Jasmonates are lipid mediators that control defence gene expression in response to wounding and other environmental stresses. These small molecules can accumulate at distances up to several cm from sites of damage and this is likely to involve cell-to-cell jasmonate transport. Also, and independently of jasmonate synthesis, transport and perception, different long-distance wound signals that stimulate distal jasmonate synthesis are propagated at apparent speeds of several cm min–1 to tissues distal to wounds in a mechanism that involves clade 3 GLUTAMATE RECEPTOR-LIKE (GLR) genes. A search for jasmonate synthesis enzymes that might decode these signals revealed LOX6, a lipoxygenase that is necessary for much of the rapid accumulation of jasmonic acid at sites distal to wounds. Intriguingly, the LOX6 promoter is expressed in a distinct niche of cells that are adjacent to mature xylem vessels, a location that would make these contact cells sensitive to the release of xylem water column tension upon wounding. We propose a model in which rapid axial changes in xylem hydrostatic pressure caused by wounding travel through the vasculature and lead to slower, radially dispersed pressure changes that act in a clade 3 GLR-dependent mechanism to promote distal jasmonate synthesis.
The strong activation of gene expression controlled by jasmonates (Browse, 2009) can lead to defence responses throughout much of the plant body (Koo & Howe, 2009). However, this comes at a price, as induced defence is frequently associated with growth inhibition (Yang et al., 2012). As defence and growth are coordinated, long-distance signals emanating from wounds need to reach regions of cell proliferation and/or cell expansion – that is, they have to travel from damaged organs (e.g. leaves) to the vicinity of meristems, as well as reaching distal undamaged tissues. The effects of localized wounds on jasmonate signalling are readily detected in both seedlings and rosettes. Fig. 1 shows plants expressing β-glucuronidase (GUS) under the control of the jasmonate-responsive JASMONATE ZIM-DOMAIN10 (JAZ10) promoter (Acosta et al., 2013). In seedlings, small wounds to individual cotyledons lead to GUS staining of the root stele. Crush wounds to leaves cause GUS expression not only in the wounded leaf but also in certain distal leaves that share vascular connections with the wounded leaf. JAZ10 activation in response to wounding requires the synthesis of jasmonic acid (JA) and its conversion to biologically active forms, chiefly jasmonoyl-isoleucine (JA-Ile, Fonseca et al., 2009). This is followed by the interaction of JA-Ile with the receptor CORONATINE INSENSITIVE1 (COI1), a process that then leads to defence gene expression (Browse, 2009; Fonseca et al., 2009), as well as to the increased synthesis of jasmonates (Wasternack & Hause, 2013). Here, we concentrate on signalling events relevant to the initial activation of jasmonate synthesis distal to wounds.
Jasmonate synthesis after wounding can be rapid. In the case of Arabidopsis, damaged leaves synthesize substantial amounts of JA and increases in the concentrations of this molecule in crushed tissue are first detectable within c. 30 s after wounding (e.g. Chauvin et al., 2013). Interestingly, despite the ability of crushed tissue to produce JA, wound-induced JAZ10 expression is at least 80 times lower within the wounded tissue than it is in the unwounded part of the same leaf (Chauvin et al., 2013). Crush wounds are particularly effective in exciting jasmonate synthesis and do this more strongly than ‘clean’ wounds, such as those produced by chewing insects, pin pricks or sharp scissors (e.g. Farmer et al., 1992; Koo et al., 2009; Mousavi et al., 2013). This raises the possibility that elicitors of jasmonate synthesis and/or jasmonates themselves may be supplied to undamaged cells from crushed tissues. Indeed, jasmonates (or their precursors) were proposed to be transported either between cells (Farmer et al., 1992) or between organs (Li et al., 2005). Moreover, the transport of chemical elicitors of the wound response may be, to some extent, coupled to fluid transport in the xylem (e.g. Alarcon & Malone, 1994; Rhodes et al., 2006) or in the phloem (Stenzel et al., 2003). In order to explain reduced jasmonate signalling in crushed tissues and high jasmonate signalling in undamaged tissues, it is also possible that molecules that inhibit jasmonate signalling are present in undamaged tissue and are drawn into a wound (Rhodes et al., 2006). One such molecule could be ethylene (Kahl et al., 2000).
The importance of the vasculature in jasmonate synthesis
Cells outside the vasculature almost certainly produce JA. For example, LIPOXYGENASE 2, encoding an enzyme producing fatty acid 13-hydroperoxide jasmonate precursors, is strongly expressed in mesophyll cells (Montillet et al., 2013). However, previous reports suggest that the vasculature has a particularly strong capacity to synthesize jasmonates (Stenzel et al., 2003; Chauvin et al., 2013; Wasternack & Hause, 2013). Therefore, damage to veins is expected to result in high concentrations of JA and JA-Ile. Additionally, JA synthesis amplification acting along veins was proposed for plants in the Solanaceae that produce the peptide wound hormone systemin (e.g. Stenzel et al., 2003). Further evidence supporting jasmonate synthesis amplification at a distance from a wound comes from Sato et al. (2011). Deuterated JA-Ile (made with [2H3]-JA that cannot be converted in vivo to [1H3]-JA) was applied to wounds on tobacco leaves, and some of it was later detected in leaves distal to wounds. Interestingly, this application also led to increases in unlabelled JA and JA-Ile concentrations in distal leaves that were over and above the increases observed in control plants that had been wounded but to which no JA-Ile was applied. These results are consistent with both JA-Ile transport and amplification of jasmonate synthesis along the vasculature to produce JA-Ile de novo. In summary, the events leading to jasmonate production in and near wounds are highly complex so it may therefore be simpler at present to look at tissues at a distance from sites of damage. In fact, the events that lead to jasmonate accumulation distal to wounds can be reduced to two principal mechanisms (Koo & Howe, 2009). One is transport of jasmonate between cells, and the other is a rapid signal (or signals) acting over long distances and, in principle, independently of jasmonate transport.
Long-distance activation of jasmonate signalling in Arabidopsis
Here we focus on a possible mechanism that controls jasmonate synthesis in veins distal to wounds and, to do so, examine events that take place in an undamaged leaf in the first 90 s after wounding another leaf. This is the approximate time required to detect the first increases in JA concentrations in Arabidopsis leaf 13 when leaf 8 is wounded (Chauvin et al., 2013). Specifically, we treat the following questions. What are the long-distance wound signals that initiate distal jasmonate synthesis? What cells do they travel through? And how do they initiate the signalling that leads to activation of the jasmonate pathway? Among the many processes that might activate jasmonate synthesis far from a wound is the generation of signals that could travel through the vasculature. For example, hydrostatic pressure changes are generated upon wounding (Malone & Stanković, 1991; Stahlberg & Cosgrove, 1992) but have not, to our knowledge, been linked to jasmonate signalling. However, electrical signals were proposed to activate jasmonate synthesis (Herde et al., 1996). This review will attempt to integrate these ‘hydraulic’ and ‘electrical’ signalling pathways with what is known about sites of jasmonate synthesis in the vasculature.
The general features of long-distance wound-related signals were elucidated before their recent genetic association with the jasmonate pathway. They are: generated a few seconds after wounding and appear to travel bidirectionally away from a wound (e.g. Davies & Schuster, 1981); associated with easily detected electrical activity (e.g. Stahlberg & Cosgrove, 1992); associated with the relief of xylem water tension (Stahlberg & Cosgrove, 1992, 1997); and correlated with defence gene expression (Wildon et al., 1992). It is now known that the activation of jasmonate synthesis by signals that meet these general criteria depends, at least in part, on clade 3 GLUTAMATE RECEPTOR-LIKE (GLR) genes (Mousavi et al., 2013).
When subjected to large crush wounds, the wounded leaves of the glr3.3 glr3.6 double mutant showed near wildtype JAZ10 expression, whereas jasmonate signalling in distal leaves was reduced relative to the wildtype. This reduction was correlated with diminished wound-activated surface potential changes that were detected with noninvasive (extracellular) electrodes (Mousavi et al., 2013). Presumably, the capacity of the vasculature to both make and transport jasmonates (or other elicitors of jasmonate synthesis) could explain why the glr3.3 glr3.6 double mutant had little impact on jasmonate responses in a crush-damaged leaf but had a greater and more easily detected impact in distal leaves. In other words, jasmonate transport coupled to autoamplification of jasmonate synthesis may override and mask the effects of other long-distance signals. However, when Spodoptera littoralis larvae feed on leaves, they typically leave very little crushed tissue and yet they efficiently trigger both propagated electrical activity (Mousavi et al., 2013) and hydraulic signals in leaves (Alarcon & Malone, 1994). In this case, the prediction is that the impact of jasmonate synthesis-independent pathways on distal jasmonate pathway activation (e.g. JAZ10 induction) may be proportionally greater than any input of elicitors, including jasmonates, because damaged cells are consumed by herbivores.
The GLR-LOX pathway
The discovery of the role of clade 3 GLR genes in long-distance ‘electrical’ signalling suggests the existence of a novel signalling pathway upstream of jasmonate synthesis. Additionally, a lipoxygenase (LOX6) responsible for rapid jasmonate synthesis in tissues distal to wounds has been identified in Arabidopsis (Chauvin et al., 2013; Grebner et al., 2013). This raises the possibility that this enzyme functions close to the interface between a ‘GLR-LOX’ pathway and the canonical jasmonate signalling pathway based on COI1 and JAZ proteins.
Plant GLRs are structurally related to vertebrate ionotropic glutamate receptors (iGluRs) (Lam et al., 1998), which mediate electrochemical signalling in animal nervous systems (Traynelis et al., 2010). Like iGluRs, GLRs contain a ligand-binding domain (LBD) whose two putative lobes (S1 and S2) are separated by a predicted ion channel portion consisting of three transmembrane domains (M1–M3) and one pore loop (P, Fig. 2). iGluRs and GLRs also have a large amino-terminal domain (ATD) that, in iGluRs, is involved in channel assembly and cofactor binding (Jin et al., 2009). In mammals, functional ligand-gated channels consist of either homo- or heterotetramers (Sobolevsky et al., 2009). Subunit composition dictates the functional properties of the channel, resulting in a large number of receptor types serving as nonselective cation channels that conduct Na+, K+ and Ca2+ in the presence of glutamate (Mayer, 2006).
In plants, calcium-permeable GLRs are known to participate in pollen tube growth (GLR1.2) and morphogenesis (GLR3.7; Michard et al., 2011). Recently, a role in lateral root initiation has been established for GLR3.2 and GLR3.4 (Vincill et al., 2013). Mutations in GLR3.3 greatly attenuate glutamate-stimulated membrane depolarizations in seedlings (Qi et al., 2006), and GLR3.3 gene product(s) can participate in a variety of different channel configurations that have distinguishable responses to different agonists (i.e. amino acids and glutathione: Qi et al., 2006; Stephens et al., 2008). Further work on GLRs will be necessary to assess the mechanism of activation, subunit compositions and ions transported, especially because clade 3 GLRs are dissimilar to iGluRs in the pore region, which typically determines the ion selectivity of glutamate receptors (Davenport, 2002; Traynelis et al., 2010). Indeed, the presence of positively charged amino acid residues in Arabidopsis clade 3 GLR pore regions (Fig. 2b) suggests that the channel specificities between iGluRs and GLRs might differ. Nevertheless, there is mounting evidence that some plant GLRs control calcium fluxes directly or indirectly (e.g. Qi et al., 2006; Michard et al., 2011). Most LOXs (including LOXs in Arabidopsis) have putative Ca2+-binding domains and, for some LOXs, Ca2+ binding is needed for enzyme activity (e.g. Oldham et al., 2005). Therefore, one possibility is that Ca2+ fluxes controlled directly or indirectly by GLRs could activate plant LOXs to stimulate de novo JA synthesis. It is also possible that LOXs build pools of preformed jasmonate precursors such as oxo-phytodienoic acid (OPDA). Depletion of basal OPDA pools in response to wounding has been observed (Koo et al., 2009; Grebner et al., 2013) and the conversion of these OPDA pools to JA could also be ion flux-regulated. In any case, LOX6 is likely to operate directly or indirectly in a mechanism that decodes long-distance GLR-dependent signals.
The importance of the xylem in long-distance jasmonate signalling
The phloem is a site of jasmonate synthesis (e.g. Hause et al., 2003; Stenzel et al., 2003) and this tissue has been implicated in jasmonate signalling as the site of production of the peptide wound hormone systemin, an activator of jasmonate synthesis in solanaceous plants (Jacinto et al., 1997). More recently, cell-specific living electrodes (aphids) were used to detect wound-induced depolarization waves in phloem sieve tubes and this electrical activity was abolished in the glr3.3 glr3.6 double mutant (Salvador-Recatalà et al., 2014). Sieve elements can propagate electrical activity along their axes (van Bel et al., 2014), although this is not necessarily a key route to long-distance wound signal dispersal (Rhodes et al., 2006). Moreover, given the ease with which massive depolarizations can be detected with surface electrodes, it is likely that sieve tubes are not the only cell type responsible for surface-detected electrical activity after wounding. Finally, blocking phloem translocation by cold girdling of the petioles did not reduce the electrical activity recorded in distal leaves of wounded tomato plants (Wildon et al., 1992). Here, we propose that xylem-associated cells, in addition to cells in the phloem, have important roles in the long-distance wound signal transmission that leads to jasmonate pathway activation. That is, important events in the initiation of jasmonate synthesis take place within the bounds of the vascular bundle sheath.
While most cells in the leaf exert strong positive pressure on their walls, the fluid contents of xylem vessels is frequently under tension (Taiz & Zeiger, 1991) and this appears to be involved in rapid long-distance wound signalling. Earlier work on a wide variety of plants has underlined the importance of long-distance signals that generate easily detected electrical activity resulting, at least in part, from pressure changes in the xylem (Malone & Stanković, 1991; Stahlberg & Cosgrove, 1992, 1995, 1997). These electrical events are known as ‘slow wave potentials’ (SWPs; reviewed in Fromm & Lautner, 2007). Importantly, rapid wound-induced pressure changes in the xylem are probably transmitted to other cells to initiate depolarization events detectable with noninvasive electrodes placed on the cuticle (Stahlberg & Cosgrove, 1997). Interestingly, the expression domain of Arabidopsis LOX6, the lipoxygenase necessary for rapid JA accumulation in leaf tissues distal to wounds, is in a group of cells that are tightly associated with mature xylem vessels. Owing to their strong association with the walls of xylem vessels (Chauvin et al., 2013), they could correspond to contact cells (Evert, 2006). Because such cells are hydrostatically coupled to the xylem (van Bel & van der Schoot, 1988), they are likely to be squeezed by pressure increases in the xylem after wounding.
The squeeze cell hypothesis
Slow wave potentials share plasma membrane depolarizations of similar overall amplitudes and durations with the electrical activity we observed in wounded Arabidopsis leaves (Mousavi et al., 2013). As stated earlier, SWP propagation depends on the release of xylem water column tension upon wounding (Malone & Stanković, 1991; Stahlberg & Cosgrove, 1992, 1995). Crucially, rather than being the direct result of rapid axial pressure change along the xylem, this electrical activity may instead reflect subsequent (and slower) radial pressure changes emanating from xylem vessels (Stahlberg & Cosgrove, 1997). That is, cells adjacent to vessels (contact cells in particular) are probably subjected to abrupt increases in turgor pressures upon wounding. How they respond would depend to a large extent on the elasticity modulus (ε) of their walls (Taiz & Zeiger, 1991). Changes in water pressure in the xylem would lead to significant changes in turgor pressure in adjacent cells and possibly in other nearby cells, especially if their walls had low elasticity. This is a result of the relationship between turgor pressure (P) changes and volume (V) changes. As ε = ΔP/(ΔV/V), the more rigid the cell wall, the higher the pressure change for a given cellular volume change (Taiz & Zeiger, 1991). Radially transmitted turgor pressure changes could literally squeeze specific cells in and near the xylem as illustrated in Fig. 3 – hence the name ‘squeeze cell hypothesis’. Xylem contact cells probably correspond to the LOX6 expression niche (Chauvin et al., 2013) and in this model they would produce jasmonate in response to rapid pressure changes.
In Fig. 4 we propose that, upon wounding, axial pressure changes passing though xylem vessels are converted to radially transmitted pressure changes distal to wounds, causing contact cell squeezing. We also propose that these pressure changes underlie much of the GLR-dependent electrical activity investigated Mousavi et al. (2013). Rapid pressure changes that result from wounding or milder insults probably activate ion and water fluxes, which may be controlled directly or indirectly by GLR proteins and lead to the promotion of jasmonate synthesis. It is possible that these ion channels function downstream of mechanosensors, as current injection alone is sufficient to induce jasmonate synthesis (Herde et al., 1996; Mousavi et al., 2013). Alternatively, clade 3 GLRs could themselves act in mechanoperception related to turgor pressure changes. To test this model, it will be necessary to determine how clade 3 GLRs are regulated during the wound response; which ions they conduct; and what other ion channels, pumps and transporters contribute to the perception of pressure changes and associated electrical activity in jasmonate-synthesizing cell types.
The hypothesis is that Ca2+ is an important component of wound-induced intravascular ion fluxes that might lead to or be part of the mechanism of membrane depolarization after wounding or herbivory (Maffei et al., 2004) and could perhaps coordinate JA synthesis in different cell types with the vasculature. Plasmodesmata are important for coupling the electrical activity of cells within the vasculature (e.g. van Bel & Ehlers, 2005), as has been confirmed in studies with herbivores (Bricchi et al., 2012). It is likely that, upon wounding, electrical activity originating from near the xylem would be transmitted through plasmodesmata to phloem sieve elements and perhaps then along sieve tubes (van Bel et al., 2014).
The squeeze cell hypothesis is consistent with the jasmonate-dependent activation of cell division in the fascicular cambium (FC) of Arabidopsis stems. FC cells are immediately proximal to xylem in the inflorescence stem, similar to LOX6-expressing cells in Arabidopsis leaves. The jasmonate-dependent proliferation of these cells during secondary growth is likely to be stimulated by mechanical stresses (Sehr et al., 2010). Also, numerous jasmonate-controlled genes are up-regulated when leaves are repeatedly bent (Chehab et al., 2012), so leaf bending might be linked to xylem-associated cell pressure changes. The hypothesis could be applicable to other extravascular cell types in plants. For example, JAZ10 expression in and near the epidermis was stimulated by gentle mechanostimulation (Sehr et al., 2010).
These considerations should lead to further testable hypotheses, although one obstacle is that current technology does not permit the noninvasive measurement of pressures of single cells below the epidermis. First, differences in xylem vessel diameter along leaves might help to explain why long-distance electrical signals in Arabidopsis have heterogeneous speeds, moving quickly through the vasculature near the centre of the leaf but apparently slowing near the centre of the plant (Mousavi et al., 2013). The expectation is that abrupt pressure changes caused by wounding may be lower in narrow vessels near the centre of the plant than in wider vessels nearer the centre of the leaf; the model would also predict that the greater the diameter of the xylem vessels damaged by wounding, the greater the activation of the jasmonate pathway. Secondly, the transpiration status of the plant should also affect surface-detected electrical activity generated upon wounding, as it would influence xylem water column tension. Thirdly, genetic approaches may be used to test the model. For example, mutants with imperfect vascular development, particularly in xylem (e.g. the xyp1 xyp2 double mutant; Motose et al., 2004), are expected to display defects in wound signal propagation. Fourthly, the possible involvement of Ca2+ could be addressed with reporters to study Ca2+ fluxes in glr, plasmodesmata and xylem mutants. Another easily testable prediction is that some clade 3 GLRs are expressed in contact cells, which are putative sites of jasmonate synthesis (Chauvin et al., 2013). Given the abolition of herbivore-induced sieve tube depolarization in a glr3.3 glr3.6 double mutant (Salvador-Recatalà et al., 2014), it is likely that at least one of these GLR genes is expressed in the phloem, a site of jasmonate synthesis (Hause et al., 2003). Finally, possible parallels with hydraulic signalling in water stress (e.g. Christmann et al., 2007), as well as with systemic signalling in pathogenesis (reviewed in Romeis & Herde, 2014), deserve further exploration.
Supported by Swiss National Science Foundation grant 31003A-138235 to E.E.F.