Elucidating the role of jasmonate metabolites in jasmonic acid signaling

Abstract

The plant hormone jasmonic acid controls immune responses against insect herbivory and regulates plant development. Jasmonic acid is synthesized upon wounding and herbivory and is converted to many derivatives collectively termed as jasmonate (JA). Jasmonoyl-isoleucine (JA-Ile), among the JA is the bioactive form that signals through a nuclear residing co-receptor complex via direct binding to the receptor and subsequent activation of downstream gene expression. Although the JA biosynthetic pathway is well known with almost all the biosynthetic enzymes identified and characterized in detail, the catabolic pathways leading to a diverse array of JA metabolites has only begun to be studied in recent years. One of those pathways catalyzed by the Arabidopsis cytochrome P450 enzymes CYP94B1 (B1) and CYP94B3 (B3) oxidizes JA-Ile to 12-hydroxy (OH)-JA-Ile. Further oxidation of 12OH-JA-Ile to 12-carboxy (COOH)-JA-Ile is carried out by CYP94C1 (C1) in the same CYP94 family. In a second pathway, the amidohydrolases ILL6 and IAR3 cleave JA-Ile at the amide bond into free jasmonic acid and -Ile. In contrast to the well-established function of JA-Ile in mediating JA-dependent responses, biological functions of other JA metabolites are less clear. Identification of additional metabolic pathways of JA and respective mutants blocked in such pathways are providing a unique opportunity to study the homeostatic regulation of JA and its biological function in plant stress responses. Two chapters of this thesis presents the physiological and signaling function of oxidized JA-Ile in plant growth and defense responses. The last chapter discusses a screening work aimed at identifying the novel enzyme group involved in JA metabolism. Collectively, the results extend our understanding of JA metabolism in plant growth and defense responses. Chapter II presents a mechanism for the inactivation of the JA pathway and its relationship with plant response to wounding. Arabidopsis cytochrome P450 enzymes in the CYP94 clade metabolize JA-Ile, a major isoform of JA responsible for many biological effects attributed to the JA signaling pathway; thus, they are expected to contribute to the attenuation of JA-dependent wound responses. To directly test this, we created 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 hyper-accumulate in the wounded leaves. Surprisingly, over accumulation of JA-Ile in the mutants did not result in a stronger wound response, but instead displayed a series of symptoms reminiscent of JA-Ile deficiency, including increased susceptibility to insect. The mutants responded normally to exogenous JA treatments, indicating that JA-perception or signaling pathways were intact. Untargeted metabolite analyses revealed a global reduction in wound-induced metabolite production in the mutants consistent with the dampened wound-response and increased susceptibility to insects. These observations raise questions about the current JA-signaling model and point toward a more complex model perhaps involving other JA-Ile derivatives and/or feedback mechanisms. Chapter III focuses on the signaling function of 12OH-JA-Ile which is the first metabolite in the JA-Ile signal catabolic pathway. Contrary to the widely held belief that 12OH-JA-Ile is largely an inactive signal, genetic analyses in chapter II, provided indirect evidence that 12OH-JA-Ile may function as an active signal. Consistently, Arabidopsis seedlings treated with 12OH-JA-Ile accumulated anthocyanin and were also increased in leaf trichome cell numbers to levels comparable to that induced by the same concentration of JA-Ile. Both anthocyanin and trichomes are anti-herbivory features known to be regulated by JA-Ile. In addition, expression of several JA-Ile responsive marker genes was upregulated by 12OH-JA-Ile. Genome-wide transcript analyses and untargeted metabolomics experiments showed that 12OH-JA-Ile could mimic a significant part of JA-Ile effects both at the transcriptional and metabolic levels. Mutation in CORONATINE INSENSITIVE 1 (COI1) blocked 12OH-JA-Ile effects on anthocyanin and trichome induction, indicating that 12OH-JA-Ile signals through the common receptor and signaling mechanism as JA-Ile. 12OH-JA-Ile was able to trigger anthocyanin accumulation in tomato seedlings in a COI1-dependent manner indicating that the 12OH-JA-Ile signaling system is likely to be conserved in eudicots. Increased endogenous 12OH-JA-Ile levels in a double T-DNA insertion mutant blocked in 12OH-JA-Ile hydrolysis, ill6iar3, and decreased 12OH-JA-Ile either by triggering hydrolysis or oxidation of 12OH-JA-Ile in transgenic line ILL6-OE or CYP94C1-OE displayed phenotypes proportionate with endogenous 12OH-JA-Ile, indicative of JA-Ile-like signaling in wounded plants. Together, these results show that 12OH-JA-Ile likely plays a prominent role in the JA-regulated wound response in plants than previously thought. Chapter IV presents a genetic screening approach aimed at identifying novel genes in JA metabolism. JA-resistant phenotypes have been observed in most transgenic lines overexpressing catabolic enzymes of the JA pathway. Screening of a mutant population randomly overexpressing Arabidopsis genes was hypothesized to reveal other catabolic enzymes. Such a transgenic mutant population generated by Ichikawa and colleagues (2006) was screened for resistance to JA-inhibited root elongation and an altered JA-Ile profile in wounded leaves. Twenty two candidates were selected and the identity of cDNAs potentially responsible for the phenotypes in ten of those candidates was determined by PCR and DNA sequencing. A candidate gene encoding UDP-glucosyltransferase 86A1 (UGT86A1) and its close homolog UGT86A2 were further characterized. Transgenic lines overexpressing UGT86A1 or UGT86A2 displayed reduced JA-Ile and increased 12-O-glucosyl-jasmonic acid levels in wounded leaves. T-DNA insertion mutants, ugt86a1 and ugt86a2, however, did not show JA profiles opposite to the overexpressing lines and a preliminary in vitro enzyme assay using purified glutathione S-transferase tagged UGT86A1 and UGT86A2 proteins failed to detect clear glucosyltransferase activities. Potential reasons for the discrepancies and future directions are discussed.