In angiosperms, wound-derived signals travel through the vasculature to systemically activate defence responses throughout the plant. In Arabidopsis thaliana, activity of vasculature-specific Clade 3 glutamate receptor-like (GLR) channels is required for the transmission of electrical signals and cytosolic Ca2+ ([Ca2+]cyt) waves from wounded leaves to distal tissues, triggering activation of oxylipin-dependent defences. Whether nonvascular plants mount systemic responses upon wounding remains unknown. To explore the evolution of systemic defence responses, we investigated electrical and calcium signalling in the nonvascular plant Marchantia polymorpha. We found that electrical signals and [Ca2+]cyt waves are generated in response to mechanical wounding and propagated to nondamaged distal tissues in M. polymorpha. Functional analysis of MpGLR, the only GLR encoded in the genome of M. polymorpha, indicates that its activity is necessary for the systemic transmission of wound-induced electrical signals and [Ca2+]cyt waves, similar to vascular plants. However, spread of these signals is neither coupled to systemic accumulation of oxylipins nor to a transcriptional defence response in the distal tissues of wounded M. polymorpha plants. Our results suggest that lack of vasculature prevents translocation of additional signalling factors that, together with electrical signals and [Ca2+]cyt waves, contribute to systemic activation of defences in tracheophytes.

Ricca assays allow the direct introduction of compounds extracted from plants or the organisms that attack them into the leaf vasculature. Using chromatographic fractionation of Arabidopsis (Arabidopsis thaliana) leaf extracts, we found glutamate was the most active low mass elicitor of membrane depolarization. However, other known elicitors of membrane depolarization are generated in the wound response. These include unstable aglycones generated by glucosinolate (GSL) breakdown. None of the aglycone-derived GSL-breakdown products, including nitriles and isothiocyanates, that we tested using Ricca assays triggered electrical activity. Instead, we found that glutathione and the GSL-derived compound sulforaphane glutathione triggered membrane depolarizations. These findings identify a potential link between GSL breakdown and glutathione in the generation of membrane depolarizing signals. Noting that the chromatographic fractionation of plant extracts can dilute or exchange ions, we found that Cl- caused glutamate receptor-like3.3-dependent membrane depolarizations. In summary, we show that, in addition to glutamate, glutathione derivatives as well as chloride ions will need to be considered as potential elicitors of wound-response membrane potential change. Finally, by introducing aphid (Brevicoryne brassicae) extracts or the flagellin-derived peptide flg22 into the leaf vasculature we extend the use of Ricca assays for the exploration of insect/plant and bacteria/plant interactions.

Whether the plant vasculature has the capacity to sense touch is unknown. We developed a quantitative assay to investigate touch-response electrical signals in the leaves and veins of Arabidopsis thaliana. Mechanostimulated electrical signaling in leaves displayed strong diel regulation. Signals of full amplitude could be generated by repeated stimulation at the same site after approximately 90 minutes. However, the signals showed intermediate amplitudes when repeatedly stimulated in shorter timeframes. Using intracellular electrodes, we detected touch-response membrane depolarizations in the phloem. On the basis of this, we mutated multiple Arabidopsis H+-ATPase (AHA) genes expressed in companion cells. We found that aha1 aha3 double mutants attenuated touch-responses, and this was coupled to growth rate reduction. Moreover, propagating membrane depolarizations could be triggered by mechanostimulating the exposed primary vasculature of wild-type plants but not of aha1 aha3 mutants. Primary veins have autonomous mechanosensory properties which depend on P-type proton pumps.

The major developmental signal in plants, auxin is perceived by TIR1/AFB receptors. It triggers transcriptional reprogramming via well-established canonical mechanism but also elusive rapid, non-transcriptional responses. Here we demonstrate that TIR1/AFB receptors have, next to recently identified adenylate cyclase, also guanylate cyclase activity. Auxin perception activates independently the cAMP and cGMP production by TIR1/AFBs in vitro and increases cAMP and cGMP levels in planta with a slow and fast dynamics, respectively. Exogenous cGMP but not cAMP application induces rapid cytosolic Ca 2+ transients and root growth inhibition, suggesting that TIR1/AFB-derived cGMP mediates rapid auxin responses. This unprecedented combination of adenylate and guanylate cyclase activities in a hormone receptor provides a new paradigm for how a single perception mechanism can mediate a multitude of diverse downstream responses. One-Sentence Summary TIR1/AFB auxin receptors have guanylate cyclase activity, whose product, cGMP mediates rapid non-transcriptional auxin responses.

The links between wound-response electrical signalling and the activation of jasmonate synthesis are unknown. We investigated damage-response remodelling of jasmonate precursor pools in the Arabidopsis thaliana leaf vasculature. Galactolipids and jasmonate precursors in primary veins from undamaged and wounded plants were analysed using MS-based metabolomics and NMR. In parallel, DAD1-LIKE LIPASEs (DALLs), which control the levels of jasmonate precursors in veins, were identified. A novel galactolipid containing the jasmonate precursor 12-oxo-phytodienoic acid (OPDA) was identified in veins: sn-2-O-(cis-12-oxo-phytodienoyl)-sn-3-O-(β-galactopyranosyl) glyceride (sn-2-OPDA-MGMG). Lower levels of sn-1-OPDA-MGMG were also detected. Vascular OPDA-MGMGs, sn-2-18:3-MGMG and free OPDA pools were reduced rapidly in response to damage-activated electrical signals. Reduced function dall2 mutants failed to build resting vascular sn-2-OPDA-MGMG and OPDA pools and, upon wounding, dall2 produced less jasmonoyl-isoleucine (JA-Ile) than the wild-type. DALL3 acted to suppress excess JA-Ile production after wounding, whereas dall2 dall3 double mutants strongly reduce jasmonate signalling in leaves distal to wounds. LOX6 and DALL2 function to produce OPDA and the non-bilayer-forming lipid sn-2-OPDA-MGMG in the primary vasculature. Membrane depolarizations trigger rapid depletion of these molecules. We suggest that electrical signal-dependent lipid phase changes help to initiate vascular jasmonate synthesis in wounded leaves.

Leaf-feeding insects trigger high-amplitude, defense-inducing electrical signals called slow wave potentials (SWPs). These signals are thought to be triggered by the long-distance transport of low molecular mass elicitors termed Ricca's factors. We sought mediators of leaf-to-leaf electrical signaling in Arabidopsis thaliana and identified them as β-THIOGLUCOSIDE GLUCOHYDROLASE 1 and 2 (TGG1 and TGG2). SWP propagation from insect feeding sites was strongly attenuated in tgg1 tgg2 mutants and wound-response cytosolic Ca2+ increases were reduced in these plants. Recombinant TGG1 fed into the xylem elicited wild-type-like membrane depolarization and Ca2+ transients. Moreover, TGGs catalyze the deglucosidation of glucosinolates. Metabolite profiling revealed rapid wound-induced breakdown of aliphatic glucosinolates in primary veins. Using in vivo chemical trapping, we found evidence for roles of short-lived aglycone intermediates generated by glucosinolate hydrolysis in SWP membrane depolarization. Our findings reveal a mechanism whereby organ-to-organ protein transport plays a major role in electrical signaling.

Abstract When attacked by herbivores, plants produce electrical signals which can activate the synthesis of the defense mediator jasmonate. These wound-induced membrane potential changes can occur in response to elicitors that are released from damaged plant cells. We list plant-derived elicitors of membrane depolarization. These compounds include the amino acid l-glutamate (Glu), a potential ligand for GLUTAMATE RECEPTOR-LIKE (GLR) proteins that play roles in herbivore-activated electrical signaling. How are membrane depolarization elicitors dispersed in wounded plants? In analogy with widespread turgor-driven cell and organ movements, we propose osmoelectric siphon mechanisms for elicitor transport. These mechanisms are based on membrane depolarization leading to cell water shedding into the apoplast followed by membrane repolarization and water uptake. We discuss two related mechanisms likely to occur in response to small wounds and large wounds that trigger leaf-to-leaf electrical signal propagation. To reduce jasmonate pathway activation, a feeding insect must cut through tissues cleanly. If their mandibles become worn, the herbivore is converted into a robust plant defense activator. Our models may therefore help to explain why numerous plants produce abrasives which can blunt herbivore mouthparts. Finally, if verified, the models we propose may be generalizable for cell to cell transport of water and pathogen-derived regulators.

Plants detect their neighbors via various cues, including reflected light and touching of leaf tips, which elicit upward leaf movement (hyponasty). It is currently unknown how touch is sensed and how the signal is transferred from the leaf tip to the petiole base that drives hyponasty. Here, we show that touch-induced hyponasty involves a signal transduction pathway that is distinct from light-mediated hyponasty. We found that mechanostimulation of the leaf tip upon touching causes cytosolic calcium ([Ca 2+ ] cyt induction in leaf tip trichomes that spreads towards the petiole. Both perturbation of the calcium response and the absence of trichomes reduce touch-induced hyponasty. Finally, using plant competition assays, we show that touch-induced hyponasty is adaptive in dense stands of Arabidopsis. We thus establish a novel, adaptive mechanism regulating hyponastic leaf movement in response to mechanostimulation by neighbors in dense vegetation.

Summary Arabidopsis Clade 3 GLUTAMATE RECEPTOR‐LIKEs (GLRs) are primary players in wound‐induced systemic signaling. Previous studies focused on dissecting their ligand‐activated channel properties involving extracellular and membrane‐related domains. Here, we report that the carboxy‐terminal tails (C‐tails) of GLRs contain key elements controlling their function in wound signaling. GLR3.3 without its C‐tail failed to rescue the glr3.3a mutant. We carried out a yeast two‐hybrid screen to identify the C‐tail interactors. We performed functional studies of the interactor by measuring electrical signals and defense responses. Then we mapped their binding sites and evaluated the impact of the sites on GLR functions. IMPAIRED SUCROSE INDUCTION 1 (ISI1) interacted with GLR3.3. Enhanced electrical activity was detected in reduced function isi1 mutants in a GLR3.3‐dependent manner. isi1 mutants were slightly more resistant to insect feeding than the wild‐type. Furthermore, a triresidue motif RFL in the GLR3.3 C‐tail binds to ISI1 in yeast. Finally, we demonstrated that FL residues were conserved across GLRs and functionally required. Our study provides new insights into the functions of GLR C‐tails, reveals parallels with the ionotropic glutamate receptor regulation in animal cells, and may enable rational design of strategies to engineer GLRs for future practical applications.

Recurrent damage by lepidopteran folivores triggers repeated leaf-to-leaf electrical signaling. We found that the ability to propagate electrical signals-called slow wave potentials-was unexpectedly robust and was maintained in plants that had experienced severe damage. We sought genes that maintain tissue excitability during group insect attack. When Arabidopsis thaliana P-Type II Ca2+-ATPase mutants were mechanically wounded, all mutants tested displayed leaf-to-leaf electrical signals. However, when the auto-inhibited Ca2+-ATPase double-mutant aca10 aca12 was attacked by Spodoptera littoralis caterpillars, electrical signaling failed catastrophically, and the insects consumed these plants rapidly. The attacked double mutant displayed petiole base deformation and chlorosis, which spread acropetally into laminas and led to senescence. A phloem-feeding aphid recapitulated these effects, implicating the vasculature in electrical signaling failure. Consistent with this, ACA10 expressed in phloem companion cells in an aca10 aca12 background rescued electrical signaling and defense during protracted S. littoralis attack. When expressed in xylem contact cells, ACA10 partially rescued these phenotypes. Extending our analyses, we found that prolonged darkness also caused wound-response electrical signaling failure in aca10 aca12 mutants. Our results lead to a model in which the plant vasculature acts as a capacitor that discharges temporarily when leaves are subjected to energy-depleting stresses. Under these conditions, ACA10 and ACA12 function allows the restoration of vein cell membrane potentials. In the absence of these gene functions, vascular cell excitability can no longer be restored efficiently. Additionally, this work demonstrates that non-invasive electrophysiology is a powerful tool for probing early events underlying senescence.

Glutamate has dual roles in metabolism and signaling; thus, signaling functions must be isolatable and distinct from metabolic fluctuations, as seen in low-glutamate domains at synapses. In plants, wounding triggers electrical and calcium (Ca 2+ ) signaling, which involve homologs of mammalian glutamate receptors. The hydraulic dispersal and squeeze-cell hypotheses implicate pressure as a key component of systemic signaling. Here, we identify the stretch-activated anion channel MSL10 as necessary for proper wound-induced electrical and Ca 2+ signaling. Wound gene induction, genetics, and Ca 2+ imaging indicate that MSL10 acts in the same pathway as the glutamate receptor–like proteins (GLRs). Analogous to mammalian NMDA glutamate receptors, GLRs may serve as coincidence detectors gated by the combined requirement for ligand binding and membrane depolarization, here mediated by stretch activation of MSL10. This study provides a molecular genetic basis for a role of mechanical signal perception and the transmission of long-distance electrical and Ca 2+ signals in plants.

Plants in nature are constantly exposed to organisms that touch them and wound them. A highly conserved response to these stimuli is a rapid collapse of membrane potential (i.e. a decrease of electrical field strength across membranes). This can be coupled to the production and/or action of jasmonate or ethylene. Here, the various types of electrical signals in plants are discussed in the context of hormone responses. Genetic approaches are revealing genes involved in wound-induced electrical signalling. These include clade 3 GLUTAMATE RECEPTOR-LIKE (GLR) genes, Arabidopsis H+-ATPases (AHAs), RESPIRATORY BURST OXIDASE HOMOLOGUEs (RBOHs), and genes that determine cell wall properties. We briefly review touch- and wound-induced increases in cytosolic Ca2+ concentrations and their temporal relationship to electrical activities. We then look at the questions that need addressing to link mechanostimulation and wound-induced electrical activity to hormone responses. Utilizing recently published results, we also present a hypothesis for wound-response leaf-to-leaf electrical signalling. This model is based on rapid electro-osmotic coupling between the phloem and xylem. The model suggests that the depolarization of membranes within the vascular matrix triggered by physical stimuli and/or chemical elicitors is linked to changes in phloem turgor and that this plays vital roles in leaf-to-leaf electrical signal propagation.

Wound-response plant growth restriction requires the synthesis of potent mediators called jasmonates (JAs). Four 13-lipoxygenases (13-LOXs) produce JA precursors in Arabidopsis (Arabidopsis thaliana) leaves, but the 13-LOXs responsible for growth restriction have not yet been identified. Through loss-of-function genetic analyses, we identified LOX3 and LOX4 as the principal 13-LOXs responsible for vegetative growth restriction after repetitive wounding. Additional genetic studies were carried out in the gain-of-function fatty acid oxygenation 2 (fou2) mutant that, even when undamaged, shows JA-dependent leaf growth restriction. The fou2 lox3 lox4 triple mutant suppressed the fou2 JA-dependent growth phenotype, confirming that LOX3 and LOX4 function in leaf growth restriction. The fou2 mutation affects the TWO PORE CHANNEL1 (TPC1) ion channel. Additional genetic approaches based on this gene were used to further investigate LOX3 function in relation to leaf growth. To activate LOX3-dependent JA production in unwounded plants, we employed hyperactive TPC1 variants. Expression of the TPC1ΔCa i variant in phloem companion cells caused strongly reduced rosette growth in the absence of wounding. Summarizing, in parallel to their established roles in male reproductive development in Arabidopsis, LOX3 and LOX4 control leaf growth rates after wounding. The process of wound-response growth restriction can be recapitulated in unwounded plants when the LOX3 pathway is activated genetically using a hyperactive vacuolar cation channel.

Most of the great diversity of oxylipins in plants is produced by a group of specialized cytochrome P450 enzymes, among which is ALLENE OXIDE SYNTHASE (AOS). Many AOSs generate precursors of the defense hormone jasmonate. As a consequence, aos mutants fail to defend themselves against herbivores and do not display restriction of vegetative growth when wounded. These links between growth and defense that are controlled by AOS-derived oxylipins are ancient. Here, we focus on oxylipin-regulated coordination of growth/defense, how this optimizes defense, and how a plant's need for light can override jasmonate activity. AOS-derived oxylipins are candidate regulators throughout land plant evolution.

Slow wave potentials (SWPs) are damage-induced electrical signals which, based on experiments in which organs are burned, have been linked to rapid increases in leaf or stem thickness. The possibility that pressure surges in injured xylem underlie these events has been evoked frequently. We sought evidence for insect feeding-induced positive pressure changes in the petioles of Arabidopsis thaliana. Instead, we found that petiole surfaces of leaves distal to insect-feeding sites subsided. We also found that insect damage induced longer-duration downward leaf movements in undamaged leaves. The transient petiole deformations were contemporary with and dependent on the SWP. We then investigated if mutants that affect the xylem, which has been implicated in SWP transmission, might modify SWP architecture. irregular xylem mutants strongly affected SWP velocity and kinetics and, in parallel, restructured insect damage-induced petiole deformations. Together, with force change measurements on the primary vein, the results suggest that extravascular water fluxes accompany the SWP. Moreover, petiole deformations in Arabidopsis mimic parts of the spectacular distal leaf collapse phase seen in wounded Mimosa pudica. We genetically link electrical signals to organ movement and deformation and suggest an evolutionary origin of the large leaf movements seen in wounded Mimosa.

Electrogenic proton pumps have been implicated in the generation of slow wave potentials (SWPs), damage-induced membrane depolarizations that activate the jasmonate (JA) defense pathway in leaves distal to wounds. However, no defined H+-ATPases have been shown to modulate these electrical signals. Pilot experiments revealed that the proton pump activator fusicoccin attenuated SWP duration in Arabidopsis. Using mutant analyses, we identified Arabidopsis H+-ATPase 1 (AHA1) as a SWP regulator. The duration of the repolarization phase was strongly extended in reduced function aha1 mutants. Moreover, the duration of SWP repolarization was shortened in the presence of a gain-of-function AHA1 allele. We employed aphid electrodes to probe the effects of the aha1 mutation on wound-stimulated electrical activity in the phloem. Relative to the wild type, the aha1-7 mutant increased the duration and reduced the amplitudes of electrical signals in sieve tube cells. In addition to affecting electrical signaling, expression of the JA pathway marker gene JAZ10 in leaves distal to wounds was enhanced in aha1-7. Consistent with this, levels of wound-response jasmonoyl-isoleucine were enhanced in the mutant, as was defense against a lepidopteran herbivore. The work identifies a discrete member of the P-type ATPase superfamily with a role in leaf-to-leaf electrical signaling and plant defense.

Plants are exposed to cellular damage by mechanical stresses, herbivore feeding, or invading microbes. Primary wound responses are communicated to neighboring and distal tissues by mobile signals. In leaves, crushing of large cell populations activates a long-distance signal, causing jasmonate production in distal organs. This is mediated by a cation channel-mediated depolarization wave and is associated with cytosolic Ca2+ transient currents. Here, we report that much more restricted, single-cell wounding in roots by laser ablation elicits non-systemic, regional surface potential changes, calcium waves, and reactive oxygen species (ROS) production. Surprisingly, laser ablation does not induce a robust jasmonate response, but regionally activates ethylene production and ethylene-response markers. This ethylene activation depends on calcium channel activities distinct from those in leaves, as well as a specific set of NADPH oxidases. Intriguingly, nematode attack elicits very similar responses, including membrane depolarization and regional upregulation of ethylene markers. Moreover, ethylene signaling antagonizes nematode feeding, delaying initial syncytial-phase establishment. Regional signals caused by single-cell wounding thus appear to constitute a relevant root immune response against small invaders.

The identity of the cell files necessary for the leaf-to-leaf transmission of wound signals plants has been debated for decades. In Arabidopsis, wounding initiates the glutamate receptor-like (GLR)-dependent propagation of membrane depolarizations that lead to defense gene activation. Using a vein extraction procedure we found pools of GLR-fusion proteins in endomembranes in phloem sieve elements and/or in xylem contact cells. Strikingly, only double mutants that eliminated GLRs from both of these spatially separated cell types strongly attenuated leaf-to-leaf electrical signaling. glr3.3 mutants were also compromised in their defense against herbivores. Since wounding is known to cause increases in cytosolic calcium, we monitored electrical signals and Ca2+ transients simultaneously. This revealed that wound-induced membrane depolarizations in the wild-type preceded cytosolic Ca2+ maxima. The axial and radial distributions of calcium fluxes were differentially affected in each glr mutant. Resolving a debate over which cell types are necessary for electrical signaling between leaves, we show that phloem sieve elements and xylem contact cells function together in this process.