Identification and characterization of GLYCEROLIPASE A1 for wound-triggered JA biosynthesis in Nicotiana benthamiana leaves.

Although many important discoveries have been made regarding the jasmonate signaling pathway, how jasmonate biosynthesis is initiated is still a major unanswered question in the field. Previous evidences suggest that jasmonate biosynthesis is limited by the availability of fatty acid precursor, such as ⍺-linolenic acid (⍺-LA). This indicates that the lipase responsible for releasing α-LA in the chloroplast, where early steps of jasmonate biosynthesis take place, is the key initial step in the jasmonate biosynthetic pathway. Nicotiana benthamiana glycerol lipase A1 (NbGLA1) is homologous to N. attenuata GLA1 (NaGLA1) which has been reported to be a major lipase in leaves for jasmonate biosynthesis. NbGLA1 was studied for its potential usefulness in a species that is more common in laboratories. Virus-induced gene silencing of both NbGLA1 and NbGLA2, another homolog, resulted in more than 80% reduction in jasmonic acid (JA) biosynthesis in wounded leaves. Overexpression of NbGLA1 utilizing an inducible vector system failed to increase JA, indicating that transcriptional induction of NbGLA1 is insufficient to trigger JA biosynthesis. However, co-treatment with wounding in addition to NbGLA1 induction increased JA accumulation several fold higher than the gene expression or wounding alone, indicating an enhancement of the enzyme activity by wounding. Domain-deletion of a 126-bp C-terminal region hypothesized to have regulatory roles increased NbGLA1-induced JA level. Together, the data show NbGLA1 to be a major lipase for wound-induced JA biosynthesis in N. benthamiana leaves and demonstrate the use of inducible promoter-driven construct of NbGLA1 in conjunction with its transient expression in N. benthamiana as a useful system to study its protein function.

Seed Priming with MeJa Prevents Salt-Induced Growth Inhibition and Oxidative Damage in Sorghum bicolor by Inducing the Expression of Jasmonic Acid Biosynthesis Genes.

Salinity is one of the major detrimental abiotic stresses at the forefront of deterring crop productivity globally. Although the exogenous application of phytohormones has formerly proven efficacious to plants, their effect on the moderately stress-tolerant crop "Sorghum bicolor" remains elusive. To investigate this, S. bicolor seeds primed with methyl jasmonate (0; 10 and 15 µM MeJa) were exposed to salt (200 mM NaCl) stress, and their morpho-physiological, biochemical, and molecular attributes were measured. Salt stress significantly decreased shoot length and fresh weight by 50%, whereas dry weight and chlorophyll content were decreased by more than 40%. Furthermore, salt-stress-induced oxidative damage was evident by the formation of brown formazan spots (indicative of H2O2 production) on sorghum leaves and a more than 30% increase in MDA content. However, priming with MeJa improved growth, increased chlorophyll content, and prevented oxidative damage under salt stress. While 15 µM MeJa maintained proline content to the same level as the salt-stressed samples, total soluble sugars were maintained under 10 µM MeJa, indicating a high degree of osmotic adjustment. Shriveling and thinning of the epidermis and xylem tissues due to salt stress was prevented by MeJa, followed by a more than 70% decrease in the Na+/K+ ratio. MeJa also reversed the FTIR spectral shifts observed for salt-stressed plants. Furthermore, salt stress induced the expression of the jasmonic acid biosynthesis genes; linoleate 92-lipoxygenase 3, allene oxide synthase 1, allene oxide cyclase, and 12-oxophytodienoate reductase 1. In MeJa-primed plants, their expression was reduced, except for the 12-oxophytodienoate reductase 1 transcript, which further increased by 67%. These findings suggest that MeJa conferred salt-stress tolerance to S. bicolor through osmoregulation and synthesis of JA-related metabolites.

On the initiation of jasmonate biosynthesis in wounded leaves.

The basal level of the plant defense hormone jasmonate (JA) in unstressed leaves is low, but wounding causes its near instantaneous increase. How JA biosynthesis is initiated is uncertain, but the lipolysis step that generates fatty acid precursors is generally considered to be the first step. Here, we used a series of physiological, pharmacological, genetic, and kinetic analyses of gene expression and hormone profiling to demonstrate that the early spiking of JA upon wounding does not depend on the expression of JA biosynthetic genes in Arabidopsis (Arabidopsis thaliana). Using a transgenic system, we showed how decoupling the responses to wounding and JA prevents the perpetual synthesis of JA in wounded leaves. We then used DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) as a model wound-responsive lipase to demonstrate that although its transient expression in leaves can elicit JA biosynthesis to a low level, an additional level of activation is triggered by wounding, which causes massive accumulation of JA. This wound-triggered boosting effect of DAD1-mediated JA synthesis can happen directly in damaged leaves or indirectly in undamaged remote leaves by the systemically transmitted wound signal. Finally, protein stability of DAD1 was influenced by wounding, α-linolenic acid, and mutation in its catalytic site. Together, the data support mechanisms that are independent of gene transcription and translation to initiate the rapid JA burst in wounded leaves and demonstrate how transient expression of the lipase can be used to reveal changes occurring at the level of activity and stability of the key lipolytic step.

Differential Regulation of the Ribosomal Association of mRNA Transcripts in an Arabidopsis Mutant Defective in Jasmonate-Dependent Wound Response.

Jasmonoyl-L-isoleucine (JA-Ile) is a powerful oxylipin responsible for the genome-wide transcriptional reprogramming in plants that results in major physiological shifts from growth to defense. The double T-DNA insertion Arabidopsis mutant, cyp94b1cyp94b3 (b1b3), defective in cytochrome p450s, CYP94B1 and CYP94B3, which are responsible for oxidizing JA-Ile, accumulates several fold higher levels of JA-Ile yet displays dampened JA-Ile-dependent wound responses-the opposite of what is expected. Transcriptomic and proteomic analyses showed that while the transcriptional response to wounding was largely unchanged in b1b3 compared to wild type (WT), many proteins were found to be significantly reduced in the mutant, which was verified by immunoblot analyses of marker proteins. To understand this protein phenotype and their hypothesized contribution to the b1b3 phenotypes, wounded rosette leaf samples from both WT and b1b3 were subject to a translating ribosome affinity purification RNA sequencing analysis. More than 1,600 genes whose transcripts do not change in abundance by wounding changed their association with the ribosomes after wounding in WT leaves. Consistent with previous observations, the total pool of mRNA transcripts was similar between WT and b1b3; however, the ribosome-associated pool of transcripts was changed significantly. Most notably, fewer transcripts were associated with the ribosome pool in b1b3 than in WT, potentially explaining the reduction of many proteins in the mutant. Among those genes with fewer ribosome-associated transcripts in b1b3 were genes relating to stress response, specialized metabolism, protein metabolism, ribosomal subunits, and transcription factors, consistent with the biochemical phenotypes of the mutant. These results show previously unrecognized regulations at the translational level that are affected by misregulation of JA homeostasis during the wound response in plants.

A light-dependent molecular link between competition cues and defence responses in plants.

Growth responses to competition1 and defence responses to the attack of consumer organisms2 are two classic examples of adaptive phenotypic plasticity in plants. However, the mechanistic and functional links between these responses are not well understood. Jasmonates, a family of lipid-derived signals, are potent growth inhibitors and central regulators of plant immunity to herbivores and pathogens3,4, with both roles being evolutionarily conserved from bryophytes5 to angiosperms6. When shade-intolerant plants perceive the proximity of competitors using the photoreceptor phytochrome B, they activate the shade-avoidance syndrome and downregulate jasmonate responses7. Despite the central implications of this light-mediated change in the growth/defence balance for plant adaptation and crop yield8,9, the mechanisms by which photoreceptors relay light cues to the jasmonate signalling pathway remain poorly understood10. Here, we identify a sulfotransferase (ST2a) that is strongly upregulated by plant proximity perceived by phytochrome B via the phytochrome B-phytochrome interacting factor signalling module. By catalysing the formation of a sulfated jasmonate derivative, ST2a acts to reduce the pool of precursors of active forms of jasmonates and represents a direct molecular link between photoreceptors and hormone signalling in plants. The metabolic step defined by this enzyme provides a molecular mechanism for prioritizing shade avoidance over defence under intense plant competition.

Use of Yellow Fluorescent Protein Fluorescence to Track OPR3 Expression in Arabidopsis Thaliana Responses to Insect Herbivory.

Feeding by chewing insects induces chemical defenses in plants that are regulated by the jasmonic acid (JA) pathway. Jasmonates are usually quantified by liquid chromatography-mass spectrometry (LC-MS) analysis of precursors and products in the biosynthetic pathway or inferred from the extraction and expression of genes known to respond to elevated levels of JA. Both approaches are costly and time consuming. To address these limitations, we developed a rapid reporter for the synthesis of JA based on the OPR3promoter:YFP-PTS1. Yellow fluorescent protein (YFP) fluorescence was increased by mechanical wounding and methyl jasmonate (MeJA) treatment and by caterpillar feeding. To develop an optimal sampling time for a quantitative bioassay, OPR3promoter:YFP-PTS1 plants were sampled at 1, 2, 3, and 24 h after treatment with 115 µM MeJA. The first increase in YFP fluorescence was detected at 2 h and remained elevated 3 and 24 h later; as a result, 3 h was chosen as the sampling time for a quantitative bioassay of jasmonate response to insect attack. Feeding by Pieris rapae caterpillars induced a 1.8-fold increase in YFP fluorescence, consistent with the known induction of JA production by this insect. We also assessed the utility of this reporter in studies of plant responses to caterpillar feeding vibrations, which are known to potentiate the JA-dependent production of chemical defenses. Pretreatment with feeding vibrations increased expression of the OPR3promoter:YFP-PTS1 in response to 14 µM MeJA. Feeding vibrations did not potentiate responses at higher MeJA concentrations, suggesting that potentiating effects of prior treatments can only be detected when plants are below a response threshold to the elicitor. The expression of OPR3 does not indicate levels of specific downstream jasmonates and quantification of specific jasmonates still requires detailed analysis by LC-MS. However, OPR3 expression does provide a rapid and inexpensive way to screen large numbers of plants for the involvement of jasmonate signaling in their response to a wide variety of treatments, and to study the induction and expression of AtOPR3.

12-Hydroxy-Jasmonoyl-l-Isoleucine Is an Active Jasmonate That Signals through CORONATINE INSENSITIVE 1 and Contributes to the Wound Response in Arabidopsis.

12-hydroxy-jasmonoyl-isoleucine (12OH-JA-Ile) is a metabolite in the catabolic pathway of the plant hormone jasmonate, and is synthesized by the cytochrome P450 subclade 94 enzymes. Contrary to the well-established function of jasmonoyl-isoleucine (JA-Ile) as the endogenous bioactive form of jasmonate, the function of 12OH-JA-Ile is unclear. Here, the potential role of 12OH-JA-Ile in jasmonate signaling and wound response was investigated. Exogenous application of 12OH-JA-Ile mimicked several JA-Ile effects including marker gene expression, anthocyanin accumulation and trichome induction in Arabidopsis thaliana. Genome-wide transcriptomics and untargeted metabolite analyses showed large overlaps between those affected by 12OH-JA-Ile and JA-Ile. 12OH-JA-Ile signaling was blocked by mutation in CORONATINE INSENSITIVE 1. Increased anthocyanin accumulation by 12OH-JA-Ile was additionally observed in tomato and sorghum, and was disrupted by the COI1 defect in tomato jai1 mutant. In silico ligand docking predicted that 12OH-JA-Ile can maintain many of the key interactions with COI1-JAZ1 residues identified earlier by crystal structure studies using JA-Ile as ligand. Genetic alternation of jasmonate metabolic pathways in Arabidopsis to deplete both JA-Ile and 12OH-JA-Ile displayed enhanced jasmonate deficient wound phenotypes and was more susceptible to insect herbivory than that depleted in only JA-Ile. Conversely, mutants overaccumulating 12OH-JA-Ile showed intensified wound responses compared with wild type with similar JA-Ile content. These data are indicative of 12OH-JA-Ile functioning as an active jasmonate signal and contributing to wound and defense response in higher plants.

Tasselseed5 overexpresses a wound-inducible enzyme, ZmCYP94B1, that affects jasmonate catabolism, sex determination, and plant architecture in maize.

Maize is monecious, with separate male and female inflorescences. Maize flowers are initially bisexual but achieve separate sexual identities through organ arrest. Loss-of-function mutants in the jasmonic acid (JA) pathway have only female flowers due to failure to abort silks in the tassel. Tasselseed5 (Ts5) shares this phenotype but is dominant. Positional cloning and transcriptomics of tassels identified an ectopically expressed gene in the CYP94B subfamily, Ts5 (ZmCYP94B1). CYP94B enzymes are wound inducible and inactivate bioactive jasmonoyl-L-isoleucine (JA-Ile). Consistent with this result, tassels and wounded leaves of Ts5 mutants displayed lower JA and JA-lle precursors and higher 12OH-JA-lle product than the wild type. Furthermore, many wounding and jasmonate pathway genes were differentially expressed in Ts5 tassels. We propose that the Ts5 phenotype results from the interruption of JA signaling during sexual differentiation via the upregulation of ZmCYP94B1 and that its proper expression maintains maize monoecy.

Glutamate triggers long-distance, calcium-based plant defense signaling.

Animals require rapid, long-range molecular signaling networks to integrate sensing and response throughout their bodies. The amino acid glutamate acts as an excitatory neurotransmitter in the vertebrate central nervous system, facilitating long-range information exchange via activation of glutamate receptor channels. Similarly, plants sense local signals, such as herbivore attack, and transmit this information throughout the plant body to rapidly activate defense responses in undamaged parts. Here we show that glutamate is a wound signal in plants. Ion channels of the GLUTAMATE RECEPTOR-LIKE family act as sensors that convert this signal into an increase in intracellular calcium ion concentration that propagates to distant organs, where defense responses are then induced.

Response of high leaf-oil Arabidopsis thaliana plant lines to biotic or abiotic stress.

Recent studies have shown that it is possible to engineer substantial increases in triacylglycerol (TAG) content in plant vegetative biomass, which offers a novel approach for increasing the energy density of food, feed, and bioenergy crops or for creating a sink for the accumulation of unusual, high-value fatty acids. However, whether or not these changes in lipid metabolism affect plant responses to biotic and/or abiotic stresses is an open question. Here we show that transgenic Arabidopsis thaliana plant lines engineered for elevated leaf oil content, as well as lines engineered for accumulation of unusual conjugated fatty acids in leaf oil, had similar short-term responses to heat stress (e.g., 3 days at 37°C) as wild-type plants, including a reduction in polyunsaturated fatty acid (PUFA)-containing polar lipids and an increase in PUFA-containing neutral lipids. At extended time periods (e.g., 14 days at 37°C), however, plant lines containing accumulated conjugated fatty acids displayed earlier senescence and plant death. Further, no-choice feeding studies demonstrated that plants with the highest leaf oil content generated cabbage looper (Trichoplusia ni) insects with significantly heavier body weights. Taken together, these results suggest that biotic and abiotic responses will be important considerations when developing and deploying high-oil-biomass crops in the field.

Modularity in Jasmonate Signaling for Multistress Resilience.

The plant hormone jasmonate coordinates immune and growth responses to increase plant survival in unpredictable environments. The core jasmonate signaling pathway comprises several functional modules, including a repertoire of COI1-JAZ (CORONATINE INSENSITIVE1-JASMONATE-ZIM DOMAIN) coreceptors that couple jasmonoyl-l-isoleucine perception to the degradation of JAZ repressors, JAZ-interacting transcription factors that execute physiological responses, and multiple negative feedback loops to ensure timely termination of these responses. Here, we review the jasmonate signaling pathway with an emphasis on understanding how transcriptional responses are specific, tunable, and evolvable. We explore emerging evidence that JAZ proteins integrate multiple informational cues and mediate crosstalk by propagating changes in protein-protein interaction networks. We also discuss recent insights into the evolution of jasmonate signaling and highlight how plant-associated organisms manipulate the pathway to subvert host immunity. Finally, we consider how this mechanistic foundation can accelerate the rational design of jasmonate signaling for improving crop resilience and harnessing the wellspring of specialized plant metabolites.

Extracellular ATP Acts on Jasmonate Signaling to Reinforce Plant Defense.

Damaged cells send various signals to stimulate defense responses. Recent identification and genetic studies of the plant purinoceptor, P2K1 (also known as DORN1), have demonstrated that extracellular ATP is a signal involved in plant stress responses, including wounding, perhaps to evoke plant defense. However, it remains largely unknown how extracellular ATP induces plant defense responses. Here, we demonstrate that extracellular ATP induces plant defense mediated through activation of the intracellular signaling of jasmonate (JA), a well-characterized defense hormone. In Arabidopsis (Arabidopsis thaliana) leaves, ATP pretreatment induced resistance against the necrotrophic fungus, Botrytis cinerea The induced resistance was enhanced in the P2K1 receptor overexpression line, but reduced in the receptor mutant, dorn1-3 Mining the transcriptome data revealed that ATP induces a set of JA-induced genes. In addition, the P2K1-associated coexpression network contains defense-related genes, including those encoding jasmonate ZIM-domain (JAZ) proteins, which play key roles as repressors of JA signaling. We examined whether extracellular ATP impacts the stability of JAZ1 in Arabidopsis. The results showed that the JAZ1 stability decreased in response to ATP addition in a proteasome-dependent manner. This reduction required intracellular signaling via second messengers-cytosolic calcium, reactive oxygen species, and nitric oxide. Interestingly, the ATP-induced JAZ1 degradation was attenuated in the JA receptor mutant, coi1, but not in the JA biosynthesis mutant, aos, or upon addition of JA biosynthesis inhibitors. Immunoprecipitation analysis demonstrated that ATP increases the interaction between COI1 and JAZ1, suggesting direct cross talk between extracellular ATP and JA in intracellular signaling events. Taken together, these results suggest that extracellular ATP signaling directly impacts the JA signaling pathway to maximize plant defense responses.

Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana.

Plants rapidly perceive tissue damage, such as that inflicted by insects, and activate several key defense responses. The importance of the fatty acid-derived hormone jasmonates (JA) in dictating these wound responses has been recognized for many years. However, important features pertaining to the regulation of the JA pathway are still not well understood. One key unknown is the inactivation mechanism of the JA pathway and its relationship with plant response to wounding. Arabidopsis cytochrome P450 enzymes in the CYP94 clade metabolize jasmonoyl-L-isoleucine (JA-Ile), a major metabolite of JA responsible for many biological effects attributed to the JA signaling pathway; thus, CYP94s are expected to contribute to the attenuation of JA-Ile-dependent wound responses. To directly test this, we created the double and triple knock-out mutants of three CYP94 genes, CYP94B1, CYP94B3, and CYP94C1. The mutations blocked the oxidation steps and caused JA-Ile to accumulate 3-4-fold the WT levels in the wounded leaves. Surprisingly, over accumulation of JA-Ile did not lead to a stronger wound response. On the contrary, the mutants displayed a series of symptoms reminiscent of JA-Ile deficiency, including resistance to wound-induced growth inhibition, decreased anthocyanin and trichomes, and increased susceptibility to insects. The mutants, however, responded normally to exogenous JA treatments, indicating that JA perception or signaling pathways were intact. Untargeted metabolite analyses revealed >40% reduction in wound-inducible metabolites in the mutants. These observations raise questions about the current JA signaling model and point toward a more complex model perhaps involving JA derivatives and/or feedback mechanisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

The Arabidopsis immune regulator SRFR1 dampens defences against herbivory by Spodoptera exigua and parasitism by Heterodera schachtii.

Plants have developed diverse mechanisms to fine tune defence responses to different types of enemy. Cross-regulation between signalling pathways may allow the prioritization of one response over another. Previously, we identified SUPPRESSOR OF rps4-RLD1 (SRFR1) as a negative regulator of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent effector-triggered immunity against the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4. The use of multiple stresses is a powerful tool to further define gene function. Here, we examined whether SRFR1 also impacts resistance to a herbivorous insect in leaves and to a cyst nematode in roots. Interestingly, srfr1-1 plants showed increased resistance to herbivory by the beet army worm Spodoptera exigua and to parasitism by the cyst nematode Heterodera schachtii compared with the corresponding wild-type Arabidopsis accession RLD. Using quantitative real-time PCR (qRT-PCR) to measure the transcript levels of salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathway genes, we found that enhanced resistance of srfr1-1 plants to S. exigua correlated with specific upregulation of the MYC2 branch of the JA pathway concurrent with suppression of the SA pathway. In contrast, the greater susceptibility of RLD was accompanied by simultaneously increased transcript levels of SA, JA and JA/ET signalling pathway genes. Surprisingly, mutation of either SRFR1 or EDS1 increased resistance to H. schachtii, indicating that the concurrent presence of both wild-type genes promotes susceptibility. This finding suggests a novel form of resistance in Arabidopsis to the biotrophic pathogen H. schachtii or a root-specific regulation of the SA pathway by EDS1, and places SRFR1 at an intersection between multiple defence pathways.

Potential targets of VIVIPAROUS1/ABI3-LIKE1 (VAL1) repression in developing Arabidopsis thaliana embryos.

Developing Arabidopsis seeds accumulate oils and seed storage proteins synthesized by the pathways of primary metabolism. Seed development and metabolism are positively regulated by transcription factors belonging to the LAFL (LEC1, AB13, FUSCA3 and LEC2) regulatory network. The VAL gene family encodes repressors of the seed maturation program in germinating seeds, although they are also expressed during seed maturation. The possible regulatory role of VAL1 in seed development has not been studied to date. Reverse genetics revealed that val1 mutant seeds accumulated elevated levels of proteins compared with the wild type, suggesting that VAL1 functions as a repressor of seed metabolism; however, in the absence of VAL1, the levels of metabolites, ABA, auxin and jasmonate derivatives did not change significantly in developing embryos. Two VAL1 splice variants were identified through RNA sequencing analysis: a full-length form and a truncated form lacking the plant homeodomain-like domain associated with epigenetic repression. None of the transcripts encoding the core LAFL network transcription factors were affected in val1 embryos. Instead, activation of VAL1 by FUSCA3 appears to result in the repression of a subset of seed maturation genes downstream of core LAFL regulators, as 39% of transcripts in the FUSCA3 regulon were derepressed in the val1 mutant. The LEC1 and LEC2 regulons also responded, but to a lesser extent. Additional 832 transcripts that were not LAFL targets were derepressed in val1 mutant embryos. These transcripts are candidate targets of VAL1, acting through epigenetic and/or transcriptional repression.

Hormone crosstalk in wound stress response: wound-inducible amidohydrolases can simultaneously regulate jasmonate and auxin homeostasis in Arabidopsis thaliana.

Jasmonate (JA) and auxin are essential hormones in plant development and stress responses. While the two govern distinct physiological processes, their signaling pathways interact at various levels. Recently, members of the Arabidopsis indole-3-acetic acid (IAA) amidohydrolase (IAH) family were reported to metabolize jasmonoyl-isoleucine (JA-Ile), a bioactive form of JA. Here, we characterized three IAH members, ILR1, ILL6, and IAR3, for their function in JA and IAA metabolism and signaling. Expression of all three genes in leaves was up-regulated by wounding or JA, but not by IAA. Purified recombinant proteins showed overlapping but distinct substrate specificities for diverse amino acid conjugates of JA and IAA. Perturbed patterns of the endogenous JA profile in plants overexpressing or knocked-out for the three genes were consistent with ILL6 and IAR3, but not ILR1, being the JA amidohydrolases. Increased turnover of JA-Ile in the ILL6- and IAR3-overexpressing plants created symptoms of JA deficiency whereas increased free IAA by overexpression of ILR1 and IAR3 made plants hypersensitive to exogenous IAA conjugates. Surprisingly, ILL6 overexpression rendered plants highly resistant to exogenous IAA conjugates, indicating its interference with IAA conjugate hydrolysis. Fluorescent protein-tagged IAR3 and ILL6 co-localized with the endoplasmic reticulum-localized JA-Ile 12-hydroxylase, CYP94B3. Together, these results demonstrate that in wounded leaves JA-inducible amidohydrolases contribute to regulate active IAA and JA-Ile levels, promoting auxin signaling while attenuating JA signaling. This mechanism represents an example of a metabolic-level crosstalk between the auxin and JA signaling pathways.

Loss of Arabidopsis thaliana Dynamin-Related Protein 2B reveals separation of innate immune signaling pathways.

Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC-, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC-. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.

Endoplasmic reticulum-associated inactivation of the hormone jasmonoyl-L-isoleucine by multiple members of the cytochrome P450 94 family in Arabidopsis.

The plant hormone jasmonate (JA) controls diverse aspects of plant immunity, growth, and development. The amplitude and duration of JA responses are controlled in large part by the intracellular level of jasmonoyl-L-isoleucine (JA-Ile). In contrast to detailed knowledge of the JA-Ile biosynthetic pathway, little is known about enzymes involved in JA-Ile metabolism and turnover. Cytochromes P450 (CYP) 94B3 and 94C1 were recently shown to sequentially oxidize JA-Ile to hydroxy (12OH-JA-Ile) and dicarboxy (12COOH-JA-Ile) derivatives. Here, we report that a third member (CYP94B1) of the CYP94 family also participates in oxidative turnover of JA-Ile in Arabidopsis. In vitro studies showed that recombinant CYP94B1 converts JA-Ile to 12OH-JA-Ile and lesser amounts of 12COOH-JA-Ile. Consistent with this finding, metabolic and physiological characterization of CYP94B1 loss-of-function and overexpressing plants demonstrated that CYP94B1 and CYP94B3 coordinately govern the majority (>95%) of 12-hydroxylation of JA-Ile in wounded leaves. Analysis of CYP94-promoter-GUS reporter lines indicated that CYP94B1 and CYP94B3 serve unique and overlapping spatio-temporal roles in JA-Ile homeostasis. Subcellular localization studies showed that CYP94s involved in conversion of JA-Ile to 12COOH-JA-Ile reside on endoplasmic reticulum (ER). In vitro studies further showed that 12COOH-JA-Ile, unlike JA-Ile, fails to promote assembly of COI1-JAZ co-receptor complexes. The double loss-of-function mutant of CYP94B3 and ILL6, a JA-Ile amidohydrolase, displayed a JA profile consistent with the collaborative action of the oxidative and the hydrolytic pathways in JA-Ile turnover. Collectively, our results provide an integrated view of how multiple ER-localized CYP94 and JA amidohydrolase enzymes attenuate JA signaling during stress responses.