Wound-response jasmonate dynamics in the primary vasculature.

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-(ß-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.

Arabidopsis H+-ATPase AHA1 controls slow wave potential duration and wound-response jasmonate pathway activation.

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.

Insect-damaged Arabidopsis moves like wounded Mimosa pudica.

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.

Identification of cell populations necessary for leaf-to-leaf electrical signaling in a wounded plant.

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.

Multilayered Organization of Jasmonate Signalling in the Regulation of Root Growth.

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

Axial and Radial Oxylipin Transport.

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.

Role of NINJA in root jasmonate signaling.

Wound responses in plants have to be coordinated between organs so that locally reduced growth in a wounded tissue is balanced by appropriate growth elsewhere in the body. We used a JASMONATE ZIM DOMAIN 10 (JAZ10) reporter to screen for mutants affected in the organ-specific activation of jasmonate (JA) signaling in Arabidopsis thaliana seedlings. Wounding one cotyledon activated the reporter in both aerial and root tissues, and this was either disrupted or restricted to certain organs in mutant alleles of core components of the JA pathway including COI1, OPR3, and JAR1. In contrast, three other mutants showed constitutive activation of the reporter in the roots and hypocotyls of unwounded seedlings. All three lines harbored mutations in Novel Interactor of JAZ (NINJA), which encodes part of a repressor complex that negatively regulates JA signaling. These ninja mutants displayed shorter roots mimicking JA-mediated growth inhibition, and this was due to reduced cell elongation. Remarkably, this phenotype and the constitutive JAZ10 expression were still observed in backgrounds lacking the ability to synthesize JA or the key transcriptional activator MYC2. Therefore, JA-like responses can be recapitulated in specific tissues without changing a plant's ability to make or perceive JA, and MYC2 either has no role or is not the only derepressed transcription factor in ninja mutants. Our results show that the role of NINJA in the root is to repress JA signaling and allow normal cell elongation. Furthermore, the regulation of the JA pathway differs between roots and aerial tissues at all levels, from JA biosynthesis to transcriptional activation.

A regulatory network for coordinated flower maturation.

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs.

Jasmonate controls polypeptide patterning in undamaged tissue in wounded Arabidopsis leaves.

Wounding initiates a strong and largely jasmonate-dependent remodelling of the transcriptome in the leaf blades of Arabidopsis (Arabidopsis thaliana). How much control do jasmonates exert on wound-induced protein repatterning in leaves? Replicated shotgun proteomic analyses of 2.5-mm-wide leaf strips adjacent to wounds revealed 106 differentially regulated proteins. Many of these gene products have not emerged as being wound regulated in transcriptomic studies. From experiments using the jasmonic acid (JA)-deficient allene oxide synthase mutant we estimated that approximately 95% of wound-stimulated changes in protein levels were deregulated in the absence of JA. The levels of two tonoplast proteins already implicated in defense response regulation, TWO-PORE CHANNEL1 and the calcium-V-ATPase ACA4 increased on wounding, but their transcripts were not wound inducible. The data suggest new roles for jasmonate in controlling the levels of calcium-regulated pumps and transporters, proteins involved in targeted proteolysis, a putative bacterial virulence factor target, a light-dependent catalyst, and a key redox-controlled enzyme in glutathione synthesis. Extending the latter observation we found that wounding increased the proportion of oxidized glutathione in leaves, but only in plants able to synthesize JA. The oxidizing conditions generated through JA signaling near wounds help to define the cellular environment in which proteome remodelling occurs.

Chloroplastic phosphoadenosine phosphosulfate metabolism regulates basal levels of the prohormone jasmonic acid in Arabidopsis leaves.

Levels of the enzymes that produce wound response mediators have to be controlled tightly in unwounded tissues. The Arabidopsis (Arabidopsis thaliana) fatty acid oxygenation up-regulated8 (fou8) mutant catalyzes high rates of alpha -linolenic acid oxygenation and has higher than wild-type levels of the alpha -linolenic acid-derived wound response mediator jasmonic acid (JA) in undamaged leaves. fou8 produces a null allele in the gene SAL1 (also known as FIERY1 or FRY1). Overexpression of the wild-type gene product had the opposite effect of the null allele, suggesting a regulatory role of SAL1 acting in JA synthesis. The biochemical phenotypes in fou8 were complemented when the yeast (Saccharomyces cerevisiae) sulfur metabolism 3'(2'), 5'-bisphosphate nucleotidase MET22 was targeted to chloroplasts in fou8. The data are consistent with a role of SAL1 in the chloroplast-localized dephosphorylation of 3'-phospho-5'-adenosine phosphosulfate to 5'-adenosine phosphosulfate or in a closely related reaction (e.g. 3',5'-bisphosphate dephosphorylation). Furthermore, the fou8 phenotype was genetically suppressed in a triple mutant (fou8 apk1 apk2) affecting chloroplastic 3'-phospho-5'-adenosine phosphosulfate synthesis. These results show that a nucleotide component of the sulfur futile cycle regulates early steps of JA production and basal JA levels.

Control of jasmonate biosynthesis and senescence by miR319 targets.

Considerable progress has been made in identifying the targets of plant microRNAs, many of which regulate the stability or translation of mRNAs that encode transcription factors involved in development. In most cases, it is unknown, however, which immediate transcriptional targets mediate downstream effects of the microRNA-regulated transcription factors. We identified a new process controlled by the miR319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes. In contrast to other miRNA targets, several of which modulate hormone responses, TCPs control biosynthesis of the hormone jasmonic acid. Furthermore, we demonstrate a previously unrecognized effect of TCPs on leaf senescence, a process in which jasmonic acid has been proposed to be a critical regulator. We propose that miR319-controlled TCP transcription factors coordinate two sequential processes in leaf development: leaf growth, which they negatively regulate, and leaf senescence, which they positively regulate.

Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response?

The fou8 loss of function allele of adenosine bisphosphate phosphatase FIERY1 results in numerous phenotypes including the increased enzymatic oxygenation of fatty acids and increased jasmonate synthesis. Here we show that the mutation causes also profound alterations of sulfur metabolism. The fou8 mutants possess lower levels of sulfated secondary compounds, glucosinolates, and accumulate the desulfo-precursors similar to previously described mutants in adenosine 5'phosphosulfate kinase. Transcript levels of genes involved in sulfate assimilation differ in fou8 compared to wild type Col-0 plants and are similar to plants subjected to sulfate deficiency. Indeed, independent microarray analyses of various alleles of mutants in FIERY1 showed similar patterns of gene expression as in sulfate deficient plants. This was not caused by alterations in signalling, as the fou8 mutants contained significantly lower levels of sulfate and glutathione and, consequently, of total elemental sulfur. Analysis of mutants with altered levels of sulfate and glutathione confirmed the correlation of sulfate deficiency-like gene expression pattern with low internal sulfate but not low glutathione. The changes in sulfur metabolism in fou8 correlated with massive increases in 3'-phosphoadenosine 5'-phosphate levels. The analysis of fou8 thus revealed that sulfate starvation response is triggered by a decrease in internal sulfate as opposed to external sulfate availability and that the presence of desulfo-glucosinolates does not induce the glucosinolate synthesis network. However, as well as resolving these important questions on the regulation of sulfate assimilation in plants, fou8 has also opened an array of new questions on the links between jasmonate synthesis and sulfur metabolism.

Nonenzymatic oxidation of trienoic fatty acids contributes to reactive oxygen species management in Arabidopsis.

In higher plants such as Arabidopsis thaliana, omega-3 trienoic fatty acids (TFAs), represented mainly by alpha-linolenic acid, serve as precursors of jasmonic acid (JA), a potent lipid signal molecule essential for defense. The JA-independent roles of TFAs were investigated by comparing the TFA- and JA-deficient fatty acid desaturase triple mutant (fad3-2 fad7-2 fad8 (fad3 fad7 fad8)) with the aos (allene oxide synthase) mutant that contains TFAs but is JA-deficient. When challenged with the fungus Botrytis, resistance of the fad3 fad7 fad8 mutant was reduced when compared with the aos mutant, suggesting that TFAs play a role in cell survival independently of being the precursors of JA. An independent genetic approach using the lesion mimic mutant accelerated cell death2 (acd2-2) confirmed the importance of TFAs in containing lesion spread, which was increased in the lines in which the fad3 fad7 fad8 and acd2-2 mutations were combined when compared with the aos acd2-2 lines. Malondialdehyde, found to result from oxidative TFA fragmentation during lesion formation, was measured by gas chromatography-mass spectrometry. Its levels correlated with the survival of the tissue. Furthermore, plants lacking TFAs overproduced salicylic acid (SA), hydrogen peroxide, and transcripts encoding several SA-regulated and SA biosynthetic proteins. The data suggest a physiological role for TFAs as sinks for reactive oxygen species.

A downstream mediator in the growth repression limb of the jasmonate pathway.

Wounding plant tissues initiates large-scale changes in transcription coupled to growth arrest, allowing resource diversion for defense. These processes are mediated in large part by the potent lipid regulator jasmonic acid (JA). Genes selected from a list of wound-inducible transcripts regulated by the jasmonate pathway were overexpressed in Arabidopsis thaliana, and the transgenic plants were then assayed for sensitivity to methyl jasmonate (MeJA). When grown in the presence of MeJA, the roots of plants overexpressing a gene of unknown function were longer than those of wild-type plants. When transcript levels for this gene, which we named JASMONATE-ASSOCIATED1 (JAS1), were reduced by RNA interference, the plants showed increased sensitivity to MeJA and growth was inhibited. These gain- and loss-of-function assays suggest that this gene acts as a repressor of JA-inhibited growth. An alternative transcript from the gene encoding a second protein isoform with a longer C terminus failed to repress jasmonate sensitivity. This identified a conserved C-terminal sequence in JAS1 and related genes, all of which also contain Zim motifs and many of which are jasmonate-regulated. Both forms of JAS1 were found to localize to the nucleus in transient expression assays. Physiological tests of growth responses after wounding were consistent with the fact that JAS1 is a repressor of JA-regulated growth retardation.

A gain-of-function allele of TPC1 activates oxylipin biogenesis after leaf wounding in Arabidopsis.

Jasmonates, potent lipid mediators of defense gene expression in plants, are rapidly synthesized in response to wounding. These lipid mediators also stimulate their own production via a positive feedback circuit, which depends on both JA synthesis and JA signaling. To date, molecular components regulating the activation of jasmonate biogenesis and its feedback loop have been poorly characterized. We employed a genetic screen capable of detecting the misregulated activity of 13-lipoxygenase, which operates at the entry point of the jasmonate biosynthesis pathway. Leaf extracts from the Arabidopsis fou2 (fatty acid oxygenation upregulated 2) mutant displayed an increased capacity to catalyze the synthesis of lipoxygenase (LOX) metabolites. Quantitative oxylipin analysis identified less than twofold increased jasmonate levels in healthy fou2 leaves compared to wild-type; however, wounded fou2 leaves strongly increased jasmonate biogenesis compared to wounded wild-type. Furthermore, the plants displayed enhanced resistance to the fungus Botrytis cinerea. Higher than wild-type LOX activity and enhanced resistance in the fou2 mutant depend fully on a functional jasmonate response pathway. The fou2 mutant carries a missense mutation in the putative voltage sensor of the Two Pore Channel 1 gene (TPC1), which encodes a Ca(2+)-permeant non-selective cation channel. Patch-clamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. The results indicate that cation fluxes exert strong control over the positive feedback loop whereby JA stimulates its own synthesis.

Selective and powerful stress gene expression in Arabidopsis in response to malondialdehyde.

The provenance, half-life and biological activity of malondialdehyde (MDA) were investigated in Arabidopsis thaliana. We provide genetic confirmation of the hypothesis that MDA originates from fatty acids containing more than two methylene-linked double bonds, showing that tri-unsaturated fatty acids are the in vivo source of up to 75% of MDA. The abundance of the combined pool of free and reversibly bound MDA did not change dramatically in stress, although a significant increase in the free MDA pool under oxidative conditions was observed. The half-life of infiltrated MDA indicated rapid metabolic turnover/sequestration. Exposure of plants to low levels of MDA using a recently developed protocol powerfully upregulated many genes on a cDNA microarray with a bias towards those implicated in abiotic/environmental stress (e.g. ROF1 and XERO2). Remarkably, and in contrast to the activities of other reactive electrophile species (i.e. small vinyl ketones), none of the pathogenesis-related (PR) genes tested responded to MDA. The use of structural mimics of MDA isomers suggested that the propensity of the molecule to act as a cross-linking/modifying reagent might contribute to the activation of gene expression. Changes in the concentration/localisation of unbound MDA in vivo could strongly affect stress-related transcription.