Mechanisms of oxidative turnover in jasmonate hormonal signaling – JASMON'OX

The project combines a number of complementary approaches, including synthetic chemistry, molecular biology, genetics, enzymology, metabolic analysis and phytopathology.
Most JA derivatives under study are not commercially available and will be prepared by the chemist partner who will implement original synthesis routes. These synthetic JA derivatives are required for important aspects of the project, namely : i) identification of novel JA derivatives in plant extracts ; ii) use as internal standards ; iii) use as substrates for candidate enzymes.
The exploration of jasmonate oxidation is based on in vitro and in planta analysis. First, candidate cytochrome P450 genes for jasmonate oxidation will be expressed in yeast and prepared as microsomes that will be incubated with potential substrates. Second, genetic analysis will be undertaken with insertion mutants in the model plant Arabidopsis thaliana. Jasmonate metabolism and signaling will be stimulated by mechanical wounding or leaf infection. The impact of jasmonate oxidation on hormone metabolism and signalisation will largely rely on comparative jasmonate profiling by liquid chromatography – mass spectrometry analysis.

A new synthetic strategy has been setup to give access to a wide array of oxidized jasmonates that are enantiomerically pure. The method is versatile and should be of general value to obtain a collection of jasmonates conjugated to a variety of aminoacids.
The project undergoes also currently the development of biochemical and genetic tools. New plant lines have been acquired and validated. Yeast-expressed CYP94 are available for enzymatic assays.

The project priority is to increase our knowledge of oxidative jasmonate catabolism, a prerequisite to understand the regulation of this class of hormones in plant development and adaptation. Our efforts will also provide methods and tools to provide original jasmonates, that can be tested for their biological properties, for example in crop protection against pathogens, or studied for anti-cancer potential, as already reported for Methyl-JA.

The first 6 months have been devoted to the generation of a number of molecular tools, that precede any scientific production.

Hormonal control is of crucial importance for plant development and their adaptation to changing environmental conditions. Molecular genetics combined with biochemical and metabolic approaches have revealed insights into the biosynthesis, transport, perception, and cellular signalling of several plant hormones. Jasmonic acid (JA) is one of such hormones, playing prominent roles in plant reproduction and their constant adaptation to environmental stresses and attacks by insects or microbial pathogens. JA is synthetized from unsaturated fatty acids and its structure is reminiscent of animal prostaglandins. Among numerous JA derivatives that were detected in plant extracts, the JA-Isoleucine (JA-Ile) conjugate has been recognized as an active endogenous ligand of the F-box receptor protein COI1. The current model for JA signalling implies that ligand binding promotes the recruitment of “JAZ” protein transcriptional repressors to be proteolytically degraded by the ubiquitin-proteasome system, resulting in the de-repression of downstream JA-responsive genes. In contrast, elucidating mechanisms for hormonal signal extinction just started to attract interest. Hydroxy- and carboxy-derivatives of JA-Ile with high potential biological significance accumulate subsequently to jasmonate signalling and we proposed that they may reflect modification of the JA-Ile hormonal activity.
We have provided recently strong biochemical and genetic evidence that these oxidized derivatives are generated upon wounding by members of the CYP94 family of cytochrome P450 enzymes, and that their formation is a major catabolic route for JA-Ile turnover. Based on our validation of this underlying hypothesis, the global objective of this proposal is to characterize the actors and processes leading to the enzymatic oxidation of jasmonates and their physiological consequences on various jasmonate-regulated responses. More specific aims include i) Characterization of the full catalytic diversity in the Arabidopsis CYP94 enzyme family in terms of oxidation of jasmonate compounds, ii) Investigation of oxidized jasmonate metabolism in newly generated cyp94 mutants upon antimicrobial defense or developmental steps, iii) Determination of the receptor-binding and signalling properties of oxidized JA-Ile derivatives, iv) Enzymatic activity and function elucidation of novel candidate genes/proteins to form unconjugated hydroxy-jasmonates. The proposed strategies should provide a deeper knowledge of the mechanisms that govern jasmonate catabolism and homeostasis for plant defense and development.