REGULATOR OF FATTY ACID SYNTHESIS proteins regulate de novo fatty acid synthesis by modulating hetACCase distribution

In plants, heteromeric acetyl-CoA carboxylase (hetACCase) initiates de novo fatty acid synthesis (FAS) by generating malonyl-CoA in the first committed step of this process. hetACCase activity is precisely regulated to meet the cellular demand for acyl chains during the plant life cycle. In this study, we performed a systematic coexpression analysis of hetACCase and its regulators in Arabidopsis (Arabidopsis thaliana) to better understand the regulatory mechanism of hetACCase. Our analysis uncovered REGULATOR OF FATTY ACID SYNTHESIS 1 (RFS1), whose expression is positively correlated with that of other regulators of hetACCase. The RFS gene family encodes two plastid inner envelope membrane proteins with undiscovered roles. Further analysis revealed that RFS1 colocalizes and directly interacts with CARBOXYLTRANSFERASE INTERACTOR 1 (CTI1). CRISPR/Cas9-mediated knockouts of RFSs exhibit enhanced hetACCase activity, higher FAS rates, and increased fatty acid contents, with particularly marked accumulation of absolute triacylglycerol levels in leaves, similar to cti mutants. The mutations of rfs and cti alter the plastid membrane distribution pattern of α-CT, leading to reduced hetACCase activity on the membrane, which could potentially be the original mechanism through which RFSs restrain hetACCase activity. Thus, we reveal a unique regulatory module that regulates de novo FAS and a genetic locus that may contribute to breeding of improved oil crops.