Abstract
Over the past decades much research focused on the biosynthesis of the plant hormone jasmonyl-isoleucine (JA-Ile). While many details about its biosynthetic pathway as well about its physiological function are established nowadays, knowledge about its catabolic fate is still scarce. Only recently, the hormonal inactivation mechanisms became a stronger research focus. Two major pathways have been proposed to inactivate JA-Ile: i) The cleavage of the jasmonyl-residue from the isoleucine moiety, a reaction that is catalyzed by specific amido-hydrolases, or ii), the sequential oxidation of the ω-end of the pentenyl side-chain. This reaction is catalyzed by specific members of the cytochrome P450 (CYP) subfamily CYP94: CYP94B1, CYP94B3 and CYP94C1. In the present study, we further investigated the oxidative fate of JA-Ile by expanding the analysis on Arabidopsis thaliana mutants, lacking only one (cyp94b1, cyp94b2, cyp94b3, cyp94c1), two (cyp94b1xcyp94b2, cyp94b1xcyp94b3, cyp94b2xcyp94b3), three (cyp94b1xcyp94b2xcyp94b3) or even four (cyp94b1xcyp94b2xcyp94b3xcyp94c1) CYP94 functionalities. The results obtained in the present study show that CYP94B1, CYP94B2, CYP94B3 and CYP94C1 are responsible for catalyzing the sequential ω-oxidation of JA-Ile in a semi-redundant manner. While CYP94B-enzymes preferentially hydroxylate JA-Ile to 12-hydroxy-JA-Ile, CYP94C1 catalyzes primarily the subsequent oxidation, yielding 12-carboxy-JA-Ile. In addition, data obtained from investigating the triple and quadruple mutants let us hypothesize that a direct oxidation of unconjugated JA to 12-hydroxy-JA is possible in planta. Using a non-targeted metabolite fingerprinting analysis, we identified unconjugated 12-carboxy-JA as novel jasmonate derivative in floral tissues. Using the same approach, we could show that deletion of CYP94-genes might not only affect JA-homeostasis but also other signaling pathways. Deletion of CYP94B1, for example, led to accumulation of metabolites that may be characteristic for plant stress responses like systemic acquired resistance. Evaluation of the in vivo function of the different CYP94-enzymes on the JA-sensitivity demonstrated that particularly CYP94B-enzymes might play an essential role for JA-response, whereas CYP94C1 might only be of minor importance.
Highlights
In plants a diverse set of different plant-specific hormones, the phytohormones, orchestrates different metabolic settings of growth, development and defense
At 2 hpw, a significantly increased JA content (2.0–2.5-fold) was detected for the triple mutant and the quadruple mutant compared to Col-0
Results from the analysis of the quadruple mutant lacking all four CYP94 functionalities indicate that besides the known and “classical” ω-oxidation of conjugated JA-Ile a “direct” ω-oxidation of its unconjugated form may occur in planta
Summary
In plants a diverse set of different plant-specific hormones, the phytohormones, orchestrates different metabolic settings of growth, development and defense. In response to wounding and necrotrophic pathogen attack, the plant hormone jasmonyl-isoleucine (JA-Ile) plays a key-role as it activates a rapid metabolic defense answer to those stress stimuli [1]. A 13S-lipoxygenase oxidizes the free 18:3(n-3) to 13S-hydroperoxy octadecatrienoic acid. In the following reaction steps this product is converted to cis(+)-12-oxo phytodienoic acid (cis(+)OPDA) by the successive action of allene-oxide synthase and allene-oxide cyclase [2]. In the peroxisome cis(+)OPDA is reduced, activated and processed by three rounds of β-oxidation yielding (+)-7-iso-JA (for reasons of simplicity this compound will be abbreviated as JA )
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