E3 ubiquitin ligase RING3 mediates AsWRKY44 degradation to promote wound-induced sesquiterpene biosynthesis in Aquilaria sinensis

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Agarwood, a resinous heartwood derived from injured Aquilaria plants, is widely used in cosmetics and pharmaceuticals. Sesquiterpenes are among the main active components of agarwood. Agarwood sesquiterpene synthase 1 (ASS1), a typical wound-inducible synthase, is a key sesquiterpene biosynthesis enzyme. Previously, we reported that the transcription repressor AsWRKY44 inhibits the expression of ASS1 and relieves this inhibition by degrading itself in response to treatment with MeJA (methyl jasmonate). Yet the molecular mechanisms underlying this wound-induced AsWRKY44 degradation remain unknown. Here, we demonstrate that a C3HC4-type E3 ubiquitin ligase, RING3, shows ubiquitin activity and regulates the degradation of WRKY44 in response to an exogenous MeJA treatment. AsWRKY44 protein degraded more slowly in the RING3-RNAi lines than the CK group, suggesting that RING3 contributes to AsWRKY44 protein stability. RING3 interacted with AsWRKY44 in vitro and in vivo, and RING3 ubiquitinated AsWRKY44 in vitro. Downregulation of RING3 remarkably reduced the levels of α-guaiene, humulene, and δ-guaiene, which are the main catalytic products of ASS1. These findings reveal a critical mechanism by which RING3 positively regulates sesquiterpene biosynthesis by mediating AsWRKY44 degradation in response to wound stimuli in A. sinensis. Here, we provide a novel view of the regulation of transcriptional inhibition in other medicinal plants. Our results might help elucidate plants’ direct induction of natural secondary metabolites.