Wounded leaves communicate their damage status to one another through a poorly understood process of long-distance signalling. This stimulates the distal production of jasmonates, potent regulators of defence responses. Using non-invasive electrodes we mapped surface potential changes in Arabidopsis thaliana after wounding leaf eight and found that membrane depolarizations correlated with jasmonate signalling domains in undamaged leaves. Furthermore, current injection elicited jasmonoyl-isoleucine accumulation, resulting in a transcriptome enriched in RNAs encoding key jasmonate signalling regulators. From among 34 screened membrane protein mutant lines, mutations in several clade 3 GLUTAMATE RECEPTOR-LIKE genes (GLRs 3.2, 3.3 and 3.6) attenuated wound-induced surface potential changes. Jasmonate-response gene expression in leaves distal to wounds was reduced in a glr3.3 glr3.6 double mutant. This work provides a genetic basis for investigating mechanisms of long-distance wound signalling in plants and indicates that plant genes related to those important for synaptic activity in animals function in organ-to-organ wound signalling.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
BMC Plant Biology Open Access 22 March 2022
Ether anesthetics prevents touch-induced trigger hair calcium-electrical signals excite the Venus flytrap
Scientific Reports Open Access 18 February 2022
Scientific Reports Open Access 18 March 2021
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Card, G. & Dickinson, M. H. Visually mediated motor planning in the escape response of Drosophila. Curr. Biol. 18, 1300–1307 (2008)
Escalante-Pérez, M. et al. A special pair of phytohormones controls excitability, slow closure, and external stomach formation in the Venus flytrap. Proc. Natl Acad. Sci. USA 108, 15492–15497 (2011)
Walters, D. R. Plant Defense (Blackwell, 2011)
Koo, A. J. K. & Howe, G. A. The wound hormone jasmonate. Phytochemistry 70, 1571–1580 (2009)
Wildon, D. C. et al. Electrical signaling and systemic proteinase-inhibitor induction in the wounded plant. Nature 360, 62–65 (1992)
Browse, J. Jasmonate passes muster: a receptor and targets for the defense hormone. Annu. Rev. Plant Biol. 60, 183–205 (2009)
Howe, G. A. & Jander, G. Plant immunity to insect herbivores. Annu. Rev. Plant Biol. 59, 41–66 (2008)
Fonseca, S. et al. (+)-7-iso-Jasmonoyl-l-isoleucine is the endogenous bioactive jasmonate. Nature Chem. Biol. 5, 344–350 (2009)
Glauser, G. et al. Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding. J. Biol. Chem. 283, 16400–16407 (2008)
Glauser, G. et al. Velocity estimates for signal propagation leading to systemic jasmonic acid accumulation in wounded Arabidopsis. J. Biol. Chem. 284, 34506–34513 (2009)
Koo, A. J. K., Gao, X., Jones, A. D. & Howe, G. A. A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis. Plant J. 59, 974–986 (2009)
Reymond, P. et al. A conserved transcript pattern in response to a specialist and a generalist herbivore. Plant Cell 16, 3132–3147 (2004)
Maffei, M., Bossi, S., Spiteller, D., Mithöfer, A. & Boland, W. Effects of feeding Spodoptera littoralis on Lima bean leaves. I. Membrane potentials, intracellular calcium variations, oral secretions, and regurgitate components. Plant Physiol. 134, 1752–1762 (2004)
Fromm, J. & Lautner, S. Electrical signals and their physiological significance in plants. Plant Cell Environ. 30, 249–257 (2007)
Stahlberg, R., Cleland, R. E. & Volkenburgh, E. V. in Communication in plants (eds Baluška, F., Mancuso, S. & Volkmann, D. ) 291–308 (Springer-Verlag, 2006)
Boller, T. & Felix, G. A Renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu. Rev. Plant Biol. 60, 379–406 (2009)
Krol, E. et al. Perception of the Arabidopsis danger signal peptide 1 involves the pattern recognition receptor AtPEPR1 and its close homologue AtPEPR2. J. Biol. Chem. 285, 13471–13479 (2010)
Schaller, A. & Oecking, C. Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 11, 263–272 (1999)
Schaller, A. & Frasson, D. Induction of wound response gene expression in tomato leaves by ionophores. Planta 212, 431–435 (2001)
Fisahn, J., Herde, O., Willmitzer, L. & Peña-Cortés, H. Analysis of the transient increase in cytosolic Ca2+ during the action potential of higher plants with high temporal resolution: requirement of Ca2+ transients for induction of jasmonic acid biosynthesis and PINII gene expression. Plant Cell Physiol. 45, 456–459 (2004)
Stahlberg, R. & Cosgrove, D. J. Comparison of electric and growth-responses to excision in cucumber and pea-seedlings. 1. Short-distance effects are a result of wounding. Plant Cell Environ. 17, 1143–1151 (1994)
Carpaneto, A. et al. Cold transiently activates calcium-permeable channels in Arabidopsis mesophyll cells. Plant Physiol. 143, 487–494 (2007)
Minorsky, P. V. Temperature sensing by plants: a review and hypothesis. Plant Cell Environ. 12, 119–135 (1989)
Dengler, N. G. The shoot apical meristem and development of vascular architecture. Can. J. Bot. 84, 1660–1671 (2006)
Yan, Y. et al. A downstream mediator in the growth repression limb of the jasmonate pathway. Plant Cell 19, 2470–2483 (2007)
Chauvin, A., Caldelari, D., Wolfender, J.-L. & Farmer, E. E. Four 13-lipoxygenases contribute to rapid jasmonate synthesis in wounded Arabidopsis leaves: a role for LOX6 in responses to long distance wound signals. New Phytol. 197, 566–575 (2013)
Zimmermann, M. R., Maischak, H., Mithofer, A., Boland, W. & Felle, H. H. System potentials, a novel electrical long-distance apoplastic signal in plants, induced by wounding. Plant Physiol. 149, 1593–1600 (2009)
Favre, P. & Agosti, R. D. Voltage-dependent action potentials in Arabidopsis thaliana. Physiol. Plant. 131, 263–272 (2007)
Herde, O. et al. Localized wounding by heat initiates the accumulation of proteinase inhibitor II in abscisic acid-deficient plants by triggering jasmonic acid biosynthesis. Plant Physiol. 112, 853–860 (1996)
Liu, Y. et al. Arabidopsis vegetative storage protein is an anti-insect acid phosphatase. Plant Physiol. 139, 1545–1556 (2005)
Xie, D.-X., Feys, B. F., James, S., Nieto-Rostro, M. & Turner, J. G. COI1: An Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280, 1091–1094 (1998)
Kilian, J. et al. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J. 50, 347–363 (2007)
Walley, J. W. et al. Mechanical stress induces biotic and abiotic stress responses via a novel cis-element. PLoS Genet. 3, e172 (2007)
Miller, G. et al. The plant NADPH oxidase RBOHD mediates rapid systemic signaling in response to diverse stimuli. Sci. Signal. 2, ra45 (2009)
Brüx, A. et al. Reduced V-ATPase activity in the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in Arabidopsis. Plant Cell 20, 1088–1100 (2008)
Bonaventure, G. et al. A gain-of-function allele of TPC1 activates oxylipin biogenesis after leaf wounding in Arabidopsis. Plant J. 49, 889–898 (2007)
Kang, S. et al. Overexpression in Arabidopsis of a plasma membrane-targeting glutamate receptor from small radish increases glutamate-mediated Ca2+ influx and delays fungal infection. Mol. Cells 21, 418–427 (2006)
Qi, Z., Stephens, N. R. & Spalding, E. P. Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile. Plant Physiol. 142, 963–971 (2006)
Stephens, N. R., Qi, Z. & Spalding, E. P. Glutamate receptor subtypes evidenced by differences in desensitization and dependence on the GLR3. 3 and GLR3. 4 genes. Plant Physiol. 146, 529–538 (2008)
Michard, E. et al. Glutamate receptor–like genes form Ca2+ channels in pollen tubes and are regulated by pistil d-serine. Science 332, 434–437 (2011)
Kwaaitaal, M., Huisman, R., Maintz, J., Reinstädler, A. & Panstruga, R. Ionotropic glutamate receptor (iGluR)-like channels mediate MAMP-induced calcium influx in Arabidopsis thaliana. Biochem. J. 440, 355–365 (2011)
Traynelis, S. F. et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol. Rev. 62, 405–496 (2010)
Chiu, J. C. et al. Phylogenetic and expression analysis of the glutamate-receptor–like gene family in Arabidopsis thaliana. Mol. Biol. Evol. 19, 1066–1082 (2002)
van Wees, S. Phenotypic analysis of Arabidopsis mutants: trypan blue stain for fungi, oomycetes, and dead plant cells. Cold Spring Harb Protoc. http://dx.doi.org/10.1101/pdb.prot4982 (2008)
Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 20, 3901–3907 (1987)
Oñate-Sánchez, L. & Vicente-Carbajosa, J. DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques. BMC Res. Notes 1, 93–100 (2008)
Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K. & Scheible, W.-R. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5–17 (2005)
Smyth, G. K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, e3 (2004)
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995)
Mueller, M. J., Mène-Saffrané, L., Grun, C., Karg, K. & Farmer, E. E. Oxylipin analysis methods. Plant J. 45, 472–489 (2006)
Kramell, R., Schneider, G. & Miersch, O. Chiral separation of amide conjugates of jasmonic acid by liquid chromatography. Chromatographia 45, 104–108 (1997)
Torres, M. A., Dangl, J. L. & Jones, J. D. Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc. Natl Acad. Sci. USA 99, 517–522 (2002)
Shimada, T. L., Shimada, T. & Hara-Nishimura, I. A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. Plant J. 61, 519–528 (2010)
Berberich, T., Takahashi, Y., Saitoh, H. & Terauchi, R. in The Handbook of Plant Functional Genomics Ch. 6 (eds Kahl, G. & Meksem K. ) 113–136 (Wiley, 2008)
Supported by a Faculty of Biology and Medicine Interdisciplinary grant (to S.K. and E.E.F.) and Swiss NSF grants 3100A0-122441 and 31003A-138235 (to E.E.F.). We thank I. Acosta, D. Gasperini, S. Stolz and A. Chételat and other Farmer lab members for critical comments and/or technical help, M. Blanchard for help with electrophysiology, and the Lausanne Genomic Technologies Facility and M. Shakhsi-Niaei for help with transcriptome analyses. We thank Y. Lee and F. Mauch for rbohD seeds, P. Schweizer and P. Reymond for insect larvae, J.-L. Wolfender for analytics support, and R. Benton, C. Fankhauser, N. Geldner, C. Hardtke and Y. Poirier for valuable comments.
The authors declare no competing financial interests.
Extended data figures and tables
a, The setup showing the ring cage around the insect (S. littoralis) and the position of the recording electrodes (e2 and e3) on leaf 8. b, Surface potential recording from electrode e2 while S. littoralis walked on the leaf. c, Typical surface potential changes recorded on electrode e3 during S. littoralis feeding. The arrowheads indicate periodicity in the signal. d, A proportion of WASPs induced by mechanical damage show periodicity. Filled arrowhead, time of wounding. The apical 40% of leaf 8 was wounded with forceps. Periodicity (unfilled arrowheads) was seen in 61% (n = 110) of experiments. e, Chilling-induced depolarization generated by gently placing water (150 μl, 0 °C) onto leaf 8 at the time indicated with the arrowhead. Chilling induced a change in surface potential in 3 out of 7 recordings. f, Typical WASP of the same polarity. For d, e and f the recording electrode was on leaf 8 at position e3 (Fig. 1a in the main text). g, Amplitude of the change in surface potential (± s.d.) induced by wounding or by cold water.
a, WASP characteristics in wounded leaf 8. b, Wound-activated surface potential changes in leaves 5, 9, 11, 13 and 16. Leaf 8 was wounded and surface potentials were monitored in distal leaves with electrodes placed on these leaves at position e3′. For leaf 8 the monitoring electrode was at position e2. W, wounded; x, number of experiments in which amplitudes of surface potentials exceeded −10 mV. Values are means ± s.d. c, Leaf-to-leaf signal speeds. Leaves 8 or 12 (the largest rosette leaves in 6-week-old plants) were chosen for estimating the apparent velocities of signals that travel within the wounded leaf. For leaf-to-leaf recordings, leaf 8 was wounded and recordings were made both on this leaf and on leaf 13. Analysis of variance (ANOVA) followed by Bonferroni post-hoc test showed that the WASP speed indicated in cm min−1 along the midrib and petiole within a leaf was not significantly different between leaves 8, 12 and 13, but was faster than the overall signalling speed from leaf 8 to leaf 13, and the signal speed from the wound to the lamina electrode (eL).
a, Electrode placements on leaves 8 (e3), 9 (e4) and 13 (e5). b, Typical changes in surface potential in leaves 8, 9 and 13 after wounding leaf 13. Arrowhead shows the time of wounding (W). c, WASP amplitudes (± s.d.) after wounding of leaf 13. d, WASP durations (± s.d.) after wounding of leaf 13. e, JAZ10 expression 1 h after wounding leaf 13 (± s.d.). U, unwounded leaves; W, wounded leaf 13. ***P < 0.001 (± s.d.).
a, Experimental design: electrodes were placed on the midrib (e2) and petiole base (e3) of leaf 8, and on leaf 9 (e4) and leaf 13 (e5). 40% of leaf 8 was wounded. b, WASP traces for leaves 9 (non-parastichious) and leaf 13 (connected) provoked by wounding leaf 8. The first pair of traces was recorded when leaf 8 was severed upon detection of a signal at e2 and before a WASP was detected at e3. The second pair of traces was recorded when the WASP generated by wounding leaf 8 was allowed to reach e3 and the leaf was then severed immediately. c, JAZ10 expression in unwounded leaves (U), wounded leaf 8 (W) and leaves 9 and 13. Left of dashed line: JAZ10 levels in leaves 8, 9 and 13 of intact control plants 1 h after wounding leaf 8. Right of the dashed line: plants in which the wounded leaf 8 was severed when WASPs were detected at e2 but were not allowed to reach electrode e3 (cut no WASP) or when WASPs were allowed to reach e3 before severing leaf 8 (cut WASP). ***P < 0.001 (± s.d.). Note: compared to crush-wounding, severing the petioles of otherwise undamaged leaves with sharp blades does not activate jasmonate signalling strongly in distal leaves.
Extended Data Figure 5 Current injection does not cause cell death in the lamina but elicits surface potential changes.
a–c, Trypan blue staining. a, Undamaged leaf. b, Pt wires inserted but no current injected. c, Current-injected leaf. Leaves were harvested 1 h after current injection. Cells were killed around the Pt wires (arrowheads) but CI did not cause increased staining of the lamina. Scale bars in boxes, 200 μm. d, Ion leakage analysis after current injection (CI). For controls leaves were either untreated or implanted with Pt wires and connected to three surface electrodes on the laminas (no CI). A further set of leaves was prepared identically but subjected to CI (40 μA, 10 s; CI) and harvested 1 h later for conductivity analyses. Positive controls: leaves infiltrated with 20 μl Triton X-100 (1% v/v in water) 1 h before harvest (‘TX-100 infiltration’). For analysis, leaves were excised at the base of the petiole and attached so that only their laminas were bathed in deionised water (25 ml) for 20 min at 22 °C. A control for the Triton X-100 infiltration was 20 μl Triton X-100 (1% v/v in water; TX-100 control), ± s.d. d, Surface potential changes in different parts of leaf 8 generated by current injection. Current (40 μA, 10 s) was injected into the petiole of leaf 8 (see Fig. 2a in the main text for electrode placements). x/n = the number of experiments in which signal amplitudes exceeded −10 mV/total number of experiments. Values are means ± s.d.
a, Typical recording from leaf 8 of the wild type after wounding the leaf tip. b, A typical recording from leaf 8 of the allene oxide synthase (aos) mutant after similar damage. In both cases the recording electrode was placed at position e3 (shown in Fig. 1a in the main text) before wounding the apical 40% of leaf 8. Arrowheads indicate the time of wound infliction (W). c, WASP amplitude (± s.d.) in wild-type and aos plants. d, Surface potential changes following CI (40 μA for 10 s) in the coronatine-insensitive 1-1 (coi1-1) mutant. Art, artefacts recorded in the leaf during CI (bar = 10 s). Note that the signal amplitude at eP reaches a maximum before that at eD and eL. For electrode placements see Fig. 2a. e, Relative JAZ10 levels in wounded WT and in the coi1-1 mutant that had been wounded or into which current (40 µA, 10 s) had been injected. Leaves were harvested 1 h after wounding or current injection. U, unwounded; W, wounded; CI, current injection. Significant differences from the unwounded wild type are indicated, *P < 0.05, ***P < 0.001 (± s.d.).
a, List of the JAZ genes that were upregulated 1 h after current injection (CI) into leaf 8 (this study), in leaf 13 at 1 h after wounding leaf 8 (this study), or in wounded leaves of 18-day-old plants (from ref. 32). b, Venn diagram showing downregulated (>twofold, P < 0.05) genes for current injected leaf 8 (this study), for leaf 13 from plants wounded on leaf 8 (this study), and for wounded rosette leaves (‘rosette after wounding’, from ref. 32). c, List of common genes that were downregulated more than twofold (P ≤ 0.05) 1 h after current injection into leaf 8 (this study), in leaf 13 1 h after wounding leaf 8 (leaf 13, this study), and in wounded leaves of 18-day-old plants (ref. 32), FC, fold change.
a–c, Inhibitors were tested for their effects on WASP generation. a, Diphenyleneiodonium chloride (DPI; 50 μM in H2O containing 1% v/v DMSO), b, catalase (100 U μl−1 in H2O) and c, lanthanum chloride (LaCl3, 2 mM in H2O) were infiltrated into leaf 8 at 25–30 min before wounding. After wounding leaf 8, WASP amplitude and duration were measured on leaf 13. For controls leaf 8 was infiltrated only with carrier. *P < 0.05 (± s.d.). d, JAZ10 transcript levels in leaf 13 following infiltration of DPI (50 μM in H2O containing 1% v/v DMSO) into leaf 8 followed 30 min later by wounding leaf 8 (± s.e.m.). Controls (CON) were infiltrated with carrier. e, Similar wound-induced expression of JAZ10 in WT and rbohD plants. Plants (wild type or rbohD-dSpm) were wounded on leaf 8 (W). After 1 h leaves 8 and 13 were harvested and JAZ10 expression measured by qRT–PCR (± s.d.). U, unwounded; W, wounded.
Extended Data Figure 9 Characterization of wound-activated surface potential changes (WASPs) in homozygous T-DNA insertion lines.
Leaf 8 was wounded and the surface potential was monitored in leaf 8 and distal leaf 13. For leaf 8, an electrode was placed 3 cm from the leaf apex wound (Fig. 1a, position e3). All measurements for leaf 13 were from electrodes placed on the petiole 1 cm from the centre of the rosette (position e3′ in Extended Data Fig. 2c). n, number of experiments. Values are means ± s.d. Mutants displaying WASP durations of <60 s in leaf 8 or <40 s in leaf 13 are highlighted.
Extended Data Figure 10 Relative expression levels of GLRs in the wild type and in T-DNA insertion lines.
a, Level of GLR3.1 transcripts in glr3.1a (Salk_063873). b, Level of GLR3.2 transcripts in glr3.2a (Salk_150710) and glr3.2b (Salk_133700). c, Level of GLR3.3 transcripts in glr3.3a (Salk_099757), glr3.3b (Salk_077608) and double mutant glr3.3a glr3.6a. d, Level of GLR3.6 transcripts in glr3.6a (Salk_091801), glr3.6b (Salk_035353) and double mutant glr3.3a glr3.6a. In all cases leaves were harvested from unwounded plant. Significant differences to the wild type are indicated, *P < 0.05, **P < 0.01, ***P < 0.001 (± s.d.). e, RT–PCR analyses of the expression pattern of GLR3.3 and GLR3.6 genes in glr3.3a glr3.6a and glr3.3b glr3.6a double mutants. UBC21 was the reference transcript.
Gene expression in response to current injection or wounding: The file contains a list of genes that were upregulated more than 2-fold (P ≤ 0.05) in leaf 8 1h after injecting current into leaf 8 (this study), in leaf 13 1h after wounding leaf 8 (this study), and in wounded leaves of 18 d-old plants 1 h after wound infliction32, FC=fold change (upregulated), ci = current injection, no ci = no current injection. (XLSX 30 kb)
Three Spodoptera littoralis larvae were placed on leaf 8 of a 5 week-old plant within a ring barrier. Surface potentials measured at the two electrodes are shown. A clock is displayed at upper right. The x-axis in ms and the y-axis is in V. Note that events that are recorded at the proximal electrode precede those recorded at the distal electrode and that the signal recorded at the distal electrode is simpler that at the proximal electrode. The video has been edited to shorten it. (MP4 14087 kb)
About this article
Cite this article
Mousavi, S., Chauvin, A., Pascaud, F. et al. GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling. Nature 500, 422–426 (2013). https://doi.org/10.1038/nature12478
This article is cited by
BMC Plant Biology (2022)
Ether anesthetics prevents touch-induced trigger hair calcium-electrical signals excite the Venus flytrap
Scientific Reports (2022)
Scientific Reports (2021)
Nature Communications (2021)
Nature Nanotechnology (2021)