A multifunctional ANL superfamily gene OsACS couples oxylipin and phenylpropanoid metabolism

Introduction The ANL (acyl-CoA synthetase/NRPS adenylation domain-luciferase) superfamily catalyzes adenylation and thioesterification. ACS (acyl-CoA synthetase) enzymes activate fatty acids, but their roles in coordinating distinct pathways remain unclear. The jasmonic acid (JA) pathway is part of the oxylipin network, where OPCL (OPC-8:0 CoA ligase) converts OPC (12-oxophytodienoic acid) to OPC‑CoA. Meanwhile, 4CL (4-coumarate:CoA ligase) channels hydroxycinnamates into phenylpropanoids (e.g., lignin). Whether JA directly regulates lignin synthesis or a shared enzyme bridges them is unknown. Here, we identified OsACS, a rice gene coupling oxylipin with lignin biosynthesis. 

Methods We performed mGWAS for 4‑coumaric acid (4-CA, a lignin precursor) in indica rice. Multi‑omics integration associated OsACS alleles with metabolite and expression traits. Recombinant OsACS was assayed in vitro with 4‑CA and OPDA. CRISPR/Cas9 knockout and overexpression lines were phenotyped under dehydration stress. Natural variants were characterized via yeast one‑hybrid, EMSA (electrophoretic mobility shift assay), and dual‑luciferase assays. 

Results mGWAS pinpointed OsACS, and phylogenetic analysis placed it close to OPCL and 4CL. OsACS localized to peroxisomes, with expression negatively correlated with phenylpropanoids but positively with jasmonates. In vitro, OsACS converted OPC and OPDA to CoA esters (OPCL activity) and accepted multiple hydroxycinnamates (4CL activity). Knockout mutants accumulated less JA land showed enhanced drought tolerance, whereas overexpressors were hypersensitive. Exogenous 4‑CA or JA rescued the phenotype. Under osmotic stress, OsACS relocalized to the cytosol and nucleus, with ERF transcription factors binding its promoter. A solo‑LTR insertion in the MH63 haplotype reduced promoter activity relative to NIP (Nipponbare).

Conclusions OsACS is a bifunctional enzyme coupling JA and lignin pathways while negatively regulating drought tolerance. Its stress‑induced relocalization may enable metabolic reprogramming. The solo‑LTR insertion offers a natural allelic variant for breeding.