Overaccumulation of γ-Glutamylcysteine in a Jasmonate-Hypersensitive Arabidopsis Mutant Causes Jasmonate-Dependent Growth Inhibition.

Glutathione (GSH) is essential for many aspects of plant biology and is associated with jasmonate signaling in stress responses. We characterized an Arabidopsis (Arabidopsis thaliana) jasmonate-hypersensitive mutant (jah2) with seedling root growth 100-fold more sensitive to inhibition by the hormone jasmonyl-isoleucine than the wild type. Genetic mapping and genome sequencing determined that the mutation is in intron 6 of GLUTATHIONE SYNTHETASE2, encoding the enzyme that converts γ-glutamylcysteine (γ-EC) to GSH. The level of GSH in jah2 was 71% of the wild type, while the phytoalexin-deficient2-1 (pad2-1) mutant, defective in GSH1 and having only 27% of wild-type GSH level, was not jasmonate hypersensitive. Growth defects for jah2, but not pad2, were also seen in plants grown to maturity. Surprisingly, all phenotypes in the jah2 pad2-1 double mutant were weaker than in jah2. Quantification of γ-EC indicated these defects result from hyperaccumulation of this GSH precursor by 294- and 65-fold in jah2 and the double mutant, respectively. γ-EC reportedly partially substitutes for loss of GSH, but growth inhibition seen here was likely not due to an excess of total glutathione plus γ-EC because their sum in jah2 pad2-1 was only 16% greater than in the wild type. Further, the jah2 phenotypes were lost in a jasmonic acid biosynthesis mutant background, indicating the effect of γ-EC is mediated through jasmonate signaling and not as a direct result of perturbed redox status.

BRAHMA ATPase of the SWI/SNF chromatin remodeling complex acts as a positive regulator of gibberellin-mediated responses in arabidopsis.

SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana) mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA) are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM) ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways.

RSOsPR10 expression in response to environmental stresses is regulated antagonistically by jasmonate/ethylene and salicylic acid signaling pathways in rice roots.

Plant roots play important roles not only in the absorption of water and nutrients, but also in stress tolerance. Previously, we identified RSOsPR10 as a root-specific pathogenesis-related (PR) protein induced by drought and salt treatments in rice. Transcripts and proteins of RSOsPR10 were strongly induced by jasmonate (JA) and the ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid (ACC), while salicylic acid (SA) almost completely suppressed these inductions. Immunohistochemical analyses showed that RSOsPR10 strongly accumulated in cortex cells surrounding the vascular system of roots, and this accumulation was also suppressed when SA was applied simultaneously with stress or hormone treatments. In the JA-deficient mutant hebiba, RSOsPR10 expression was up-regulated by NaCl, wounding, drought and exogenous application of JA. This suggested the involvement of a signal transduction pathway that integrates JA and ET signals in plant defense responses. Expression of OsERF1, a transcription factor in the JA/ET pathway, was induced earlier than that of RSOsPR10 after salt, JA and ACC treatments. Simultaneous SA treatment strongly inhibited the induction of RSOsPR10 expression and, to a lesser extent, induction of OsERF1 expression. These results suggest that JA/ET and SA pathways function in the stress-responsive induction of RSOsPR10, and that OsERF1 may be one of the transcriptional factors in the JA/ET pathway.

12-oxo-phytodienoic acid-glutathione conjugate is transported into the vacuole in Arabidopsis.

While exogenous toxic compounds such as herbicides are thought to be sequestered into vacuoles in the form of glutathione (GSH) conjugates, little is understood about natural plant products conjugated with GSH. To identify natural products conjugated with GSH in plants, metabolites in the Arabidopsis γ-glutamyl transpeptidase (ggt) 4 knockout mutants that are blocked in the degradation of GSH conjugates in the vacuole were compared with those in wild-type plants. Among the metabolites identified, one was confirmed to be the 12-oxo-phytodienoic acid (OPDA)-GSH conjugate, indicating that OPDA, a precursor of jasmonic acid (JA), is transported into the vacuole as a GSH conjugate.

Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis.

Auxins are hormones that regulate many aspects of plant growth and development. The main plant auxin is indole-3-acetic acid (IAA), whose biosynthetic pathway is not fully understood. Indole-3-acetaldoxime (IAOx) has been proposed to be a key intermediate in the synthesis of IAA and several other indolic compounds. Genetic studies of IAA biosynthesis in Arabidopsis have suggested that 2 distinct pathways involving the CYP79B or YUCCA (YUC) genes may contribute to IAOx synthesis and that several pathways are also involved in the conversion of IAOx to IAA. Here we report the biochemical dissection of IAOx biosynthesis and metabolism in plants by analyzing IAA biosynthesis intermediates. We demonstrated that the majority of IAOx is produced by CYP79B genes in Arabidopsis because IAOx production was abolished in CYP79B-deficient mutants. IAOx was not detected from rice, maize, and tobacco, which do not have apparent CYP79B orthologues. IAOx levels were not significantly altered in the yuc1 yuc2 yuc4 yuc6 quadruple mutants, suggesting that the YUC gene family probably does not contribute to IAOx synthesis. We determined the pathway for conversion of IAOx to IAA by identifying 2 likely intermediates, indole-3-acetamide (IAM) and indole-3-acetonitrile (IAN), in Arabidopsis. When (13)C(6)-labeled IAOx was fed to CYP79B-deficient mutants, (13)C(6) atoms were efficiently incorporated to IAM, IAN, and IAA. This biochemical evidence indicates that IAOx-dependent IAA biosynthesis, which involves IAM and IAN as intermediates, is not a common but a species-specific pathway in plants; thus IAA biosynthesis may differ among plant species.

Antagonistic interaction between systemic acquired resistance and the abscisic acid-mediated abiotic stress response in Arabidopsis.

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR development in dicotyledonous plants, such as tobacco (Nicotiana tabacum) and Arabidopsis thaliana, is mediated by salicylic acid (SA). Here, using two types of SAR-inducing chemicals, 1,2-benzisothiazol-3(2H)-one1,1-dioxide and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, which act upstream and downstream of SA in the SAR signaling pathway, respectively, we show that treatment with abscisic acid (ABA) suppresses the induction of SAR in Arabidopsis. In an analysis using several mutants in combination with these chemicals, treatment with ABA suppressed SAR induction by inhibiting the pathway both upstream and downstream of SA, independently of the jasmonic acid/ethylene-mediated signaling pathway. Suppression of SAR induction by the NaCl-activated environmental stress response proved to be ABA dependent. Conversely, the activation of SAR suppressed the expression of ABA biosynthesis-related and ABA-responsive genes, in which the NPR1 protein or signaling downstream of NPR1 appears to contribute. Therefore, our data have revealed that antagonistic crosstalk occurs at multiple steps between the SA-mediated signaling of SAR induction and the ABA-mediated signaling of environmental stress responses.

Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8'-hydroxylase in rice.

A rapid decrease of the plant hormone ABA under submergence is thought to be a prerequisite for the enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA), an oxidized form of ABA, increased with decreasing ABA level during submergence. The oxidation of ABA to PA is catalyzed by ABA 8'-hydroxylase, which is possibly encoded by three genes (OsABA8ox1, -2 and -3) in rice. The ABA 8'-hydroxylase activity was confirmed in microsomes from yeast expressing OsABA8ox1. OsABA8ox1-green fluorescent protein (GFP) fusion protein in onion cells was localized to the endoplasmic reticulum. The mRNA level of OsABA8ox1, but not the mRNA levels of other OsABA8ox genes, increased dramatically within 1 h after submergence. On the other hand, the mRNA levels of genes involved in ABA biosynthesis (OsZEP and OsNCEDs) decreased after 1-2 h of submergence. Treatment of aerobic seedlings with ethylene and its precursor, 1-aminocyclopropane-1-carboxylate (ACC), rapidly induced the expression of OsABA8ox1, but the ethylene treatment did not strongly affect the expression of ABA biosynthetic genes. Moreover, pre-treatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, partially suppressed induction of OsABA8ox1 expression under submergence. The ABA level was found to be negatively correlated with OsABA8ox1 expression under ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA levels in submerged rice shoots is controlled partly by ethylene-induced expression of OsABA8ox1 and partly by ethylene-independent suppression of genes involved in the biosynthesis of ABA.

Methylation of gibberellins by Arabidopsis GAMT1 and GAMT2.

Arabidopsis thaliana GAMT1 and GAMT2 encode enzymes that catalyze formation of the methyl esters of gibberellins (GAs). Ectopic expression of GAMT1 or GAMT2 in Arabidopsis, tobacco (Nicotiana tabacum), and petunia (Petunia hybrida) resulted in plants with GA deficiency and typical GA deficiency phenotypes, such as dwarfism and reduced fertility. GAMT1 and GAMT2 are both expressed mainly in whole siliques (including seeds), with peak transcript levels from the middle until the end of silique development. Within whole siliques, GAMT2 was previously shown to be expressed mostly in developing seeds, and we show here that GAMT1 expression is also localized mostly to seed, suggesting a role in seed development. Siliques of null single GAMT1 and GAMT2 mutants accumulated high levels of various GAs, with particularly high levels of GA(1) in the double mutant. Methylated GAs were not detected in wild-type siliques, suggesting that methylation of GAs by GAMT1 and GAMT2 serves to deactivate GAs and initiate their degradation as the seeds mature. Seeds of homozygous GAMT1 and GAMT2 null mutants showed reduced inhibition of germination, compared with the wild type, when placed on plates containing the GA biosynthesis inhibitor ancymidol, with the double mutant showing the least inhibition. These results suggest that the mature mutant seeds contained higher levels of active GAs than wild-type seeds.

A mammalian steroid action inhibitor spironolactone retards plant growth by inhibition of brassinosteroid action and induces light-induced gene expression in the dark.

We screened steroid derivatives and found that spironolactone, an inhibitor of both 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and aldosterone receptor, is an inhibitor of phytohormone brassinosteroid (BR) action in plants. Under both dark and light growing conditions, spironolactone induced morphological changes in Arabidopsis, characteristic of brassinosteroid-deficient mutants. Spironolactone-treated plants were also nearly restored to the wild-type phenotype by treatment with additional BRs. In the spironolactone-treated Arabidopsis, the CPD gene in the BR biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR-deficiency. Spironolactone-treated tobacco plants grown in the dark showed expression of light-regulated genes as was observed in the deficient mutant. These data suggest that spironolactone inhibits brassinosteroid action probably due to the blockage of biosynthesis and exerts its activity against plants. Thus, spironolactone, in conjunction with brassinosteroid-deficient mutants, can be used to clarify the function of BRs in plants and characterize mutants. The spironolactone action site was also investigated by feeding BR biosynthesis intermediates to Arabidopsis grown in the dark, and the results are discussed.