RESUMO
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 ß-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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Animais , Glicosídeo Hidrolases/metabolismo , Glucosinolatos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , InsetosRESUMO
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.
Assuntos
Arabidopsis , Cloretos , Cloretos/metabolismo , Arabidopsis/metabolismo , Glutationa/farmacologia , Glutationa/metabolismo , Xilema , Glutamatos/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Etilenos/biossíntese , Nematoides/metabolismo , Raízes de Plantas/metabolismo , Estresse Mecânico , Estresse Fisiológico/fisiologia , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Sinalização do Cálcio/fisiologia , Ciclopentanos/metabolismo , Etilenos/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Oxilipinas/metabolismo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Análise de Célula Única , Imagem com Lapso de TempoRESUMO
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.
Assuntos
Arabidopsis , Oxilipinas , Ciclopentanos , Arabidopsis/fisiologiaRESUMO
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.
Assuntos
Plantas , Água , Água/metabolismo , Plantas/metabolismo , Oxilipinas/metabolismo , Ciclopentanos/metabolismo , HerbivoriaRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Saccharomyces cerevisiae/metabolismo , Arabidopsis/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Ciclopentanos/metabolismo , Potenciais da Membrana/genética , Redes e Vias Metabólicas , Oxilipinas/metabolismo , ATPases Translocadoras de Prótons/genética , Transdução de Sinais , Proteínas de Arabidopsis/metabolismo , Fenômenos Eletrofisiológicos , Herbivoria , Fenótipo , Bombas de Próton/genética , Bombas de Próton/metabolismo , ATPases Translocadoras de Prótons/metabolismoRESUMO
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.
Assuntos
Arabidopsis/fisiologia , Arabidopsis/parasitologia , Insetos/fisiologia , Mimosa/fisiologia , Folhas de Planta/fisiologia , Folhas de Planta/parasitologia , Animais , Estimulação Elétrica , Eletricidade , Cinética , Larva/fisiologia , Lepidópteros/fisiologia , Fenômenos Fisiológicos Vegetais , XilemaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/parasitologia , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Spodoptera/parasitologia , Animais , Arabidopsis/crescimento & desenvolvimento , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Lipoxigenase/genética , Lipoxigenase/metabolismo , Mutação , FenótipoRESUMO
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.
Assuntos
Arabidopsis/metabolismo , Sinalização do Cálcio , Potenciais da Membrana , Doenças das Plantas , Folhas de Planta/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Hormônios , Floema , Folhas de PlantaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Genes de Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismo , Receptores de Glutamato , Transdução de Sinais , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Condutividade Elétrica , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Herbivoria/fisiologia , Isoleucina/análogos & derivados , Isoleucina/metabolismo , Modelos Animais , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Doenças das Plantas/etiologia , Doenças das Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/efeitos dos fármacos , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sinapses/metabolismo , Transmissão Sináptica , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
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.
Assuntos
Arabidopsis/metabolismo , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Raízes de Plantas/crescimento & desenvolvimento , Sistemas do Segundo Mensageiro , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismoRESUMO
Genetic evidence suggests that membranes rich in polyunsaturated fatty acids (PUFAs) act as supramolecular antioxidants that capture reactive oxygen species, thereby limiting damage to proteins. This process generates lipid fragmentation products including malondialdehyde (MDA), an archetypal marker of PUFA oxidation. We observed transient increases in levels of endogenous MDA in wounded Arabidopsis thaliana leaves, raising the possibility that MDA is metabolized. We developed a rigorous ion exchange method to purify enzymatically generated (13)C- and (14)C-MDA. Delivered as a volatile to intact plants, MDA was efficiently incorporated into lipids. Mass spectral and genetic analyses identified the major chloroplast galactolipid: α-linolenic acid (18:3)-7Z,10Z,13Z-hexadecatrienoic acid (16:3)-monogalactosyldiacylglycerol (18:3-16:3-MGDG) as an end-product of MDA incorporation. Consistent with this, the fad3-2 fad7-2 fad8 mutant that lacks tri-unsaturated fatty acids incorporated (14)C-MDA into 18:2-16:2-MGDG. Saponification of (14)C-labeled 18:3-16:3-MGDG revealed 84% of (14)C-label in the acyl groups with the remaining 16% in the head group. 18:3-16:3-MGDG is enriched proximal to photosystem II and is likely a major in vivo source of MDA in photosynthetic tissues. We propose that nonenzymatically generated lipid fragments such as MDA are recycled back into plastidic galactolipids that, in their role as cell protectants, can again be fragmented into MDA.
Assuntos
Antioxidantes/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Cloroplastos/metabolismo , Ácidos Graxos/metabolismo , Malondialdeído/metabolismo , Acetatos/metabolismo , Metabolismo dos Lipídeos , Lipídeos de Membrana/fisiologia , Oxirredução , Estresse Oxidativo , Folhas de Planta/metabolismoRESUMO
Unknown mechanisms tightly regulate the basal activity of the wound-inducible defence mediator jasmonate (JA) in undamaged tissues. However, the Arabidopsis fatty acid oxygenation upregulated2 (fou2) mutant in vacuolar two-pore channel 1 (TPC1D454N ) displays high JA pathway activity in undamaged leaves. This mutant was used to explore mechanisms controlling basal JA pathway regulation. fou2 was re-mutated to generate novel 'ouf' suppressor mutants. Patch-clamping was used to examine TPC1 cation channel characteristics in the ouf suppressor mutants and in fou2. Calcium (Ca2+ ) imaging was used to study the effects fou2 on cytosolic Ca2+ concentrations. Six intragenic ouf suppressors with near wild-type (WT) JA pathway activity were recovered and one mutant, ouf8, affected the channel pore. At low luminal calcium concentrations, ouf8 had little detectable effect on fou2. However, increased vacuolar Ca2+ concentrations caused channel occlusion, selectively blocking K+ fluxes towards the cytoplasm. Cytosolic Ca2+ concentrations in unwounded fou2 were found to be lower than in the unwounded WT, but they increased in a similar manner in both genotypes following wounding. Basal JA pathway activity can be controlled solely by manipulating endomembrane cation flux capacities. We suggest that changes in endomembrane potential affect JA pathway activity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Cátions/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Canais de Cálcio/genética , Citosol/metabolismoRESUMO
The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono- and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl-MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl-MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12-oxo-phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA-containing galactolipids in the plant kingdom. While acyl-MGDG was found to be ubiquitous in green tissue of plants ranging from non-vascular plants to angiosperms, OPDA-containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non-oxidized and OPDA-containing acyl-MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl-MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.
Assuntos
Aciltransferases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Galactolipídeos/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Aciltransferases/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Galactolipídeos/química , Deleção de Genes , Regulação da Expressão Gênica de Plantas/fisiologia , Filogenia , Nicotiana/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Lipoxigenase/metabolismo , Lipoxigenases/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Lipoxigenase/genética , Lipoxigenases/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/enzimologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Estresse Fisiológico , Xilema/enzimologia , Xilema/genética , Xilema/fisiologiaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas Nucleares/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo , Plântula/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Análise de Variância , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Primers do DNA , Flores/genética , Flores/crescimento & desenvolvimento , Componentes do Gene , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Nucleares/genética , Raízes de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Plântula/metabolismoRESUMO
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.