Functional Analysis of Rice Long-Chain Acyl-CoA Synthetase 9 (OsLACS9) in the Chloroplast Envelope Membrane

The long-chain acyl-CoA synthetases (LACSs) are involved in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. These enzymes catalyze the critical reaction of fatty acyl chains to fatty acyl-CoAs for the triacylglycerol biosynthesis used as carbon and energy reserves. In Arabidopsis, LACSs are encoded by a family of nine genes, with LACS9 being the only member located in the chloroplast envelope membrane. However, the comprehensive role of LACS9 and its contribution to plant metabolism have not been explored thoroughly. In this study, we report on the identification and characterization of LACS9 mutants in rice plants. Our results indicate that the loss-of-function mutations in OsLACS9 affect the architecture of internodes resulting in dwarf plants with large starch granules in the chloroplast, showing the suppression of starch degradation. Moreover, the plastid localization of α-amylase I-1 (AmyI-1)-a key enzyme involved in starch breakdown in plastids-was suppressed in the lacs9 mutant line. Immunological and confocal laser scanning microscopy analyses showed that OsLACS9-GFP is located in the chloroplast envelope in green tissue. Microscopic analysis showed that OsLACS9s interact with each other in the plastid envelope membrane. Furthermore, OsLACS9 is also one of the proteins transported to plastids without a transit peptide or involvement of the Toc/Tic complex system. To identify the plastid-targeting signal of OsLACS9, the transient expression and localization of a series of N-terminal truncated OsLACS9-green fluorescent protein (GFP) fusion proteins were examined. Truncation analyses identified the N-terminal 30 amino acid residues to be required for OsLACS9 plastid localization. Overall, the data in this study provide an advanced understanding of the function of OsLACS9 and its role in starch degradation and plant growth.