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Plant rhabdoviruses heavily rely on insect vectors for transmission between sessile plants. However, little is known about the underlying mechanisms of insect attraction and transmission of plant rhabdoviruses. In this study, we used an arthropod-borne cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), to demonstrate the molecular mechanisms of a rhabdovirus accessory protein in improving plant attractiveness to insect vectors. Here, we found that BYSMV-infected barley (Hordeum vulgare L.) plants attracted more insect vectors than mock-treated plants. Interestingly, overexpression of BYSMV P6, an accessory protein, in transgenic wheat (Triticum aestivum L.) plants substantially increased host attractiveness to insect vectors through inhibiting the jasmonic acid (JA) signaling pathway. The BYSMV P6 protein interacted with the constitutive photomorphogenesis 9 signalosome subunit 5 (CSN5) of barley plants in vivo and in vitro, and negatively affected CSN5-mediated deRUBylation of cullin1 (CUL1). Consequently, the defective CUL1-based Skp1/Cullin1/F-box ubiquitin E3 ligases could not mediate degradation of jasmonate ZIM-domain proteins, resulting in compromised JA signaling and increased insect attraction. Overexpression of BYSMV P6 also inhibited JA signaling in transgenic Arabidopsis (Arabidopsis thaliana) plants to attract insects. Our results provide insight into how a plant cytorhabdovirus subverts plant JA signaling to attract insect vectors.

Plant adjustment to environmental changes involves complex crosstalk between extrinsic and intrinsic cues. In the past two decades, extensive research has elucidated the key roles of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and the phytohormone auxin in thermomorphogenesis. In this study, we identified a previously unexplored role of jasmonate (JA) signaling components, the Mediator complex, and their integration with auxin signaling during thermomorphogenesis in Arabidopsis (Arabidopsis thaliana). Warm temperature induces expression of JA signaling genes including MYC2, but, surprisingly, this transcriptional activation is not JA dependent. Warm temperature also promotes accumulation of the JA signaling receptor CORONATINE INSENSITIVE1 (COI1) and degradation of the JA signaling repressor JASMONATE-ZIM-DOMAIN PROTEIN9, which probably leads to de-repression of MYC2, enabling it to contribute to the expression of MEDIATOR SUBUNIT17 (MED17). In response to warm temperature, MED17 occupies the promoters of thermosensory genes including PIF4, YUCCA8 (YUC8), INDOLE-3-ACETIC ACID INDUCIBLE19 (IAA19), and IAA29. Moreover, MED17 facilitates enrichment of H3K4me3 on the promoters of PIF4, YUC8, IAA19, and IAA29 genes. Interestingly, both occupancy of MED17 and enrichment of H3K4me3 on these thermomorphogenesis-related promoters are dependent on PIF4 (or PIFs). Altered accumulation of COI1 under warm temperature in the med17 mutant suggests the possibility of a feedback mechanism. Overall, this study reveals the role of the Mediator complex as an integrator of JA and auxin signaling pathways during thermomorphogenesis.

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.

Cultivated grapevine (Vitis) is a highly valued horticultural crop, and cold stress affects its growth and productivity. Wild Amur grape (Vitis amurensis) PAT1 (Phytochrome A signal transduction 1, VaPAT1) is induced by low temperature, and ectopic expression of VaPAT1 enhances cold tolerance in Arabidopsis (Arabidopsis thaliana). However, little is known about the molecular mechanism of VaPAT1 during the cold stress response in grapevine. Here, we confirmed the overexpression of VaPAT1 in transformed grape calli enhanced cold tolerance. Yeast two-hybrid and bimolecular fluorescence complementation assays highlighted an interaction between VaPAT1 with INDETERMINATE-DOMAIN 3 (VaIDD3). A role of VaIDD3 in cold tolerance was also indicated. Transcriptome analysis revealed VaPAT1 and VaIDD3 overexpression and cold treatment coordinately modulate the expression of stress-related genes including lipoxygenase 3 (LOX3), a gene encoding a key jasmonate biosynthesis enzyme. Co-expression network analysis indicated LOX3 might be a downstream target of VaPAT1. Both electrophoretic mobility shift and dual luciferase reporter assays showed the VaPAT1-IDD3 complex binds to the IDD-box (AGACAAA) in the VaLOX3 promoter to activate its expression. Overexpression of both VaPAT1 and VaIDD3 increased the transcription of VaLOX3 and JA levels in transgenic grape calli. Conversely, VaPAT1-SRDX (dominant repression) and CRISPR/Cas9-mediated mutagenesis of PAT1-ED causing the loss of the C-terminus in grape calli dramatically prohibited the accumulation of VaLOX3 and JA levels during cold treatment. Together, these findings point to a pivotal role of VaPAT1 in the cold stress response in grape by regulating JA biosynthesis.

The world-wide distribution ofArabidopsis thaliana(Arabidopsis) accessions imposes different types of evolutionary pressures, which contributes to various responses of these accessions to environmental stresses. Drought stress responses have been well studied, particularly in Columbia, a common Arabidopsis accession. However, the reactions to drought stress are complex and our understanding of which of these responses contribute to the plant’s tolerance to mild drought is very limited. Here, we studied the mechanisms by which natural accessions react to mild drought at a physiological and molecular level during early leaf development. We documented variations in mild drought tolerance among natural accessions and used transcriptome sequencing of a drought-sensitive accession, ICE163, and a drought-tolerant accession, Yeg-1, to get insights into the mechanisms underlying this tolerance. This revealed that ICE163 preferentially induces jasmonates and anthocyanin-related pathways, which are beneficial in biotic stress defense, while Yeg-1 has a more pronounced activation of abscisic acid signaling, the classical abiotic stress response. Related physiological traits, including content of proline, anthocyanins and ROS, stomatal closure and cellular leaf parameters, were investigated and linked to the transcriptional responses. We conclude that most of these processes constitute general drought response mechanisms that are regulated similarly in drought-tolerant and -sensitive accessions. However, the capacity to close stomata and maintain cell expansion under mild drought appeared to be major factors that contribute to a better leaf growth under mild drought.One-sentence summaryThis paper demonstrates that an efficient closure of stomata and maintenance of cell expansion during drought conditions are crucial to maximally preserve plant growth during water deficit.

Jasmonates (JAs) are phytohormones with crucial roles in plant defense. Plants accumulate JAs in response to wounding or herbivore attack, but how JA biosynthesis is triggered remains poorly understood. Here we show that herbivory by cotton bollworm (Helicoverpa armigera) induced both ethylene (ET) and JA production in tomato (Solanum lycopersicum) leaves. Using RNA-seq, ET mutants, and inhibitors of ET signaling, we identified ET-induced ETHYLENE RESPONSE FACTOR 15 (ERF15) and ERF16 as critical regulators of JA biosynthesis in tomato plants. Transcripts of ERF15 and ERF16 were markedly upregulated and peaked at 60 and 15 min, respectively, after simulated herbivore attack. While mutation in ERF16 resulted in the attenuated expression of JA biosynthetic genes and decreased JA accumulation 15 min after the simulated herbivory treatment, these changes were not observed in erf15 mutants until 60 min after treatment. Electrophoretic mobility shift assays and dual-luciferase assays demonstrated that both ERFs15 and 16 are transcriptional activators of LIPOXYGENASE D, ALLENE OXIDE CYCLASE, and 12-OXO-PHYTODIENOIC ACID REDUCTASE 3, key genes in JA biosynthesis. Furthermore, JA-activated MYC2 and ERF16 also function as the transcriptional activators of ERF16, contributing to dramatic increases in ERF16 expression. Taken together, our results demonstrated that ET signaling is involved in the rapid induction of the JA burst. ET-induced ERF15 and ERF16 function as powerful transcriptional activators that trigger the JA burst in response to herbivore attack.

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.

Argonautes (AGOs) associate with noncoding RNAs to regulate gene expression during development and stress adaptation. Their role in plant immunity against hemibiotrophic fungal infection remains poorly understood. Here, we explore the function of AGOs in the interaction of wild tobacco (Nicotiana attenuata) with a naturally occurring hemibiotrophic pathogen, Fusarium brachygibbosum. Among all AGOs, only transcripts of AGO4 were elicited after fungal infection. The disease progressed more rapidly in AGO4-silenced (irAGO4) plants than in wild type, and small RNA (smRNA) profiling revealed that 24-nucleotide smRNA accumulation was severely abrogated in irAGO4 plants. Unique microRNAs (miRNAs: 130 conserved and 208 novel, including 11 canonical miRNA sequence variants known as “isomiRs”) were identified in infected plants; silencing of AGO4 strongly changed miRNA accumulation dynamics. Time-course studies revealed that infection increased accumulation of abscisic acid, jasmonates, and salicylic acid in wild type; in irAGO4 plants, infection accumulated lower jasmonate levels and lower transcripts of jasmonic acid (JA) biosynthesis genes. Treating irAGO4 plants with JA, methyl jasmonate, or cis-(+)-12-oxo-phytodienoic acid restored wild-type levels of resistance. Silencing expression of RNA-directed RNA polymerases RdR1 and RdR2 (but not RdR3) and Dicer-like3 (DCL3, but not DCL2 or DCL4) increased susceptibility to F. brachygibbosum. The relevance of AGO4, RdR1, RdR2, and DCL3 in a natural setting was revealed when plants individually silenced in their expression (and their binary combinations) were planted in a diseased field plot in the Great Basin Desert of Utah. These plants were more susceptible to infection and accumulated lower JA levels than wild type. We infer that AGO4-dependent smRNAs play a central role in modulating JA biogenesis and signaling during hemibiotrophic fungal infections.

The plant hormone jasmonate (JA) promotes resistance to biotic stress by stimulating the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins, which relieves repression on MYC transcription factors that execute defense programs. JA-triggered depletion of JAZ proteins in Arabidopsis (Arabidopsis thaliana) is also associated with reduced growth and seed production, but the mechanisms underlying these pleiotropic growth effects remain unclear. Here, we investigated this question using an Arabidopsis JAZ-deficient mutant (jazD; jaz1–jaz7,jaz9, jaz10, andjaz13) that exhibits high levels of defense and strong growth inhibition. Genetic suppressor screens for mutations that uncouple growth-defense tradeoffs in the jazD mutant identified nine independent causal mutations in the red-light receptor phytochrome B (phyB). Unlike the ability of the phyB mutations to completely uncouple the mild growth-defense phenotypes in a jaz mutant (jazQ) defective in JAZ1, JAZ3, JAZ4, JAZ9, and JAZ10, phyB null alleles only weakly alleviated the growth and reproductive defects in the jazD mutant. phyB-independent growth restriction of the jazD mutant was tightly correlated with upregulation of the Trp biosynthetic pathway but not with changes in central carbon metabolism. Interestingly, jazD and jazD phyB plants were insensitive to a chemical inhibitor of Trp biosynthesis, which is a phenotype previously observed in plants expressing hyperactive MYC transcription factors that cannot bind JAZ repressors. These data provide evidence that the mechanisms underlying JA-mediated growth-defense balance depend on the level of defense, and they further establish an association between growth inhibition at high levels of defense and dysregulation of Trp biosynthesis.

Fibrillins (FBNs) are the major structural proteins of plastoglobules (PGs) in chloroplasts. PGs are associated with defense against abiotic and biotic stresses, as well as lipid storage. Although FBN2 is abundant in PGs, its independent function under abiotic stress has not yet been identified. In this study, the targeting of FBN2 to PGs was clearly demonstrated using an FBN2-YFP fusion protein. FBN2 showed higher expression in green photosynthetic tissues and was upregulated at the transcriptional level under high-light stress. The photosynthetic capacity of fbn2 knockout mutants generated using CRISPR/Cas9 technology decreased rapidly compared with that of wild-type (WT) plants under high-light stress. In addition to the photoprotective function of FBN2, fbn2 mutants had lower levels of plastoquinone-9 and plastochromanol-8. The fbn2 mutants were highly sensitive to methyl jasmonate (MeJA) and exhibited root growth inhibition and a pale-green phenotype due to reduced chlorophyll content. Consistently, upon MeJA treatment, the fbn2 mutants showed faster leaf senescence and more rapid chlorophyll degradation with decreased photosynthetic ability compared with the WT plants. The results of this study suggest that FBN2 is involved in protection against high-light stress and acts as an inhibitor of jasmonate-induced senescence in Arabidopsis (Arabidopsis thaliana).

MYB transcription factors play essential roles in regulating plant secondary metabolism and jasmonate (JA) signaling. Putrescine N-methyltransferase is a key JA-regulated step in the biosynthesis of nicotine, an alkaloidal compound highly accumulated in Nicotiana spp. Here we report the identification of NtMYB305a in tobacco (Nicotiana tabacum) as a regulatory component of nicotine biosynthesis and demonstrate that it binds to the JA-responsive GAG region, which comprises a G-box, an AT-rich motif, and a GCC-box-like element, in the NtPMT1a promoter. Yeast one-hybrid analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed that NtMYB305a binds to the GAG region in vitro and in vivo. Binding specifically occurs at the ∼30-bp AT-rich motif in a G/C-base-independent manner, thus defining the AT-rich motif as previously unknown MYB-binding element. NtMYB305a localized in the nucleus of tobacco cells where it is capable of activating the expression of a 4×GAG-driven GUS reporter in an AT-rich motif-dependent manner. NtMYB305a positively regulates nicotine biosynthesis and the expression of NtPMT and other nicotine pathway genes. NtMYB305a acts synergistically with NtMYC2a to regulate nicotine biosynthesis, but no interaction between these two proteins was detected. This identification of NtMYB305a provides insights into the regulation of nicotine biosynthesis and extends the roles played by MYB transcription factors in plant secondary metabolism.

The phytohormone JA-Ile regulates many stress responses and developmental processes in plants. A co-receptor complex formed by the F-box protein COI1 (Coronatine Insensitive 1) and a JAZ (Jasmonate ZIM-domain) repressor perceives the hormone. JA-Ile antagonists are invaluable tools for exploring the role of JA-Ile in specific tissues and developmental stages, and for identifying regulatory processes of the signalling pathway. Using two complementary chemical screens, we identified three compounds that exhibit a robust inhibitory effect on both the hormone-mediated COI-JAZ interaction and degradation of JAZ1 and JAZ9in vivo. One molecule, J4, also restrains specific JA-induced physiological responses in different angiosperm plants, including JA-mediated gene expression, growth inhibition, chlorophyll degradation and anthocyanin accumulation. Interaction experiments with purified proteins indicate that J4 directly interferes with the formation of the Arabidopsis (Arabidopsis thaliana)COI1-JAZ complex otherwise induced by JA. The antagonistic effect of J4 on COI1- JAZ also occurs in the liverwortMarchantia polymorpha, suggesting the mode of action is conserved in land plants. Besides JA signalling, J4 works as an antagonist of the closely-related auxin signalling pathway, preventing TIR1/Aux-IAA interaction and auxin responsesin planta,including hormone-mediated degradation of an auxin repressor, gene expression and gravitropic response. However, J4 does not affect other hormonal pathways. Altogether, our results show that this dual antagonist competes with JA-Ile and auxin, preventing the formation of phylogenetically related receptor complexes. J4 may be a useful tool to dissect both the JA-Ile and auxin pathways in particular tissues and developmental stages since it reversibly inhibits these pathways.One sentence summaryA chemical screen identified a molecule that antagonizes jasmonate perception by directly interfering with receptor complex formation in phylogenetically distant vascular and non-vascular plants.

Sphingolipids are structural components of the lipid bilayer that acts as signaling molecules in many cellular processes, including cell death. Ceramides, key intermediates in sphingolipid metabolism, are phosphorylated by the ceramide kinase ACCELERATED CELL DEATH5 (ACD5). The loss of ACD5 function leads to ceramide accumulation and spontaneous cell death. Here, we report that the jasmonate (JA) pathway is activated in the Arabidopsis (Arabidopsis thaliana) acd5 mutant and that methyl JA treatment accelerates ceramide accumulation and cell death in acd5. Moreover, the double mutants of acd5 with jasmonate resistant1-1 and coronatine insensitive1-2 exhibited delayed cell death, suggesting that the JA pathway is involved in acd5-mediated cell death. Quantitative sphingolipid profiling of plants treated with methyl JA indicated that JAs influence sphingolipid metabolism by increasing the levels of ceramides and hydroxyceramides, but this pathway is dramatically attenuated by mutations affecting JA pathway proteins. Furthermore, we showed that JAs regulate the expression of genes encoding enzymes in ceramide metabolism. Together, our findings show that JAs accelerate cell death in acd5 mutants, possibly by modulating sphingolipid metabolism and increasing ceramide levels.

Brassinosteroids (BRs) and jasmonates (JAs) regulate plant growth, development, and defense responses, but how these phytohormones mediate the growth-defense tradeoff is unclear. Here, we identified the Arabidopsis (Arabidopsis thaliana) dwarf at early stages1 (dwe1) mutant, which exhibits enhanced expression of defensin genes PLANT DEFENSIN1.2a (PDF1.2a) and PDF1.2b. The dwe1 mutant showed increased resistance to herbivory by beet armyworms (Spodoptera exigua) and infection by botrytis (Botrytis cinerea). DWE1 encodes ROTUNDIFOLIA3, a cytochrome P450 protein essential for BR biosynthesis. The JA-inducible transcription of PDF1.2a and PDF1.2b was significantly reduced in the BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) gain-of-function mutant bes1- D, which was highly susceptible to S. exigua and B. cinerea. BES1 directly targeted the terminator regions of PDF1.2a/PDF1.2b and suppressed their expression. PDF1.2a overexpression diminished the enhanced susceptibility of bes1- D to B. cinerea but did not improve resistance of bes1- D to S. exigua. In response to S. exigua herbivory, BES1 inhibited biosynthesis of the JA-induced insect defense-related metabolite indolic glucosinolate by interacting with transcription factors MYB DOMAIN PROTEIN34 (MYB34), MYB51, and MYB122 and suppressing expression of genes encoding CYTOCHROME P450 FAMILY79 SUBFAMILY B POLYPEPTIDE3 (CYP79B3) and UDP-GLUCOSYL TRANSFERASE 74B1 (UGT74B1). Thus, BR contributes to the growth-defense tradeoff by suppressing expression of defensin and glucosinolate biosynthesis genes.

Chlorophyll degradation is one of the most visible signs of leaf senescence. During senescence, chlorophyll is degraded in the multistep pheophorbide a oxygenase (PAO)/phyllobilin pathway. This pathway is tightly regulated at the transcriptional level, allowing coordinated and efficient remobilization of nitrogen toward sink organs. Using a combination of transcriptome and metabolite analyses during dark-induced senescence of Arabidopsis (Arabidopsis thaliana) mutants deficient in key steps of the PAO/phyllobilin pathway, we show an unanticipated role for one of the pathway intermediates, i.e. pheophorbide a. Both jasmonic acid-related gene expression and jasmonic acid precursors specifically accumulated in pao1, a mutant deficient in PAO. We propose that pheophorbide a, the last intact porphyrin intermediate of chlorophyll degradation and a unique pathway “bottleneck,” has been recruited as a signaling molecule of chloroplast metabolic status. Our work challenges the assumption that chlorophyll breakdown is merely a result of senescence, and proposes that the flux of pheophorbide a through the pathway acts in a feed-forward loop that remodels the nuclear transcriptome and controls the pace of chlorophyll degradation in senescing leaves.

APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene clusters regulate the biosynthesis of diverse specialized metabolites, including steroidal glycoalkaloids in tomato (Solanum lycopersicum) and potato (Solanum tuberosum), nicotine in tobacco (Nicotiana tabacum), and pharmaceutically valuable terpenoid indole alkaloids in Madagascar periwinkle (Catharanthus roseus). However, the regulatory relationships between individual AP2/ERF genes within the cluster remain unexplored. We uncovered intracluster regulation of the C. roseus AP2/ERF regulatory circuit, which consists of ORCA3, ORCA4, and ORCA5. ORCA3 and ORCA5 activate ORCA4 by directly binding to a GC-rich motif in the ORCA4 promoter. ORCA5 regulates its own expression through a positive autoregulatory loop and indirectly activates ORCA3. In determining the functional conservation of AP2/ERF clusters in other plant species, we found that GC-rich motifs are present in the promoters of analogous AP2/ERF clusters in tobacco, tomato, and potato. Intracluster regulation is evident within the tobacco NICOTINE2 (NIC2) ERF cluster. Moreover, overexpression of ORCA5 in tobacco and of NIC2 ERF189 in C. roseus hairy roots activates nicotine and terpenoid indole alkaloid pathway genes, respectively, suggesting that the AP2/ERFs are functionally equivalent and are likely to be interchangeable. Elucidation of the intracluster and mutual regulation of transcription factor gene clusters advances our understanding of the underlying molecular mechanism governing regulatory gene clusters in plants.

Partial root-zone irrigation (PRI), a water-saving technique, improves water uptake in hydrated roots by inducing specific responses that are thought to be regulated by signals originating from leaves; however, this signaling is poorly understood. Using a split-root system and polyethylene glycol 6000 to simulate PRI in cotton (Gossypium hirsutum), we showed that increased root hydraulic conductance (L) and water uptake in the hydrated roots may be due to the elevated expression of cotton plasma membrane intrinsic protein (PIP) genes. Jasmonate (jasmonic acid [JA] and jasmonic acid-isoleucine conjugate [JA-Ile]) content and the expression of three JA biosynthesis genes increased in the leaves of the PRI plants compared with those of the polyethylene glycol-free control. JA/JA-Ile content also increased in the hydrated roots, although the expression of the three JA genes was unaltered, compared with the control. The JA/JA-Ile contents in leaves increased after the foliar application of exogenous JA and was followed by an increase in both JA/JA-Ile content and L in the hydrated roots, whereas the silencing of the three JA genes had the opposite effect in the leaves. Ring-barking the hydrated hypocotyls increased the JA/JA-Ile content in the leaves but decreased the JA/JA-Ile content and L in the hydrated roots. These results suggested that the increased JA/JA-Ile in the hydrated roots was mostly transported from the leaves through the phloem, thus increasing L by increasing the expression of GhPIP in the hydrated roots under PRI. We believe that leaf-derived JA/JA-Ile, as a long-distance signal, positively mediates water uptake from the hydrated roots of cotton under PRI.

HOPs (HSP70–HSP90 organizing proteins) are a highly conserved family of HSP70 and HSP90 co-chaperones whose role in assisting the folding of various hormonal receptors has been extensively studied in mammals. In plants, HOPs are mainly associated with stress response, but their potential involvement in hormonal networks remains completely unexplored. In this article we describe that a member of the HOP family, HOP3, is involved in the jasmonic acid (JA) pathway and is linked to plant defense responses not only to pathogens, but also to a generalist herbivore. The JA pathway regulates responses to Botrytis cinerea infection and to Tetranychus urticae feeding; our data demonstrate that the Arabidopsis (Arabidopsis thaliana) hop3-1 mutant shows an increased susceptibility to both. The hop3-1 mutant exhibits reduced sensitivity to JA derivatives in root growth assays and downregulation of different JA-responsive genes in response to methyl jasmonate, further revealing the relevance of HOP3 in the JA pathway. Interestingly, yeast two-hybrid assays and in planta co-immunoprecipitation assays found that HOP3 interacts with COI1, suggesting that COI1 is a target of HOP3. Consistent with this observation, COI1 activity is reduced in the hop3-1 mutant. All these data strongly suggest that, specifically among HOPs, HOP3 plays a relevant role in the JA pathway by regulating COI1 activity in response to JA and, consequently, participating in defense signaling to biotic stresses., One-sentence summary: The co-chaperone protein HOP3 (HSP70-HSP90 ORGANIZING PROTEIN 3) regulates the activity of jasmonic acid co-receptor CORONATINE INSENSITIVE 1 and functions in plant defense.

Pyrethrins are synthesized by the plant pyrethrum (Tanacetum cinerariifolium), a chrysanthemum relative. These compounds possess efficient insecticidal properties and are not toxic to humans and most vertebrates. Pyrethrum flowers, and to a smaller extent leaves, synthesize six main types of pyrethrins, which are all esters of a monoterpenoid acid moiety and an alcohol moiety derived from jasmonic acid. Here, we identified and characterized the enzyme responsible for the conversion of jasmone, a derivative of jasmonic acid, to jasmolone. Feeding pyrethrum flowers with jasmone resulted in a 4-fold increase in the concentration of free jasmolone as well as smaller but significant proportional increases in free pyrethrolone and all three type I pyrethrins. We used floral transcriptomic data to identify cytochrome P450 genes whose expression patterns were most highly correlated with that of a key gene in pyrethrin biosynthesis, T. cinerariifolium chrysanthemyl diphosphate synthase The candidate genes were screened for jasmone hydroxylase activity through transient expression in Nicotiana benthamiana leaves fed with jasmone. The expression of only one of these candidate genes produced jasmolone; therefore, this gene was named T. cinerariifolium jasmolone hydroxylase (TcJMH) and given the CYP designation CYP71AT148. The protein encoded by TcJMH localized to the endoplasmic reticulum, and microsomal preparations from N. benthamiana leaves expressing TcJMH were capable of catalyzing the hydroxylation of jasmone to jasmolone in vitro, with a Km value of 53.9 µm TcJMH was expressed almost exclusively in trichomes of floral ovaries and was induced in leaves by jasmonate.

Plant root systems are indispensable for water uptake, nutrient acquisition, and anchoring plants in the soil. Previous studies using auxin inhibitors definitively established that auxin plays a central role regulating root growth and development. Most auxin inhibitors affect all auxin signaling at the same time, which obscures an understanding of individual events. Here, we report that jasmonic acid (JA) functions as a lateral root (LR)-preferential auxin inhibitor in Arabidopsis (Arabidopsis thaliana) in a manner that is independent of the JA receptor, CORONATINE INSENSITIVE1 (COI1). Treatment of wild-type Arabidopsis with either (−)-JA or (+)-JA reduced primary root length and LR number; the reduction of LR number was also observed in coi1 mutants. Treatment of seedlings with (−)-JA or (+)-JA suppressed auxin-inducible genes related to LR formation, diminished accumulation of the auxin reporter DR5::GUS, and inhibited auxin-dependent DII-VENUS degradation. A structural mimic of (−)-JA and (+)-coronafacic acid also inhibited LR formation and stabilized DII-VENUS protein. COI1-independent activity was retained in the double mutant of transport inhibitor response1 and auxin signaling f-box protein2 (tir1 afb2) but reduced in the afb5 single mutant. These results reveal JAs and (+)-coronafacic acid to be selective counter-auxins, a finding that could lead to new approaches for studying the mechanisms of LR formation.

Cereal endosperms produce a vast array of metabolites, including the essential amino acid lysine (Lys). Enhanced accumulation of Lys has been achieved via metabolic engineering in cereals, but the potential connection between metabolic engineering and Lys fortification is unclear. In mature seeds of engineered High Free Lysine (HFL) rice (Oryza sativa), the endosperm takes on a characteristic dark-brown appearance. In this study, we use an integrated metabolomic and transcriptomic approach combined with functional validation to elucidate the key metabolites responsible for the dark-brown phenotype. Importantly, we found that serotonin biosynthesis was elevated dramatically and closely linked with dark-brown endosperm color in HFL rice. A functional connection between serotonin and endosperm color was confirmed via overexpression of TDC3, a key enzyme of serotonin biosynthesis. Furthermore, we show that both the jasmonate signaling pathway and TDC expression were strongly induced in the late stage of endosperm development of HFL rice, coinciding with serotonin accumulation and dark-brown pigmentation. We propose a model for the metabolic connection between Lys and serotonin metabolism in which elevated 2-aminoadipate from Lys catabolism may play a key role in the connection between the jasmonate signaling pathway, serotonin accumulation, and the brown phenotype in rice endosperm. Our data provide a deeper understanding of amino acid metabolism in rice. In addition, the finding that both Lys and serotonin accumulate in HFL rice grains should promote efforts to create a nutritionally favorable crop.

Wounding is a primary trigger of organ regeneration, but how wound stress reactivates cell proliferation and promotes cellular reprogramming remains elusive. In this study, we combined transcriptome analysis with quantitative hormonal analysis to investigate how wounding induces callus formation in Arabidopsis (Arabidopsis thaliana). Our time course RNA-seq analysis revealed that wounding induces dynamic transcriptional changes, starting from rapid stress responses followed by the activation of metabolic processes and protein synthesis and subsequent activation of cell cycle regulators. Gene ontology analyses further uncovered that wounding modifies the expression of hormone biosynthesis and response genes, and quantitative analysis of endogenous plant hormones revealed accumulation of cytokinin prior to callus formation. Mutants defective in cytokinin synthesis and signaling display reduced efficiency in callus formation, indicating that de novo synthesis of cytokinin is critical for wound-induced callus formation. We further demonstrate that type-B ARABIDOPSIS RESPONSE REGULATOR-mediated cytokinin signaling regulates the expression of CYCLIN D3;1 (CYCD3;1) and that mutations in CYCD3;1 and its homologs CYCD3;2 and 3 cause defects in callus formation. In addition to these hormone-mediated changes, our transcriptome data uncovered that wounding activates multiple developmental regulators, and we found novel roles of ETHYLENE RESPONSE FACTOR 115 and PLETHORA3 (PLT3), PLT5, and PLT7 in callus generation. All together, these results provide novel mechanistic insights into how wounding reactivates cell proliferation during callus formation.

Submergence induces hypoxia in plants; exposure to oxygen following submergence, termed reoxygenation, produces a burst of reactive oxygen species. The mechanisms of hypoxia sensing and signaling in plants have been well studied, but how plants respond to reoxygenation remains unclear. Here, we show that reoxygenation in Arabidopsis (Arabidopsis thaliana) involves rapid accumulation of jasmonates (JAs) and increased transcript levels of JA biosynthesis genes. Application of exogenous methyl jasmonate improved tolerance to reoxygenation in wild-type Arabidopsis; also, mutants deficient in JA biosynthesis and signaling were very sensitive to reoxygenation. Moreover, overexpression of the transcription factor gene MYC2 enhanced tolerance to posthypoxic stress, and myc2 knockout mutants showed increased sensitivity to reoxygenation, indicating that MYC2 functions as a key regulator in the JA-mediated reoxygenation response. MYC2 transcriptionally activates members of the VITAMIN C DEFECTIVE (VTC) and GLUTATHIONE SYNTHETASE (GSH) gene families, which encode rate-limiting enzymes in the ascorbate and glutathione synthesis pathways. Overexpression of VTC1 and GSH1 in the myc2-2 mutant suppressed the posthypoxic hypersensitive phenotype. The JA-inducible accumulation of antioxidants may alleviate oxidative damage caused by reoxygenation, improving plant survival after submergence. Taken together, our findings demonstrate that JA signaling interacts with the antioxidant pathway to regulate reoxygenation responses in Arabidopsis.

Temperature has a large impact on plant immune responses. Earlier studies identified intracellular immune receptor nucleotide-binding leucine-rich repeat (NLR) genes and salicylic acid (SA) as targets of high-temperature inhibition of plant immunity. Here, we report that moderately low temperature enhances immunity to the bacterial pathogen Pseudomonas syringae in Arabidopsis (Arabidopsis thaliana). This enhancement is dependent on SA signaling and is accompanied by up-regulation of multiple SA biosynthesis and signaling genes at lower temperature. SA signaling is repressed by jasmonic acid and ethylene at both normal and low temperatures. The inhibition of SA biosynthesis by ethylene, while mainly through ISOCHORISMATE SYNTHASE1/SALICYLIC ACID-INDUCTION DEFICIENT2 (ICS1/SID2) at normal temperature, is through ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5)/SID1, ICS2, and ICS1/SID2 at lower temperature. The repression by ethylene is mediated by a direct regulation of the ethylene response transcription factor ETHYLENE INSENSITIVE3 (EIN3) on multiple SA biosynthesis and signaling genes. Thus, low temperature enhances the SA pathway to promote immunity and at the same time uses ethylene to repress multiple SA regulators to achieve fine-tuned immune responses.

Calmodulin (CaM) regulates plant disease responses through its downstream calmodulin-binding proteins (CaMBPs) often by affecting the biosynthesis or signaling of phytohormones, such as jasmonic acid (JA) and salicylic acid. However, how these CaMBPs mediate plant hormones and other stress resistance-related signaling remains largely unknown. In this study, we conducted analyses in Arabidopsis (Arabidopsis thaliana) on the functions of AtIQM1 (IQ-Motif Containing Protein1), a Ca(2+)-independent CaMBP, in JA biosynthesis and defense against the necrotrophic pathogen Botrytis cinerea using molecular, biochemical, and genetic analyses. IQM1 directly interacted with and promoted CATALASE2 (CAT2) expression and CAT2 enzyme activity and indirectly increased the activity of the JA biosynthetic enzymes ACX2 and ACX3 through CAT2, thereby positively regulating JA content and B. cinerea resistance. In addition, in vitro assays showed that in the presence of CaM5, IQM1 further enhanced the activity of CAT2, suggesting that CaM5 may affect the activity of CAT2 by combining with IQM1 in the absence of Ca(2+). Our data indicate that IQM1 is a key regulatory factor in signaling of plant disease responses mediated by JA. The study also provides new insights that CaMBP may play a critical role in the cross talk of multiple signaling pathways in the context of plant defense processes.

The conversion of oleic acid (C18:1) to linoleic acid (C18:2) in the endoplasmic reticulum is critical to the accumulation of polyunsaturated fatty acids in seeds and other tissues, and this reaction is catalyzed by a Δ12-desaturase, FATTY ACID DESATURASE2 (FAD2). Here, we report that the tomato (Solanum lycopersicum) genome harbors two genes, SlFAD2-1 and SlFAD2-2, which encode proteins with in vitro Δ12-desaturase activity. In addition, tomato has seven divergent FAD2 members that lack Δ12-desaturase activity and differ from canonical FAD2 enzymes at multiple amino acid positions important to enzyme function. Whereas SlFAD2-1 and SlFAD2-2 are downregulated by biotic stress, the majority of divergent FAD2 genes in tomato are upregulated by one or more stresses. In particular, SlFAD2-7 is induced by the potato aphid (Macrosiphum euphorbiae) and has elevated constitutive expression levels in suppressor of prosystemin-mediated responses2 (spr2), a tomato mutant with enhanced aphid resistance and altered fatty acid profiles. Virus-induced gene silencing of SlFAD2-7 in spr2 results in significant increases in aphid population growth, indicating that a divergent FAD2 gene contributes to aphid resistance in this genotype. Thus, the FAD2 gene family in tomato is important both to primary fatty acid metabolism and to responses to biotic stress.

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development., We acknowledge financial support from the Spanish Ministry of Economy and Competitiveness through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV-2015-0533). This work was supported by the Spanish Government (grant nos. BIO2009–06984 and AGL2013–43522-R to A.F.) and the Generalitat de Catalunya (grant no. 2014SGR–1434).

Inositol polyphosphate binding specificity of the jasmonate receptor is largely determined by the F-box protein COI1.

AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense.

Gibberellins are a class of tetracyclic plant hormones that are well known to promote plant growth by inducing the degradation of a class of nuclear growth-repressing proteins, called DELLAs. In recent years, GA and DELLAs are also increasingly implicated in plant responses to pathogen attack, although our understanding of the underlying mechanisms is still limited, especially in monocotyledonous crop plants. Aiming to further decipher the molecular underpinnings of GA- and DELLA-modulated plant immunity, we studied the dynamics and impact of GA and DELLA during infection of the model crop rice (Oryza sativa) with four different pathogens exhibiting distinct lifestyles and infection strategies. Opposite to previous findings in Arabidopsis (Arabidopsis thaliana), our findings reveal a prominent role of the DELLA protein Slender Rice1 (SLR1) in the resistance toward (hemi)biotrophic but not necrotrophic rice pathogens. Moreover, contrary to the differential effect of DELLA on the archetypal defense hormones salicylic acid (SA) and jasmonic acid (JA) in Arabidopsis, we demonstrate that the resistance-promoting effect of SLR1 is due at least in part to its ability to boost both SA- and JA-mediated rice defenses. In a reciprocal manner, we found JA and SA treatment to interfere with GA metabolism and stabilize SLR1. Together, these findings favor a model whereby SLR1 acts as a positive regulator of hemibiotroph resistance in rice by integrating and amplifying SA- and JA-dependent defense signaling. Our results highlight the differences in hormone defense networking between rice and Arabidopsis and underscore the importance of GA and DELLA in molding disease outcomes.

The ecological success of diatoms requires a remarkable ability to survive many types of stress, including variations in temperature, light, salinity, and nutrient availability. On exposure to these stresses, diatoms exhibit common responses, including growth arrest, impairment of photosynthesis, production of reactive oxygen species, and accumulation of triacylglycerol (TAG). We studied the production of cyclopentane oxylipins derived from fatty acids in the diatom Phaeodactylum tricornutum in response to oxidative stress. P. tricornutum lacks the enzymatic pathway for producing cyclopentane-oxylipins, such as jasmonate, prostaglandins, or thromboxanes. In cells subjected to increasing doses of hydrogen peroxide (H(2)O(2)), we detected nonenzymatic production of isoprostanoids, including six phytoprostanes, three F(2t)-isoprostanes, two F(3t)-isoprostanes, and three F(4t)-neuroprostanes, by radical peroxidation of α-linolenic, arachidonic, eicosapentaenoic, and docosahexanoic acids, respectively. H(2)O(2) also triggered photosynthesis impairment and TAG accumulation. F(1t)-phytoprostanes constitute the major class detected (300 pmol per 1 million cells; intracellular concentration, ∼4 µm). Only two glycerolipids, phosphatidylcholine and diacylglycerylhydroxymethyl-trimethyl-alanine, could provide all substrates for these isoprostanoids. Treatment of P. tricornutum with nine synthetic isoprostanoids produced an effect in the micromolar range, marked by the accumulation of TAG and reduced growth, without affecting photosynthesis. Therefore, the emission of H(2)O(2) and free radicals upon exposure to stresses can lead to glycerolipid peroxidation and nonenzymatic synthesis of isoprostanoids, inhibiting growth and contributing to the induction of TAG accumulation via unknown processes. This characterization of nonenzymatic oxylipins in P. tricornutum opens a field of research on the study of processes controlled by isoprostanoid signaling in various physiological and environmental contexts in diatoms.

Plants respond to herbivory with the induction of resistance, mediated by distinct phytohormonal signaling pathways and their interactions. Phloem feeders are known to induce plant resistance via the salicylic acid pathway, whereas biting-chewing herbivores induce plant resistance mainly via the jasmonate pathway. Here, we show that a specialist caterpillar (biting-chewing herbivore) and a specialist aphid (phloem feeder) differentially induce resistance against Pieris brassicae caterpillars in Arabidopsis (Arabidopsis thaliana) plants. Caterpillar feeding induces resistance through the jasmonate signaling pathway that is associated with the induction of kaempferol 3,7-dirhamnoside, whereas aphid feeding induces resistance via a novel mechanism involving sinapoyl malate. The role of sinapoyl malate is confirmed through the use of a mutant compromised in the biosynthesis of this compound. Caterpillar-induced resistance is associated with a lower cost in terms of plant growth reduction than aphid-induced resistance. A strong constitutive resistance against P. brassicae caterpillars in combination with a strong growth attenuation in plants of a transfer DNA (T-DNA) insertion mutant of WRKY70 (wrky70) suggest that the WRKY70 transcription factor, a regulator of downstream responses mediated by jasmonate-salicylic acid signaling cross talk, is involved in the negative regulation of caterpillar resistance and in the tradeoff between growth and defense. In conclusion, different mechanisms of herbivore-induced resistance come with different costs, and a functional WRKY70 transcription factor is required for the induction of low-cost resistance.

Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula.

Signaling networks among multiple phytohormones fine-tune plant defense responses to insect herbivore attack. Previously, it was reported that the synergistic combination of ethylene (ET) and jasmonic acid (JA) was required for accumulation of the maize insect resistance1 (mir1) gene product, a cysteine (Cys) proteinase that is a key defensive protein against chewing insect pests in maize (Zea mays). However, this study suggests that mir1-mediated resistance to corn leaf aphid (CLA; Rhopalosiphum maidis), a phloem sap-sucking insect pest, is independent of JA but regulated by the ET-signaling pathway. Feeding by CLA triggers the rapid accumulation of mir1 transcripts in the resistant maize genotype, Mp708. Furthermore, Mp708 provided elevated levels of antibiosis (limits aphid population)- and antixenosis (deters aphid settling)-mediated resistance to CLA compared with B73 and Tx601 maize susceptible inbred lines. Synthetic diet aphid feeding trial bioassays with recombinant Mir1-Cys Protease demonstrates that Mir1-Cys Protease provides direct toxicity to CLA. Furthermore, foliar feeding by CLA rapidly sends defensive signal(s) to the roots that trigger belowground accumulation of the mir1, signifying a potential role of long-distance signaling in maize defense against the phloem-feeding insects. Collectively, our data indicate that ET-regulated mir1 transcript accumulation, uncoupled from JA, contributed to heightened resistance to CLA in maize. In addition, our results underscore the significance of ET acting as a central node in regulating mir1 expression to different feeding guilds of insect herbivores.

Polyamines are involved in key developmental processes and stress responses. Copper amine oxidases oxidize the polyamine putrescine (Put), producing an aldehyde, ammonia, and hydrogen peroxide (H2O2). The Arabidopsis (Arabidopsis thaliana) amine oxidase gene At4g14940 (AtAO1) encodes an apoplastic copper amine oxidase expressed at the early stages of vascular tissue differentiation in roots. Here, its role in root development and xylem differentiation was explored by pharmacological and forward/reverse genetic approaches. Analysis of the AtAO1 expression pattern in roots by a promoter::green fluorescent protein-beta-glucuronidase fusion revealed strong gene expression in the protoxylem at the transition, elongation, and maturation zones. Methyl jasmonate (MeJA) induced AtAO1 gene expression in vascular tissues, especially at the transition and elongation zones. Early protoxylem differentiation was observed upon MeJA treatment along with Put level decrease and H2O2 accumulation in wild-type roots, whereas Atao1 loss-of-function mutants were unresponsive to the hormone. The H2O2 scavenger N,N(1)-dimethylthiourea reversed the MeJA-induced early protoxylem differentiation in wild-type seedlings. Likewise, Put, which had no effect on Atao1 mutants, induced early protoxylem differentiation in the wild type, this event being counteracted by N,N(1)-dimethylthiourea treatment. Consistently, AtAO1-overexpressing plants showed lower Put levels and early protoxylem differentiation concurrent with H2O2 accumulation in the root zone where the first protoxylem cells with fully developed secondary wall thickenings are found. These results show that the H2O2 produced via AtAO1-driven Put oxidation plays a role in MeJA signaling leading to early protoxylem differentiation in root.

Priming refers to a mechanism whereby plants are sensitized to respond faster and/or more strongly to future pathogen attack. Here, we demonstrate that preexposure to the green leaf volatile Z-3-hexenyl acetate (Z-3-HAC) primed wheat (Triticum aestivum) for enhanced defense against subsequent infection with the hemibiotrophic fungus Fusarium graminearum. Bioassays showed that, after priming with Z-3-HAC, wheat ears accumulated up to 40% fewer necrotic spikelets. Furthermore, leaves of seedlings showed significantly smaller necrotic lesions compared with nonprimed plants, coinciding with strongly reduced fungal growth in planta. Additionally, we found that F. graminearum produced more deoxynivalenol, a mycotoxin, in the primed treatment. Expression analysis of salicylic acid (SA) and jasmonic acid (JA) biosynthesis genes and exogenous methyl salicylate and methyl jasmonate applications showed that plant defense against F. graminearum is sequentially regulated by SA and JA during the early and later stages of infection, respectively. Interestingly, analysis of the effect of Z-3-HAC pretreatment on SA- and JA-responsive gene expression in hormone-treated and pathogen-inoculated seedlings revealed that Z-3-HAC boosts JA-dependent defenses during the necrotrophic infection stage of F. graminearum but suppresses SA-regulated defense during its biotrophic phase. Together, these findings highlight the importance of temporally separated hormone changes in molding plant health and disease and support a scenario whereby the green leaf volatile Z-3-HAC protects wheat against Fusarium head blight by priming for enhanced JA-dependent defenses during the necrotrophic stages of infection.

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.