Oxylipins play important roles in plant defense against biotic and abiotic cues. Because all oxylipins are derived from the same pathway, we investigated how their synthesis might be regulated, focusing on two closely related cytochrome P450 enzymes designated AOS and HPL, respectively.
Oxylipins are plant secondary metabolites of widespread occurrence and ubiquitous function in plants. The group of oxylipins includes jasmonic acid (JA) and its derivatives, which play important roles in plant stress responses. A number of key steps in JA biosynthesis occur in chloroplasts. Within the chloroplast, the metabolic pathway to JA is, however, not linear but rather contains a number of metabolic branching points, where different enzymes compete for substrates. Among those, the competition of two closely related atypical cytochrome P450 enzymes designated CYP74A (allene oxide synthase, AOS) and CYP74B (hydroperoxide lyase, HPL), respectively, for the reaction intermediate 13-HPOT has to be highlighted. At this particular branching point, it is decided in which proportions the stress hormone JA is synthesized via the Vick-Zimmerman pathway and volatile cis-3-hexanal and its derivatives are produced as herbivore deterrent, respectively. Intriguingly, plants can direct the metabolic flux nearly exclusively into JA, when they are wounded by herbivores, which implies an effective and tight control of the metabolic flux. In this context, the intimate regulation of the competing biochemical reactions is of greatest importance, in order to facilitate adequate plant responses to the biotic stress stimulus.
In collaboration with scientists from France, Germany and the USA, CBGP researcher have taken diverse biochemical and genetic approaches to investigate how the tight regulation of the metabolic flux is achieved in the model plant Arabidopsis thaliana. The obtained data revealed that both enzymes, AOS and HPL, are imported into chloroplasts. While AOS can be found associated to the inner chloroplast membrane, HPL was localized in the outer envelope fraction. Apart from the different localization of AOS and HPL, the researchers were also able to demonstrate that AOS forms higher molecular weight complexes with LOX2 and AOC2. The association of LOX2, AOS, and AOC2 facilitates effective substrate channeling, converting linolenic acid (LeA) into 12-oxo-phytodienoic acid (OPDA) without the liberation of the intermediate 13-HPOT. On the basis of their results, they concluded that the formation of OPDA from LeA is strictly compartmentalised, in order to prevent competing side reactions, such as that from 13-HPOT catalysed by HPL or by chemical decay. Taking the obtained stoichiometric data into account, and using available structural data for LOX, AOS, and AOC enzymes, the authors were also able to propose a 3D model of the LOX2-AOS-AOC2 interaction in the chloroplast inner envelope.
This work provides important new insight into a highly specific cellular mechanism for controlling the flow of metabolites through key regulatory pathways and preventing unfavorable competing reactions by the compartmentalisation of enzymes as well as organization into multi-protein complexes.
Pollmann, S., Springer, A., Rustgi, S., Wettstein, D. von, Kang, C., Reinbothe, C., Reinbothe, S. 2019. Substrate channeling in oxylipin biosynthesis through a protein complex in the plastid envelope of Arabidopsis thaliana. Journal of Experimental Botany erz015. DOI: 10.1093/jxb/erz015