Unlike plants, animals rely on rapid nervous systems to escape predation. A stationary fly that perceives danger takes less than 300 ms to take off, and this process requires complex whole- to ion fluxes in cell populations in wounded Arabidopsis plants. As summarised in Supplementary Fig. 1, we show that electrical signalling activates jasmonate biosynthesis in leaves distal to wounds and we identify genes involved in electrical signal propagation. Wound-induced surface potential changesTo investigate patterns of electrical activity and gene expression in 5 week-old rosettes, individual leaves were numbered from oldest to youngest. Electrodes placed on leaf 8 at the midrib/petiole junction (e2 electrode position) and on the petiole (position e3) did not detect changes in electrical activity and such changes were not elicited by walking S. littoralis larvae (Fig. 1b). When recordings were extended, they often showed periodicity ( Supplementary Fig. 3). We used three parameters to characterise these signals: latency (time from wounding to arrival at the amplitude midpoint), amplitude and duration (Fig. 1b). To gain more information on the spread of WASPs within a wounded leaf, four electrodes were placed on the leaf surface (Fig. 1a). After damage, WASPs were detected first at e1, then several seconds later at e2, and finally at e3. An electrode on the lamina also detected damage-elicited electrical activity and, in each case (Fig. 1c), the changes in amplitude were typically close to -70 mV (SupplementaryTable 1). The signals we measured had the same polarity as those produced after a chilling treatment known to cause plasma membrane depolarisation 24,25 . Therefore WASPs in leaf 8 were due to plasma membrane depolarisation ( Supplementary Fig. 4). The WASPs detected on WT plants were indistinguishable to those on wounded plants that lacked the ability to synthesize jasmonates ( Supplementary Fig. 5).This suggests that the mechanism that produces WASPs is upstream or independent of jasmonate synthesis. WASP territories and speedsSignals generated by wounding leaf tips first move towards the centre of the rosette and then disperse away from the apex into a restricted number of distal leaves to initiate distal JA accumulation and signalling 11 . In order to map the spatial distribution of WASPs in the rosette after wounding leaf 8 we placed electrodes in the e3 position of leaves 5 through 18. Leaves 5, 11, 13 and 16 showed responses similar to those in the wounded leaf (Fig. 1d, Supplementary Table 2). For example, after wounding leaf 8, a WASP with a duration of 78±20 s and a peak amplitude of -51 ± 9 mV was reached in leaf 13 after a latency of 66 ± 13 s (n=61 plants). Other leaves (7, 9, 10, 12, 14, 15,17 and 18) showed small positive surface potential changes. For example, leaf 9 showed a 20±5 mV change in surface potential with a latency of 54±12 s (n=46 plants). Most of these observations fit a developmental pattern: In adult-phase Arabidopsis rosettes, leaf 'n' shares direct vascular connections to leave...

SummaryDamage-inducible defenses in plants are controlled in part by jasmonates, fatty acidderived regulators that start to accumulate within 30 s of wounding a leaf.Using liquid chromatography-tandem mass spectrometry, we sought to identify the 13-lipoxygenases (13-LOXs) that initiate wound-induced jasmonate synthesis within a 190-s timeframe in Arabidopsis thaliana in 19 single, double, triple and quadruple mutant combinations derived from the four 13-LOX genes in this plant.All four 13-LOXs were found to contribute to jasmonate synthesis in wounded leaves: among them LOX6 showed a unique behavior. The relative contribution of LOX6 to jasmonate synthesis increased with distance from a leaf tip wound, and LOX6 was the only 13-LOX necessary for the initiation of early jasmonate synthesis in leaves distal to the wounded leaf.Herbivory assays that compared Spodoptera littoralis feeding on the lox2-1 lox3B lox4A lox6A quadruple mutant and the lox2-1 lox3B lox4A triple mutant revealed a role for LOX6 in defense of the shoot apical meristem. Consistent with this, we found that LOX6 promoter activity was strong in the apical region of rosettes. The LOX6 promoter was active in and near developing xylem cells and in expression domains we term subtrichomal mounds.

Jasmonates are oxygenated lipids (oxylipins) that control defense gene expression in response to cell damage in plants. How mobile are these potent mediators within tissues? Exploiting a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis (Arabidopsis thaliana) that displays impaired jasmonic acid (JA) synthesis in specific cell types and using JA-inducible reporters, we mapped the extent of the transport of endogenous jasmonates across the plant vegetative growth phase. In seedlings, we found that jasmonate (or JA precursors) could translocate axially from wounded shoots to unwounded roots in a LOX2-dependent manner. Grafting experiments with the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin transport. Next, we used rosettes to investigate radial cell-to-cell transport of jasmonates. After finding that the LOX6 protein localized to xylem contact cells was not wound inducible, we used the lox234 triple mutant to genetically isolate LOX6 as the only JA precursor-producing LOX in the plant. When a leaf of this mutant was wounded, the JA reporter gene was expressed in distal leaves. Leaf sectioning showed that JA reporter expression extended from contact cells throughout the vascular bundle and into extravascular cells, revealing a radial movement of jasmonates. Our results add a crucial element to a growing picture of how the distal wound response is regulated in rosettes, showing that both axial (shoot-to-root) and radial (cell-to-cell) transport of oxylipins plays a major role in the wound response. The strategies developed herein provide unique tools with which to identify intercellular jasmonate transport routes.

Embryophyte genomes typically encode multiple 13-lipoxygenases (13-LOXs) that initiate the synthesis of wound-inducible mediators called jasmonates. Little is known about how the activities of these different LOX genes are coordinated. We found that the four 13-LOX genes in Arabidopsis thaliana have different basal expression patterns. LOX2 expression was strong in soft aerial tissues, but was excluded both within and proximal to maturing veins. LOX3 was expressed most strongly in circumfasicular parenchyma. LOX4 was expressed in phloem-associated cells, in contrast to LOX6, which is expressed in xylem contact cells. To investigate how the activities of these genes are coordinated after wounding, we carried out gene expression analyses in 13-lox mutants. This revealed a two-tiered, paired hierarchy in which LOX6, and to a lesser extent LOX2, control most of the early-phase of jasmonate response gene expression. Jasmonates precursors produced by these two LOXs in wounded leaves are converted to active jasmonates that regulate LOX3 and LOX4 gene expression. Together with LOX2 and LOX6, and working downstream of them, LOX3 and LOX4 contribute to jasmonate synthesis that leads to the expression of the defense gene VEGETATIVE STORAGE PROTEIN2 (VSP2). LOX3 and LOX4 were also found to contribute to defense against the generalist herbivore Spodoptera littoralis. Our results reveal that 13-LOX genes are organised in a regulatory network, and the data herein raise the possibility that other genomes may encode LOXs that act as pairs.

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