissue, starting with a potential of about 137 mV, producing volatile organic compounds able to reduce and V from a leaf under attack by S. littoralis.Itis wounding by attracting predators of the attacking m herbivore. This type of induced defense requires re- cognition of the attacking herbivore and the specific interactions of the plant physiology with the oral secretions. The primary candidate for intercellular signaling in higher plants is the stimulus-induced (herbivore wounding) change in Vm (Shvetsova et al., 2001). The results of the measurement of Vm after mechan- ical wounding and herbivore attack indicate a specific response of the leaf tissue. Lima bean leaf Vm varies according to the cell type. Preliminary tests on intact leaves allowed evaluating the average Vm of epider- mal, guard cell, palisade, and spongy parenchyma cells. Epidermal cells have an average Vm of 50 mV (65.7 mV), guard cells have an average Vm of 200 mV (612.2 mV), palisade cells have an average Vm of 140 mV (69.8 mV), and spongy parenchyma cells have an average Vm of 100 mV (610.5 mV). Different trials Figure 2. Time-course of the Vm variations in palisade cells distant 5, demonstrated that Lima bean palisade cells are the 30, and 60 mm from the bite activity of S. littoralis. The feeding larva most responsive cells when leaf tissues are attacked by induces a series of Vm variations leading to Vm depolarization after larvae of S. littoralis. about 15 min from the onset of the feeding activity. In particular, when V was taken at an average distance of 5 mm, a strong and transient To study the early effects at the bite zone and m subsequent signal spreading, V was evaluated at in- hyperpolarization occurred within 5 min after the herbivore bite, m followed by a constant depolarization. The same pattern was observed creasing distances from the site of damage. The re- when Vm was measured at a distance of 30 mm from the bite zone, but sponse was a strong Vm depolarization in the bite depolarization was higher than in cells at 5 mm distance. In cells zone, followed by a transient V hyperpolarization m 60 mm distant from the bite zone, Vm depolarization occurred within 2 and, finally, a constant Vm depolarization throughout to 3 min after the bite, and no hyperpolarization was observed. MW ¼ Vm the rest of the attacked leaf. Figure 1 shows the Vm value after mechanical wounding; OL ¼ Vm value of the opposite leaf. 1754 Plant Physiol. Vol. 134, 2004 Effects of Feeding Spodoptera littoralis on Lima Bean Leaves

to concentration. In fact, perfusion with 100 mgmL1 R depolarized Vm more than perfusion with 250 mg mL1, but less than perfusion with 500 mgmL1. Interestingly, when R was washed out with fresh buffer, palisade Vm experienced a hyperpolarization for all concentrations, with an opposite trend as ob- served during depolarization (Fig. 3). Since previous studies have demonstrated that R of S. littoralis contains several surface active, amphiphilic compounds, especially N-acyl Gln conjugates (Alborn et al., 1997; Pohnert et al., 1999a; Spiteller and Boland,

Figure 3. Effect of S. littoralis oral secretions and regurgitants (R) on the Vm of Lima bean palisade cells. At the lowest concentration 1 (100 mgmL ) R caused an intermediate Vm depolarization when com- pared to concentrations of 250 and 300 mgmL1. After washing the tis- sues with fresh buffer (double arrow) Vm was hyperpolarized at all concentrations, and once again R at 100 mgmL1 had an intermediate value. The single arrow indicates start of perfusion after Vm stabili- zation. Metric bars ¼ SD.

evident that feeding activity starts a series of Vm variations eventually leading to Vm depolarization within the first 15 min after the onset of the feeding activity. In particular, when Vm was taken from palisade cells at an average distance of 5 mm a strong and transient hyperpolarization occurred within 5 min after the herbivore bite, followed by a constant depolarization. The same pattern was observed when Vm palisade cell was measured at a distance of 30 mm from the bite zone, but depolarization was higher than in cells at 5 mm distance. Finally, in palisade cells that were 60 mm distant from the bite zone Vm depolarization occurred within 2 to 3 min from the bite event and no hyperpolarization was observed (Fig. 2). From Figure 2 it is evident that 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 leaves (OL) resulted in no obvious variations as did mechanical wounding (MW) on the same leaf (Fig. 2). Figure 4. A, Vm changes after perfusion of Lima bean leaves with 17-R,S-volicitin (¼ N-[17R,S-hydroxylinoleoyl]-L-Gln) and the natu- rally occurring 17S-volicitin (¼ N-[17S-hydroxylinoleoyl]-L-Gln). At all concentrations, both the racemic mixture and the natural volicitin Effect of S. littoralis Regurgitate and Regurgitate caused no variation of Vm. B, Effect on the Vm of different N-palmito- 1 Components on Lima Bean Leaf Vm leoyl-L-Gln concentrations. The highest concentration (300 mgmL ) 1 had lower Vm hyperpolarization effects than 100 mgmL , whereas at In order to evaluate which molecule may be re- 1 25 mgmL N-palmitoleoyl-L-Gln caused a constant Vm depolarization sponsible of Vm variations a series of experiments was even after washing with fresh buffer (double arrow). C, Effect of carried out using regurgitate (R) collected from larvae different N-linolenoyl-L-Gln concentrations on Vm. Clear effects were previously feeding on Lima bean leaves for 24 h. only caused by concentrations at 100 mgmL1. A single arrow indicates

Perfusion with R caused a Vm depolarization; start of perfusion after Vm stabilization. The double arrow indicates however, the effect was found not to be linearly linked washing with fresh buffer. Metric bars ¼ SD.

Plant Physiol. Vol. 134, 2004 1755 Maffei et al.

Effect of Feeding S. littoralis and Individual R Components on Lima Bean Leaf Cytosolic 21 Calcium Concentration [Ca ]c Intracellular calcium variations may depend on both the entry of Ca21 in the cytoplasm upon release from cell organelles and the entry from the apoplasm. Since plant cells respond to extracellular stimuli with changes in cytosolic calcium concentration that ulti- mately controls many integrated physiological pro- cesses (Bush, 1995), the impact of feeding on cytosolic 21 calcium concentration, [Ca ]c, was investigated. Using the membrane-permeant Ca21-selective fluores- 21 cent dye, Fluo-3 AM, the [Ca ]c were determined by confocal laser scanning microscopy in mechanically- Figure 5. Effect of increasing concentrations of the detergent SDS on and herbivore-wounded leaf tissue, as well as in 1 tissues treated with linolenoyl-L-Gln. Verapamil, a cal- Vm. Lower concentrations (from 50–100 mgmL ) had no effect, whereas at higher concentration (500 mgmL1) a considerable V cium channel blocker, was additionally used in all m 21 depolarization was observed, even after washing with fresh buffer experiments. [Ca ]c was expressed as the percentage (double arrow). The single arrow indicates start of perfusion after Vm of calcium variation in image analysis where the stabilization. Metric bars ¼ SD. lowest value (0%) refers to zero fluorescence (value 0 on the 0–256 gray level scale) and the highest values (100%) refer to the maximum fluorescence (value 256 on the 0–256 gray level scale). Figures 6 and 7 show the results of the experiments 2003a, 2003b) these compounds were used to study Vm variations. performed incubating Lima bean leaves with 5 mM The effect of racemic volicitin (Alborn et al., 1997, 2000) and that of the naturally occurring (17S)-N- (17-hydroxylinoleoyl)-L-Gln (Spiteller et al., 2001) on Vm was low (Fig. 4A). In order to assess whether fatty acid chain length and degree of saturation have an impact on Vm, leaves were perfused with N-palmito- leoyl-L-GlnandN-linolenoyl-L-Gln.Perfusingcellswith N-palmitoleoyl-L-Gln caused a Vm depolarization at 1 the lowest concentration used (25 mgmL ), but a Vm hyperpolarization when higher concentrations (100– 300 mgmL1) were applied (Fig. 4B). Removal of con- jugates with fresh buffer had no effect on 25 and 300 mg 1 mL concentrations. A Vm depolarization was, how- ever, observed in perfusion with 100 mgmL1 (Fig. 4B). Perfusion with N-linolenoyl-L-Gln caused no Vm variation when used at 50 and 500 mgmL1, whereas the strongest Vm depolarization was observed at 100 mgmL1 (Fig. 4C). To study the impact of the fatty acid and amino acid building blocks of the conjugates, Lima bean leaves Figure 6. False-color image analysis reconstructions from confocal were individually treated with linolenic acid and Gln. laser scanning microscope observations and fluochemical intracellular 21 Linolenic acid caused no obvious effect on Vm at low Ca determination. The green fluorescence refers to binding of Fluo-3 1 21 concentrations (10 and 50 mgmL ), while a weak Vm AM with Ca , whereas the chloroplasts are evidenced by a bright red depolarization was observed when leaf tissues were color caused by chlorophyll fluorescence. A, Portion of a Lima bean perfused with L-Gln (data not shown). leaf incubated with 5 mM Fluo-3 AM in 50 mM MES buffer in the 21 Owing to their molecular architecture N-acyl Glns presence of 0.5 mM Ca attacked by the herbivore S. littoralis.B, Portion of a Lima bean leaf bite zone after incubation with 5 mM Fluo-3 are amphiphilic compounds with a pronounced ability 21 to form micelles (Asselineau, 1991), similar to known AM in 50 mM MES buffer deprived of Ca . The green fluorescence is detergents such as SDS. In order to test whether considerably lower than in A. C, Portion of a Lima bean leaf bite zone incubated with 5 mM Fluo-3 AM in 50 mM MES buffer in the presence a detergent has an effect on V , increasing concen- 21 m of both 0.5 mM Ca and 100 mM Verapamil. The fluorescence is trations of SDS were applied to Lima bean leaves. Low decreased in the presence of the channel blocker Verapamil, with concentrations had no effect, whereas at high concen- respect to A. D, Portion of a Lima bean leaf mechanically wounded tration (500 mM) a clear Vm depolarization was after incubation with 5 mM Fluo-3 AM in 50 mM MES buffer in the 21 observed, even after washing with fresh buffer (Fig. 5). presence of 0.5 mM Ca . Metric bars are indicated on pictures.

1756 Plant Physiol. Vol. 134, 2004 Effects of Feeding Spodoptera littoralis on Lima Bean Leaves

Plant Physiol. Vol. 134, 2004 1757 Maffei et al.

21 Fluo-3 AM in 50 mM MES buffer both in the presence compounds or the Ca -sensitive fluorescent dyes of 0.5 mM calcium and without calcium in the could be represented by a low permeability of the cell incubation medium and in the presence or absence wall as well as by a massive cuticle. This would allow of 100 mM of Verapamil. As a general consideration, in the penetration of only a limited number of cell layers all experiments performed in the presence of Ca21 probably near the infection zone. Thus, in order to a clear peak of Fluo-3 AM fluorescence was observed study dose dependent effects of N-acyl-Gln-triggered between 30 and 200 mm from the bite zone, with Ca21 concentration changes in the plant cytosol, a sharp decrease at distances greater than 230 mm transgenic soybean suspension cells expressing the (Figs. 6 and 7). Ca21 sensitive aequorin system were used for fur- Mechanical wounding caused an influx of Ca21 ther experiments (Mitho¨fer et al., 1999; Mitho¨fer and (Figs. 6D and 7, AI). When Ca21 was absent from the Mazars, 2002). Although among independent experi- 21 incubation medium (Fig. 7, AII) or when Verapamil ments the maximum values of the [Ca ]c varied to was present (Fig. 7, AIII and AIV) a reduced Ca21 some extent, in a single set of measurements using the influx was observed. The average percentage of same population of suspension cells the same day, the fluorescence in mechanically wounded leaves was relative activities of the effectors analyzed have always 25% near the damage in the presence of Ca21 and 5% to been comparable. The Ca21 response was determined 10% without Ca21. An average of 10% to 15% was in a concentration-dependent fashion for the most recorded in the presence of Ca21 at increasing common N-acyl conjugate N-linolenoyl-Gln. For this 21 distances from the bite zone, whereas only 5% to compound the transiently accumulating [Ca ]c ap- 10% was found without Ca21. When mechanically peared to be linearly correlated with the amount of wounded leaves were treated with the larval R, an effector applied (Fig. 8A). Moreover, it is interesting to increase in Fluo-3 AM fluorescence was found in the note that the configuration of the amino acid moiety absence of Ca21 (Fig. 7, BII) whereas in the presence of had no effect on the Ca21-response. Both conjugates, 21 Verapamil the occurrence of Ca increased Fluo-3 AM containing either L-Gln or D-Gln joined to linolenic fluorescence (Fig. 7, BIII). When N-linolenoyl-Gln (100 acid were able to trigger the Ca21 response (Fig. 8B). 1 21 mgmL ) was given as a 0.5 mM Ca -containing Similar results were observed with the correspond- solution to mechanically wounded leaves, no differ- ing N-acyl Glus, which are typical constituents of the ences were observed between tissues treated with (Fig. regurgitant of the tobacco horn worm Manduca sexta 7, CI) or without (Fig. 7, CII) Ca21 in the medium, and (Halitschke et al., 2001). Again, both conjugates of the same was observed for Verapamil (Fig. 7, CIII linolenic acid with either D-orL-Glu proved to be and CIV). Almost the same results were found when active (Fig. 8B). A direct comparison among volicitin, N-linolenoyl-Gln was applied as a Ca21 free solution N-linolenoyl Gln, and the recently identified 15,16- (Fig. 7, DI–IV). On the other hand, when larvae were epoxyoctadeca-9,12-dienyl-Gln (Spiteller and Boland, allowed to feed on tissues incubated with Fluo-3 AM, 2003a, 2003b) at the same concentration, showed that a strong Fluo-3 AM fluorescence was observed. In the the nonfunctionalized N-linolenoyl-Gln is the most presence of Ca21, in both larvae reared on artificial diet effective compound (Fig. 8C); however, differences (Fig. 7, EI) and larvae reared on Lima bean leaves were small. For comparison, we also tested the (Figs. 6A and 7, FI), a sharp and consistent peak of detergent SDS (25 mgmL1), which showed a highly fluorescence was observed up to 100 mm from the bite similar pattern of Ca21-influx (Fig. 8D) demonstrating zone. The absence of Ca21 dramatically decreased that the influx of Ca21 is a rather unspecific effect Fluo-3 AM fluorescence in both feeding experiments linked to the intrinsic molecular properties of amphi- (Figs. 6B, 7EII, and 7FII). When Verapamil was added philic compounds. in the presence of Ca21 (Figs. 6C, 7EIII, and 7FIII) it almost halved Fluo-3 AM fluorescence, whereas the 21 absence of Ca in Verapamil treated tissues (Fig. 7, DISCUSSION EIV and FIV) showed a decreased Fluo-3 AM fluorescence. Plant responses to herbivore attack are complex and involve an array of signals, leading to activation of Quantification of Ca21-Release by N-acyl Glns multiple defenses. Feeding herbivores cause extensive in Suspension Cultures of Soybean and irreversible wounding along with an introduction of salivary secretions. Both wounding and compo- Loading of Ca21-sensitive fluorescent probes into nents from the insects’ secretions have an obvious, but plant cells is an essential step to measuring activities of clearly different impact on the plants’ response cytoplasmic free Ca21 ions with a fluorescent imaging (Schittko et al., 2001, and references cited therein). In technique. However, barriers to the loading of the test the model system Nicotiana attenuata and its specialist

Figure 7. Percentage of Fluo-3 AM fluorescence (y axis) as a function of the distance (in mm; x axis) from the bite zone. The experiment was designed in order to evaluate the effect of the presence or absence of exogenous Ca21 in the incubation media and the presence or absence of the Ca21 channel blocker Verapamil. Mechanical wounding was inferred with a razor blade. Values are the mean of at least five repetitions (see text for description).

1758 Plant Physiol. Vol. 134, 2004 Effects of Feeding Spodoptera littoralis on Lima Bean Leaves

uration-independent effect concerning the stereo- chemistry of the amino acid (Gln); (3) a slightly reduced influx of Ca21 after modification of the fatty acid backbone by functionalization with oxygen; and (4) a comparable effect with the detergent SDS. In general, Vm variations depend on unbalanced ion distribution across the plasma membrane and de- polarization occurs when cations (such as K1 and Ca21) are allowed to enter the cell or upon anion efflux. On the other hand, hyperpolarization mainly depends on the activity of the plasma membrane H1-ATPase or when inward anion channels (or outward cation channels) are opened. The primary candidate for intercellular signaling in higher plants is the stimu- lus-induced change in Vm (Shvetsova et al., 2001). According to Volkov and coworkers (Volkov, 2000; Shvetsova et al., 2001, and references cited therein) insects induce electrical signals that can influence both biophysical and biochemical processes in remote 21 21 Figure 8. Monitoring of cytosolic Ca concentration ([Ca ]c) changes tissues. Moreover, excitation waves transmit informa- 21 in soybean cell suspension cultures expressing the Ca sensing ae- tion from one part of the plant to another with a speed quorin system. A, Enhancement of [Ca21] determined upon treat- c of propagation of the action potential that in soybean m 1 ment with increasing concentrations (2, 10, 50, and 200 gmL ) can reach 40 m s1 (Shvetsova et al., 2001). of N-linolenoyl-Gln. B, Comparison of the amino acid configurations Since ion fluxes through channels directly influence (L-orD-Gln and L-orD-Glu, respectively) of the linolenic acid 1 21 V , it seems reasonable to assume that molecules able conjugates (50 mgmL ) on their [Ca ]c increasing activity. C, Effect m 1 21 to act on channel activity might be considered as of various N-acyl Glns (50 mgmL ) on the [Ca ]c increase: N-linolenoyl-Gln, N-(15,16-epoxyoctadeca-9,12-dienoyl)-L-Gln, (15, important factors inducing electrical signals. Among 16-epoxy-Gln), 17-hydroxylinolenoyl-L-Gln (volicitin). D, Effect of the the various channels, calcium channels are predomi- 1 21 detergent SDS (25 mgmL ) on the [Ca ]c increase in soybean cell nantly involved in cell signaling and the specificity of cultures. the cytosolic calcium concentration signal in triggering a response depends on amplitude, temporal, and spatial changes (White, 2000). In fact, the longer- herbivore Manduca sexta, feeding elicits a jasmonate lasting influxes are expected to penetrate farther into burst, a large transcriptional reorganization of the the cytoplasm and therefore encounter more centrally plant host and, after hours, a systemic release of located Ca21-dependent enzymes (Trewavas, 2000). terpenoids (Halitschke et al., 2001, 2003). Principally Recent studies have raised the challenging idea that the same sequence is passed through in the interaction depolarization-activated calcium channels can be between Lima bean and spider mites (Arimura et al., a sensor for stimuli (such as touch) and might be an 2000a, 2000b, 2002), and in the interaction of maize exclusive signaling pathway element (Miedema et al., (Zea mays) plants with the beet armyworm (Spodoptera 2001). exigua; Schmelz et al., 2003; for review, see Gatehouse, Oral secretions and some of its components such as 2002). the fatty acid-amino acid conjugates of, for example, The results of the present work add novel facets to Manduca sexta have been shown to be necessary and the previously known sequence and demonstrates that sufficient to elicit a set of herbivore-specific responses herbivore attack onto a Lima bean leaf is associated in tobacco (Halitschke et al., 2003). Our results with (1) a strong Vm depolarization at the bite zone demonstrate that regurgitants and N-acyl-amino acid causing a wave of Vm depolarization spreading conjugates interact with the plasma membrane and throughout the entire attacked leaf; and (2) a consistent alter Vm. R from Lima bean reared larvae altered Vm in influx of Ca21 at the very edge of the bite, which is a concentration-independent fashion and its effect is 21 halved by application of the Ca channel blocker clearly different from that observed in Vm studies with Verapamil. Although none of the amphiphilic N-acyl the individual compounds. Especially, the nonlinear 21 Glns had a visible impact on the influx of Ca when response of Vm to the concentration of R and R-factors monitored by fluorescence microscopy in the presence remains to be clarified. Possibly the effects are related of Fluo-3 AM, the more sensitive assay with the to different modes of membrane Vm depolarization by soybean suspension cultures expressing aequorin either micellar transport of ions or pore-formations by revealed that these compounds, at least to a certain the conjugates and other components of R (Abramson extent, may be also involved in the Ca21-signaling. The and Shamoo, 1979). The most common fatty acid quantitative study of the dose dependence effects of conjugate, namely N-linolenoyl-Gln, exhibits Vm de- N-acyl Gln conjugates upon Ca21-influx revealed (1) polarization up to a concentration of 200 mgmL1, a concentration-dependent influx of Ca21; (2) a config- representing the natural concentration in lepidopteran

Plant Physiol. Vol. 134, 2004 1759 Maffei et al. larvae R (Pohnert et al., 1999a) and this reflects the defense genes in the receiver cells requires the calcium magnitude of Vm depolarization observed in R. A typi- influx into the cells (Arimura et al., 2000a). cal minor component of lepidopteran R is N-pal- Signals induced by herbivore attack rapidly spread 1 21 mitoleoyl-Gln. At low concentration (25 mgmL ), over the leaf leading to a strong Ca -dependent Vm corresponding to a typical R concentration, this com- depolarization in the bite zone followed by a transient pound shows Vm depolarization comparable to Vm hyperpolarization in the close vicinity and a con- N-linolenoyl-Gln and to R. Application of the fatty stant depolarization in distances greater than 6 to acid or amino acid components of the conjugates 7 mm. At the long distance (6–7 cm) the overall pro- shows virtually no effect for linolenic acid but a clear cess takes not more than 5 to 6 min which requires V depolarization for Gln. The latter effect could play for signal molecules traveling with the same speed m 1 a role during larval feeding after enzymatic cleavage (approximately 1 cm min ). of the conjugates and may rely on transport processes Another interesting target is the analysis of the (e.g. symport) of the amino acid (Delrot et al., 2001) early events in the interaction of volatiles (including and/or interaction of free Gln with receptors. How- ethylene, H2O2, and NO) emitted from wounded ever, as yet, nothing is known about the stability of plants and/or perceived by neighbored healthy plants. N-acyl amino acids in the plant cells. Preliminary results already indicate compound-spe- The time-course and distance-dependence spread- cific variations in Vm. Studies are under way and will be reported soon. ing of the Vm depolarization upon herbivore attack in intact leaves (Fig. 2) is probably associated with a molecule able to disperse within tissues at a relatively high speed. Preliminary results perfusing leaves with MATERIALS AND METHODS H2O2 and Ethephon (the ethylene releasing agent) Plant Material indicate a Vm depolarizing effect of these molecules (unpublished data). Feeding experiments were carried out using the Lima bean Phaseolus 21 lunatus (cv Ferry Morse var Jackson Wonder Bush). Individual plants were The typical response pattern to different Ca grown from seed in a plastic pot with sterilized potting soil at 238C and 60% concentrations lie in the ability of cells to generate humidity using daylight fluorescent tubes at approximately 270 mEm2 s1 21 specific cytosolic Ca concentration signatures. They with a photophase of 16 h. Experiments were conducted with 12- to 16-d-old may be unique, in terms of spatio-temporal character- seedlings showing two fully developed primary leaves, which were found to istics and in response to an individual stimulus be the most responsive leaves. (McAinsh and Hetherington, 1998). In the bite zone, there is a dramatic Ca21 influx limited to few cell Animal Material layers (Figs. 6A, 7EI, and 7FI). Usually stimulus- Larvae of Spodoptera littoralis (Boisd.; Lepidoptera, Noctuidae) were grown 21 induced increases of Ca concentration occur in the in petri dishes at long photoperiod (14–16 h photophase) and 228Cto248C. 1 1 form of oscillations or in the form of spikes (McAinsh They were fed an artificial diet consisting of 300 g L agar, 400 g L bean flour, 3 g sodium ascorbate, 3 g ethyl p-hydroxybenzoate, and 1 g and Hetherington, 1998). In the case of larvae feeding formaldehyde (Bergomaz and Boppre´, 1986). Small cubes of the diet were on Lima bean leaves a spike is observed and depends placed into rearing dishes on pieces of aluminum foil. Alternatively, larvae 21 on Ca channel activity, since the response can be were fed with Lima bean leaves. The dishes were lined with filter paper to reduced by Verapamil. The involvement of N-acyl- reduce humidity and sawdust was provided for pupation when the larvae 21 terminated feeding. Adults were allowed to emerge in glass containers Glns in Ca influx is independently confirmed with supplied with water and honey solution. Eggs were deposited on strips of the test system of aequorin-transformed soybean cells. filter paper. As previously shown for the established elicitors cryptogenin, oligogalacturonides (Lecourieux et al., V 2002), and b-(1,3)-(1,6)-glucans (Mitho¨fer et al., 1999), m 21 V was determined in leaf segments. The V was determined with glass the Ca influx increases with increasing N-linolenoyl- m m micropipettes with a tip resistance of 4 to 10 MV and filled with 3 M KCl. Gln concentration. Moreover, in our assay, no differ- Micropipettes were used as micro-salt bridges to Ag/AgCl electrodes ence is found between D- and L-amino acid building obtained with a Narishighe PE-21 puller and inserted vertically in the tissue blocks of N-linolenoyl conjugate, and, thus, the effect by means of a micromanipulator (Maffei et al., 2001). Leaf segments were of induced Ca21 influx is, in the first instance, linked to always equilibrated for 60 to 120 min in 5 mM MES-NaOH (pH 6.0). Perfusion of solutions was granted by a multichannel Ismatec Reglo peristaltic pump the overall physico-chemical properties of the amphi- 1 (flow rate 1 mL min ). Vm variations were recorded both on a pen recorder philic compounds, as confirmed by the action of SDS. and through a digital port of a PC using a data logger. Measurements were It should be not ignored, however, that only the performed at increasing distances from the leaf wounding caused by conjugate with L-Gln has been shown to induce herbivore feeding and the data were plotted with respect to controls. specific defense responses in maize (Alborn et al., Measurements were also performed after perfusion with the compounds 21 listed below as well as during larvae feeding on intact plants connected to the 1997), demonstrating that, besides triggering the Ca apparatus. influx, additional interactions between N-acyl-Glns All chemicals were dissolved in 1% methanol, which was present in the and the plant are involved in the elicitation process of control solutions, and perfused in a 50 mM MES buffered solution (pH 6.0) maize plants. Overall, our findings are in agreement containing 0.5 mM calcium sulfate and 2.5 mM 3-(3,4-dichlorophenyl)-1,1- dimethylurea, used to poison photosynthetic electron transfer. After a period with previous work demonstrating that in Lima bean of Vm stabilization, saturation of the well where leaf tissues have been placed the signaling pathway(s) mediating expression of occurred in 7 min, after which perfusion was carried out for a variable time

1760 Plant Physiol. Vol. 134, 2004 Effects of Feeding Spodoptera littoralis on Lima Bean Leaves

(until stabilization of the Vm), washing of the well was done by perfusing with Statistics fresh buffer. Saturation with fresh buffer took 20 to 25 min and then the solution was allowed to perfuse until Vm reached a constant value. At least five repetitions were used for the statistical treatment of the data. More than five repetitions contributed to the mean values given in Figures 3, 4A to C, and 5. The data are expressed as mean values; metric bars indicate the SD. To evaluate the difference significance of the control and the treatments at Oral Secretions and Regurgitant Collection the given concentrations, variance analysis (ANOVA) was performed for all data using the Tukey test. Five-day-old larvae (approximately 2–3 cm long) were grown on artificial diet and reared on Lima bean leaves for 24 h prior to collection of R. Oral secretions were collected into glass capillaries connected to an evacuated sterile vial (peristaltic pump) by gently squeezing the larva with a forceps ACKNOWLEDGMENTS behind the head which caused immediate regurgitation. Secretions were stored at (208C) until perfusion. M.M. and S.B. are thankful to the graduated students who gave their technical support (particularly D. Salvetto). We are thankful to Dr. A. Elbert (Bayer AG, D-40789 Monheim) for supplying us with egg clutches of Intracellular Calcium Variation Determination lepidopteran larvae, and to Angelika Berg for caterpillar rearing. We thank Dr. G. Pohnert for synthesis of 17S-volicitin and proofreading of the Fluo-3 AM (acetoxy-methyl ester of Fluo-3, more permeant for cells) manuscript. purchased in vials containing the molecule as a stock solution in dimethyl Received October 22, 2003; returned for revision January 19, 2004; accepted sulfoxide (Fluka, Milwaukee, WI), was diluted in 50 mM MES buffer, pH 6.0, January 19, 2004. with the addition of 0.5 mM calcium sulfate, 2.5 mM 3-(3,4-dichlorophenyl)-1,1- dimethylurea to reach the concentration of 5 mM. This resulting solution was used for an initial treatment of Lima bean leaves not separated from the plant; the leaf was gently fixed over a glass slide, and a drop (about 20 mL) of 5 mM LITERATURE CITED Fluo-3 AM solution was applied and covered with another glass slide. Thirty minutes after treatment with Fluo-3 AM, the leaf was fixed on an Olympus Abramson JJ, Shamoo AE (1979) Anionic detergents as divalent-cation (Tokyo) FLUOview confocal scanning laser microscope stative without ionophores across black lipid-membranes. J Membr Biol 50: 241–255 separating the leaf from the plant. Measurements were taken in intact leaves, Agrawal AA (2000) Mechanisms, ecological consequences and agricul- with leaves wounded mechanically and after herbivore feeding, both in the tural implications of tri-trophic interactions. Curr Opin Plant Biol 3: presence and absence of exogenous calcium. In addition the leaves were 329–335 1 perfused with undiluted R and N-linolenoyl-Gln (at 100 mgmL ), as well as Alborn HT, Jones TH, Stenhagen GS, Tumlinson JH (2000) Identification with the calcium channel blocker Verapamil (100 mM; White, 2000). The and synthesis of volicitin and related components from beet armyworm microscope is operated with a Krypton/Argon laser at 488 nm and 568 nm oral secretions. J Chem Ecol 26: 203–220 wavelengths; the first wavelength excitates the Fluo-3 dye, resulting in Alborn HT, Turlings TCJ, Jones TH, Stenhagen G, Loughrin JH, emission of green light and the second mostly excitates chloroplasts, which Tumlin so n JH (1997) An elicitor of plant volatiles from beet armyworm emit red fluorescence. Images generated by the FluoView software were oral secretion. Science 276: 873–949 analyzed using the public domain NIH Image program (developed at the Allen DG, Blinks JR, Prendergast FG (1977) Aequorin luminescence: United States National Institutes of Health and available on the Internet at relation of light emission to calcium concentration – a calcium- http://rsb.info.nih.gov/nih-image). independent component. Science 195: 996–998 Arimura G-I, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J (2000a) Herbivory-induced volatiles elicit defence genes in lima bean Aequorin Dependent Luminescence Measurements leaves. Nature 406: 512–515 Arimura G-I, Tashiro K, Kuhara S, Nishioka T, Ozawa R, Takabayashi J Transgenic soybean 6.6.12 cell lines carrying the stably integrated plasmid (2000b) Gene responses in bean leaves induced by herbivory and pGNAequ/neo2 and expressing apoaequorin (Mitho¨fer et al., 1999) were used to by herbivore-induced volatiles. Biochem Biophys Res Commun 277: reconstitute aequorin in vivo with 10 mM synthetic coelenterazine (Calbio- 305–310 chem, Bad Soden, Germany) on a shaker (125 rpm) in the dark for 24 h. The ArimuraG-I,OzawaR,NishiokaT,BolandW,KochT,Ku¨ hnemann F, Ca21-specific luminescence (470 nm) was measured in a final volume of 200 Takabayashi J (2002) Herbivore-induced volatiles induce the emission mL using a digital luminometer (Bio-Orbit 1250, Turku, Finland) as described of ethylene in neighboring lima bean plants. Plant J 29: 87–98 (Mitho¨fer et al., 1999; Mitho¨fer and Mazars, 2002). Treatments with various Asselineau J (1991) Bacterial lipids containing amino acids or peptides compounds were performed by adding 1 to 10 mL of different concentrations linked by amide bonds. In WHerz,HGrisebach,GWKirby,CThamm, of aqueous stock solutions to the cell suspension culture. Mixing time for the eds, Progress in the Chemistry of Organic Natural Products. Springer- addition of any compound was 5 to 7 s. In each experiment, the concentration Verlag, Vienna, New York, pp 1–85 of reconstituted aequorin was not limiting under any of the experimental Baldwin IT, Halitschke R, Kessler A, Schittko U (2001) Merging molecular conditions, with a maximal consumption not exceeding 10%. The residual and ecological approaches in plant-insect interactions. Curr Opin Plant aequorin was completely discharged by adding 200 mL of 20% ethanol Biol 4: 351–358 containing 2 M CaCl2 (final concentration 10% and 1 M, respectively). The Bergomaz B, Boppre´ M (1986) A simple insect diet for rearing Arctiidae resulting luminescence was used to estimate the total amount of aequorin and other moths. Journal of Lepidopterists’ Society 40: 131–137 present in various experiments in order to determine the rate of aequorin Blumwald E, Aharon GS, Lam BC-H (1998) Early signal transduction 21 consumption for the calculation of the cytosolic Ca concentrations according pathways in plant-pathogen interactions. Trends Plant Sci 3: 342–346 to Allen et al. (1977). Bush DS (1995) Calcium regulation in plant cells and its role in signaling. Annu Rev Plant Physiol Plant Mol Biol 46: 95–122 Cheong J-J, Choi YD (2003) Methyl jasmonate as a vital substance in plants. Chemicals Used for Perfusion Trends Genet 19: 409–413 Delrot S, Atanassova R, Gome`sE,Coutos-The´venot P (2001) Plasma Natural volicitin, 17S-(17-hydoxylinolenoyl)-L-Gln was synthesized as membrane transporters: a machinery for uptake of organic solutes and described (Pohnert et al., 1999b). N-linolenoyl-L-Gln, N-linolenoyl-D-Gln, the stress resistance. Plant Sci 161: 391–404 corresponding Glus and N-palmitoleoyl-L-Gln were available according to the DeMoraes CM, Lewis WJ, Pare´ PW, Alborn HT, Tumlinson JH (1998) protocol of Pohnert et al. (1999a). N-(15,16-epoxy-linoleoyl)-Gln and N-(15,16- Herbivore-infested plants selectively attract parasitoids. Nature 393: dihydroxylinoleoyl)-Gln were synthesized as described by Spiteller and 570–573 Boland (2003a). Linolenic acid, SDS, and L-Gln were purchased from Sigma- Dicke M, Sabelis MW (1992) Costs and benefits of chemical information Aldrich (St. Louis). conveyance: proximate and ultimate factors. In BD Roitberg, MB Isman,

Plant Physiol. Vol. 134, 2004 1761 Maffei et al.

eds, Insect Chemical Ecology: An Evolutionary Approach. Chapman Pin˜ eros M, Tester M (1995) Characterization of a voltage-dependent Ca21 and Hall, New York, pp 122–155 selective channel from wheat roots. Planta 195: 478–488 Engelberth J, Koch T, Schu¨ ler G, Bachmann N, Rechtenbach J, Boland W Pin˜ eros M, Tester M (1997) Calcium channels in higher plant cells: (2001) Ion channel-forming alamethicin is a potent elicitor of volatile selectivity, regulation and pharmacology. J Exp Bot 48: 551–577 biosynthesis and tendril coiling. Cross talk between jasmonate and Pohnert G, Jung V, Haukioja E, Lemoa K, Boland W (1999a) New fatty salicylate signaling in Lima bean. Plant Physiol 125: 369–377 acid amides from caterpillar (Noctuide, Geometridae) regurgitants. 21 Evans DE, Briars SA, Williams LE (1991) Active Ca transport by plant Tetrahedron 55: 11275–11280 cell membranes. J Exp Bot 42: 285–303 Pohnert G, Koch T, Boland W (1999b) Synthesis of volicitin: stereoselective Gatehouse JA (2002) Plant resistance towards insects herbivores: a dynamic one pot formation of 17-hydroxylinolenic acid. Chem Commun 12: interaction. New Phytol 156: 145–169 1087–1088 21 Gelli A, Blumwald E (1997) Hyperpolarization-activated Ca -permeable Poppy GM (1999) The raison d’eˆtre of secondary plant chemicals? Trends channels in the plasma membrane of tomato cells. J Membr Biol 155: Plant Sci 4: 82–83 35–45 Roh MH, Shingles R, Cleveland MJ, McCarty RE (1998) Direct Halitschke R, Gase K, Hui D, Schmidt DD, Baldwin IT (2003) Molecular measurement of calcium transport across chloroplast inner-envelope interactions between the specialist herbivore Manduca sexta (Lepidop- vesicles. Plant Physiol 118: 1447–1454 tera, Sphingidae) and its natural host Nicotiana attenuata.VI.Microarray Schittko U, Hermsmeier D, Baldwin IT (2001) Molecular interactions analysis reveals that most herbivore-specific transcriptional changes between the specialist herbivore Manduca sexta (lepidoptera, Sphin- are mediated by fatty acid-amino acid conjugates. Plant Physiol 131: gidae) and its natural host Nicotiana attenuata. II. Accumulation of 1894–1902 plant mRNAs in response to insect-derived cues. Plant Physiol 125: Halitschke R, Schittko U, Pohnert G, Boland W, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta 701–710 (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata.III. Schmelz EA, Alborn HT, Tumlinson JH (2003) Synergistic interactions Fatty acid-amino acid conjugates in herbivore oral secretions are between volicitin, jasmonic acid and ethylene mediate insect induced necessary and sufficient for herbivore-specific plant responses. Plant volatile emission in Zea mays. Physiol Plant 117: 403–412 Physiol 125: 711–717 Shah J (2003) The salicylic acid loop in plant defense. Curr Opin Plant Biol Hui D, Iqbal J, Lehmann K, Gase K, Saluz HP, Baldwin IT (2003) 6: 365–371 Molecular interactions between the specialist herbivore Manduca sexta Shvetsova T, Mwesigwa J, Volkov AG (2001) Plant electrophysiology: (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata:V. FCCP induces action potential and excitation waves in soybean. Plant Microarray analysis and further characterization of large-scale changes Sci 161: 901–909 in herbivore-induced mRNAs. Plant Physiol 131: 1877–1893 Spiteller D, Boland W (2003a) N-(15,16-dihydroxylinoleoyl)-glutamine Koch T, Krumm T, Jung V, Engelberth J, Boland W (1999) Differential and N-(15,16-epoxylinoleoyl)-glutamine isolated from oral secretions of induction of plant volatile biosynthesis in the Lima bean by early and lepidopteran larvae. Tetrahedron 59: 135–139 late intermediates of the octadecanoid-signaling pathway. Plant Physiol Spiteller D, Boland W (2003b) Identification and synthesis of 121: 153–162 N-(17-acyloxyacyl)-glutamine conjugates from oral secretion of lepi- Labady A, Thomas DJ, Shvetsova T, Volkov AG (2002) Plant bioelec- dopteran larvae. J Org Chem 68: 8743–8749 trochemistry: effects of CCCP on electrical signaling in soybean. Spiteller D, Pohnert G, Boland W (2001) Absolute configuration of Bioelectrochemistry 57: 47–53 volicitin; an elicitor of plant volatile biosynthesis from Lepidopteran Lecourieux D, Mazars C, Pauly N, Ranjeva R, Pugin A (2002) Analysis and larvae. Tetrahedron Lett 42: 1483–1485 effects of cytosolic free calcium increases in response to elicitors in Stratmann JW (2003) Long distance run in the wound response – jasmonate Nicotiana plumbaginifolia cells. Plant Cell 11: 2627–2641 acid is pulling ahead. Trends Plant Sci 8: 247–250 Maffei M, Camusso W, Sacco S (2001) Effect of Mentha x piperita essential Thaler JS (1999) Jasmonate-inducible plant defences cause increased oil and monoterpenes on cucumber root membrane potential. Phyto- parasitism of herbivores. Nature 399: 686–688 chemistry 58: 703–707 Thuleau P, Schroeder JI, Ranjeva R (1998) Recent advances in the 21 McAinsh MR, Hetherington AM (1998) Encoding specificity in Ca regulation of plant calcium channels: evidence for regulation by signalling system. Trends Plant Sci 3: 32–36 G-proteins, the cytoskeleton and second messengers. Curr Opin Plant Miedema H, Bothwell JHF, Brownlee C, Davies JM (2001) Calcium uptake Biol 1: 424–427 by plant cells: channels and pumps acting in concert. Trends Plant Sci 6: Trewavas A (2000) Signal perception and transduction. In Biochemistry 514–519 and Molecular Biology of Plants. B Buchanan, W Gruissem, R Jones, eds. Mishra NS, Mallik BN, Sopory SK (2001) Electrical signal from root to American Society of Plant Physiologists. pp 930–987 shoot in Sorghum bicolor: induction of leaf opening and evidence for fast Turlings TCJ, Loughrin JH, McCall PJ, Ro¨se USR, Lewis WJ, Tumlinson extracellular propagation. Plant Sci 160: 237–245 JH (1995) How caterpillar-wounded plants protect themselves by Mitho¨ fer A, Ebel J, Bhagwat AA, Boller T, Neuhaus-Url G (1999) 92: Transgenic aequorin monitors cytosolic calcium transients in soybean attracting parasitic wasps. Proc Natl Acad Sci USA 4169–4174 Volkov AG (2000) Green plants: electrochemical interfaces. J Electroanal cells challenged with b-glucanorchitinelicitors.Planta207: 566–574 Mitho¨ fer A, Mazars C (2002) Aequorin-based measurements of intracel- Chem 483: 150–156 lular Ca21-signatures in plant cells. Biol Proced Online 4: 105–118 Warber S (1998) Modes of action at target sites. In PB Kaufman, JC Leland, Orozco-Ca´rdenas M, Narva´ez-Va´squez J, Ryan CA (2001) Hydrogen S Warber, JA Duke, HL Brielmann, eds, Natural Products from Plants. peroxide acts as a second messenger for the induction of defense genes CRC Press, Boca Raton, FL, pp 157–182 in tomato plants in response to wounding, systemin, and methyl White PJ (2000) Calcium channels in higher plants. Biochim Biophys Acta jasmonate. Plant Cell 13: 179–181 1465: 171–189 Pare´ PW,AlbornHT,TumlinsonJH(1998) Conterted biosynthesis of an Winz RA, Baldwin IT (2001) Molecular interactions between the specialist insect elicitor of plant volatiles. Proc Natl Acad Sci USA 95: 13971–13975 herbivore Manduca sexta (lepidoptera, Sphingidae) and its natural host Pellinen RI, Minna-Sisko K, Tauriainen AA, Palva ET, Kangasja¨rvi J Nicotiana attenuata. IV. Insect-induced ethylene reduces jasmonate- (2002) Hydrogen peroxide activates cell death and defense gene induced nicotine accumulation by regulating putrescine N-methyl- expression in birch. Plant Physiol 130: 549–560 transferase transcripts. Plant Physiol 125: 2189–2202

1762 Plant Physiol. Vol. 134, 2004