The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis

Despite its importance in a variety of plant defense responses, our understanding of how jasmonic acid (JA) functions at the biochemical level is limited. Several amino acid conjugates of JA were tested for their ability to complement the JA-insensitive Arabidopsis thaliana mutant jar1-1. Unlike free JA, JA-Ile inhibited root growth in jar1-1 to the same extent as in the wild type, whereas JA-Val, JA-Leu, and JA-Phe were ineffective inhibitors in both genotypes. Thin-layer chromatography and gas chromatography-mass spectrometry (GC-MS) analysis of products produced in vitro by recombinant JAR1 demonstrated that this enzyme forms JA-amido conjugates with several amino acids, including JA-Ile. JA-Val, -Leu, -Ile, and -Phe were each quantified in Arabidopsis seedlings by GC-MS. JA-Ile was found at 29.6 pmole g(-1) fresh weight (FW) in the wild type but was more than sevenfold lower in two jar1 alleles. JA-Leu, -Val, and -Phe were present at only low levels in both genotypes. Expression of wild-type JAR1 in transgenic jar1-1 plants restored sensitivity to JA and elevated JA-Ile to the same level as in the wild type. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) conjugated to JA was also found in plant tissue at 18.4 pmole g(-1) FW. JA-ACC was determined not be an effective jasmonate root inhibitor, and surprisingly, was twofold higher in the mutants than in the wild type. This suggests that another JA-conjugating enzyme(s) is present in Arabidopsis. Synthesis of JA-ACC might provide a mechanism to coregulate the availability of JA and ACC for conversion to the active hormones JA-Ile and ethylene, respectively. We conclude that JAR1 is a JA-amino synthetase that is required to activate JA for optimal signaling in Arabidopsis. Plant hormone activation by conjugation to amino acids and the enzymes involved in their formation were previously unknown.     

Structural, biochemical, and phylogenetic analyses suggest that indole-3-acetic acid methyltransferase is an evolutionarily ancient member of the SABATH family

The plant SABATH protein family encompasses a group of related small-molecule methyltransferases (MTs) that catalyze the S-adenosyl-L-methionine-dependent methylation of natural chemicals encompassing widely divergent structures. Indole-3-acetic acid (IAA) methyltransferase (IAMT) is a member of the SABATH family that modulates IAA homeostasis in plant tissues through methylation of IAA's free carboxyl group. The crystal structure of Arabidopsis (Arabidopsis thaliana) IAMT (AtIAMT1) was determined and refined to 2.75 A resolution. The overall tertiary and quaternary structures closely resemble the two-domain bilobed monomer and the dimeric arrangement, respectively, previously observed for the related salicylic acid carboxyl methyltransferase from Clarkia breweri (CbSAMT). To further our understanding of the biological function and evolution of SABATHs, especially of IAMT, we analyzed the SABATH gene family in the rice (Oryza sativa) genome. Forty-one OsSABATH genes were identified. Expression analysis showed that more than one-half of the OsSABATH genes were transcribed in one or multiple organs. The OsSABATH gene most similar to AtIAMT1 is OsSABATH4. Escherichia coli-expressed OsSABATH4 protein displayed the highest level of catalytic activity toward IAA and was therefore named OsIAMT1. OsIAMT1 exhibited kinetic properties similar to AtIAMT1 and poplar IAMT (PtIAMT1). Structural modeling of OsIAMT1 and PtIAMT1 using the experimentally determined structure of AtIAMT1 reported here as a template revealed conserved structural features of IAMTs within the active-site cavity that are divergent from functionally distinct members of the SABATH family, such as CbSAMT. Phylogenetic analysis revealed that IAMTs from Arabidopsis, rice, and poplar (Populus spp.) form a monophyletic group. Thus, structural, biochemical, and phylogenetic evidence supports the hypothesis that IAMT is an evolutionarily ancient member of the SABATH family likely to play a critical role in IAA homeostasis across a wide range of plants.     

Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein

Since auxin was first isolated and characterized as a plant hormone, the underlying molecular mechanism of auxin signaling has been elucidated primarily in dicot plants represented by Arabidopsis. In monocot plants, the molecular mechanism of auxin signaling has remained unclear, despite various physiological experiments. To understand the function and mechanism of auxin signaling in rice ( Oryza sativa ), we focused on the IAA gene, a well-studied gene in Arabidopsis that serves as a negative regulator of auxin signaling. We found 24 IAA gene family members in the rice genome. OsIAA3 is one of these family members whose expression is rapidly increased in response to auxin. We produced transgenic rice harboring m OsIAA3 - GR , which can overproduce mutant OsIAA3 protein containing an amino acid change in domain II to cause a gain-of-function phenotype, by treatment with dexamethasone. The transgenic rice was insensitive to auxin and gravitropic stimuli, and exhibited short leaf blades, reduced crown root formation, and abnormal leaf formation. These results suggest that , in rice, auxin is important for development and its signaling is mediated by IAA genes.     

Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. A family of seven related proteins named AtGPX1- AtGPX7 in Arabidopsis was identified, and the genomic organization of this family was reported. The putative subcellular localizations of the encoded proteins are the cytosol, chloroplast, mitochondria, and endoplasmic reticulum. Expressed sequence tags (ESTs) for all the genes except AtGPX7 were identified. Expression analysis of AtGPX genes in Arabidopsis tissues was performed, and different patterns were detected. Interestingly, several genes were up-regulated coordinately in response to abiotic stresses. AtGPX6, like human phospholipid hydroperoxide GPX (PHGPX), possibly encodes mitochondrial and cytosolic isoforms by alternative initiation. In addition, this gene showed the strongest responses under most abiotic stresses tested. AtGPX6::GUS analysis in transgenic Arabidopsis showed that AtGPX6 is highly expressed throughout development in most tissues, thus supporting an important role for this gene in protection against oxidative damage. The different effects of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and auxin on the expression of the genes indicate that the AtGPX family is regulated by multiple signaling pathways. Analysis of the upstream region of the AtGPX genes revealed the presence of multiple conserved motifs, and some of them resembled antioxidant-responsive elements found in plant and human promoters. The potential regulatory role of specific sequences is discussed.     

Dual regulation role of GH3.5 in salicylic acid and auxin signaling during Arabidopsis-Pseudomonas syringae interaction

Salicylic acid (SA) plays a central role in plant disease resistance, and emerging evidence indicates that auxin, an essential plant hormone in regulating plant growth and development, is involved in plant disease susceptibility. GH3.5, a member of the GH3 family of early auxin-responsive genes in Arabidopsis (Arabidopsis thaliana), encodes a protein possessing in vitro adenylation activity on both indole-3-acetic acid (IAA) and SA. Here, we show that GH3.5 acts as a bifunctional modulator in both SA and auxin signaling during pathogen infection. Overexpression of the GH3.5 gene in an activation-tagged mutant gh3.5-1D led to elevated accumulation of SA and increased expression of PR-1 in local and systemic tissues in response to avirulent pathogens. In contrast, two T-DNA insertional mutations of GH3.5 partially compromised the systemic acquired resistance associated with diminished PR-1 expression in systemic tissues. The gh3.5-1D mutant also accumulated high levels of free IAA after pathogen infection and impaired different resistance-gene-mediated resistance, which was also observed in the GH3.6 activation-tagged mutant dfl1-D that impacted the auxin pathway, indicating an important role of GH3.5/GH3.6 in disease susceptibility. Furthermore, microarray analysis showed that the SA and auxin pathways were simultaneously augmented in gh3.5-1D after infection with an avirulent pathogen. The SA pathway was amplified by GH3.5 through inducing SA-responsive genes and basal defense components, whereas the auxin pathway was derepressed through up-regulating IAA biosynthesis and down-regulating auxin repressor genes. Taken together, our data reveal novel regulatory functions of GH3.5 in the plant-pathogen interaction.     

The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.     

Indole-3-acetic acid protein conjugates: novel players in auxin homeostasis

Indole-3-acetic acid (IAA) is found in plants in both free and conjugated forms. Within the group of conjugated IAA there is a unique class of proteins and peptides where IAA is attached directly to the polypeptide structure as a prosthetic group. The first gene, IAP1, encoding for a protein with IAA as a prosthetic group, was cloned from bean (Phaseolus vulgaris). It was shown that the expression of IAP1 as a major IAA modified protein in bean seed (PvIAP1) was correlated to a developmental period of rapid growth during seed development. Moreover, this protein underwent rapid degradation during germination. Since further molecular analysis was difficult in bean, the IAP1 gene was transformed into Arabidopsis thaliana and Medicago truncatula. Expression of the bean IAP1 gene in both plant species under the control of its native promoter targeted protein expression to the seeds. In Arabidopsis no IAA was found to be attached to PvIAP1. These results show that there is specificity to protein modification by IAA and suggests that protein conjugation may be catalyzed by species specific enzymes. Furthermore, subcellular localization showed that in Arabidopsis PvIAP1 was predominantly associated with the microsomal fraction. In addition, a related protein and several smaller peptides that are conjugated to IAA were identified in Arabidopsis. Further research on this novel class of proteins from Arabidopsis will both advance our knowledge of IAA proteins and explore aspects of auxin homeostasis that were not fully revealed by studies of free IAA and lower molecular weight conjugates.     

NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles. In this study, we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling. Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA signaling. Analysis of the Arabidopsis mutant npr1, which is impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for the suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.     

机械伤害诱导的植物防御机制和信号转导

茉莉酸是植物伤反应的特异激素, 在植物伤反应中具有核心作用, 其下游调控机制已经比较清晰。在番茄(Lycopersicon esculentum)伤反应中, 系统素和茉莉酸协同启动相关基因的表达, 行使系统性防御功能。拟南芥(Arabidopsis thaliana)信号肽是新发现的一类信号物质, 可以激活植物的初始免疫反应, 但其在伤反应中的作用机制有待进一步研究。脱落酸位于茉莉酸上游, 单独或者协同茉莉酸参与植物的防御反应。另外, 植物中还存在以核糖核酸酶为代表的且不依赖于茉莉酸的伤反应信号转导途径。该文对植物伤反应的防御机制和信号转导做了详细概述。     

The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death

Salicylic acid (SA) has been proposed to antagonize jasmonic acid (JA) biosynthesis and signaling. We report, however, that in salicylate hydroxylase-expressing tobacco (Nicotiana tabacum) plants, where SA levels were reduced, JA levels were not elevated during a hypersensitive response elicited by Pseudomonas syringae pv phaseolicola. The effects of cotreatment with various concentrations of SA and JA were assessed in tobacco and Arabidopsis (Arabidopsis thaliana). These suggested that there was a transient synergistic enhancement in the expression of genes associated with either JA (PDF1.2 [defensin] and Thi1.2 [thionin]) or SA (PR1 [PR1a-beta-glucuronidase in tobacco]) signaling when both signals were applied at low (typically 10-100 microm) concentrations. Antagonism was observed at more prolonged treatment times or at higher concentrations. Similar results were also observed when adding the JA precursor, alpha-linolenic acid with SA. Synergic effects on gene expression and plant stress were NPR1- and COI1-dependent, SA- and JA-signaling components, respectively. Electrolyte leakage and Evans blue staining indicated that application of higher concentrations of SA + JA induced plant stress or death and elicited the generation of apoplastic reactive oxygen species. This was indicated by enhancement of hydrogen peroxide-responsive AoPR10-beta-glucuronidase expression, suppression of plant stress/death using catalase, and direct hydrogen peroxide measurements. Our data suggests that the outcomes of JA-SA interactions could be tailored to pathogen/pest attack by the relative concentration of each hormone.