Substrate channeling in oxylipin biosynthesis through a protein complex in the plastid envelope of Arabidopsis thaliana

Abstract

ABSTRACTOxygenated membrane fatty acid derivatives dubbed oxylipins play important roles in the plant’s defense against biotic and abiotic cues. Plants challenged by insect pests, for example, synthesize a blend of different defense compounds that, amongst others, comprise volatile aldehydes and jasmonic acid (JA). Because all oxylipins are derived from the same pathway, we asked how their synthesis might be regulated and focused on two closely related, atypical cytochrome P450 enzymes designated CYP74A and CYP74B, i.e., allene oxide synthase (AOS) and hydroperoxide lyase (HPL). Both enzymes compete for the same substrate but give rise to different products. While the final product of the AOS branch is JA, those of the HPL branch comprise volatile aldehydes and alcohols. AOS and HPL are plastid envelope enzymes in Arabidopsis thaliana but accumulate at different locations. Biochemical experiments identified AOS as constituent of complexes also containing lipoxygenase 2 (LOX2) and allene oxide cyclase (AOC), which catalyze consecutive steps in JA precursor biosynthesis, while excluding the concurrent HPL reaction. Based on published X-ray data, the structure of this complex could be modelled and amino acids involved in catalysis and subunit interactions identified. Genetic studies identified the microRNA 319 (miR319)-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes and CORONATINE INSENSITIVE 1 (COI1) to control JA production through the AOS-LOX2-AOC2 complex. Together, our results define a molecular branch point in oxylipin biosynthesis that allows fine-tuning the plant’s defense machinery in response to biotic and abiotic stimuli.