Characterization of acyl coenzyme a binding protein 4 in rice

Abstract

Acyl-CoA binding proteins (ACBPs) are a group of proteins conserved at the acyl-CoA binding (ACB) domain which bind long-chain acyl-CoA esters. In Arabidopsis thaliana, six ACBP (AtACBPs) have been reported to participate in various activities. In particular, the expression of AtACBP2 is induced by drought stress, and overexpression of AtACBP2 in Arabidopsis enhances drought tolerance to the plants. In Oryza sativa (rice), six ACBPs (OsACBPs) have also been identified but their functions remain largely unknown. OsACBP4 is a homolog of AtACBP2, and the stress response mechanism in rice is an important and heated research ground. This study aims to investigate the roles of OsACBP4 in drought stress response and explore the effect of AtACBP2 heterologously expressed in rice. In this study, transgenic rice overexpressing AtACBP2, OsACBP4, or RNA-interfering OsACBP4 were generated and characterized. Histochemical assay on the OsACBP4pro:GUS lines revealed that, in line with the microarray data, the expression of OsACBP4 is high in endosperm and modest in leaf. On drought stress test, the OsACBP4-OE and AtACBP2-OE rice lines displayed enhanced recovery than the control lines. The OE lines also exhibited hypersensitivity to exogenous ABA at the post-germination stage and showed reduced water loss rate, suggesting that OsACBP4 is likely to participate in the ABA-dependent stress signaling and regulation of stomatal closure. EMSA assay identified one MYC-box (CANNTG) in the promoter region of OsACBP4 involved in drought response, indicating the interaction of OsACBP4 with some drought-responsive trans-acting factor. Altogether, OsACBP4 is positively involved in drought response, which provides potential application in agriculture for enhancing drought tolerance. In ITC binding assay with CoAs, OsACBP4 recombinant protein had a prefered and high binding affinity towards linolenoyl-CoA (18:3, Kd = 1.55 μm), which is the precursor involved in the repair of the phospholipid membrane. An optimized crystallization condition for OsACBP4 was screened out [1.2 M lithium sulfate monohydrate and 0.1 M HEPES sodium (pH 7.5)], and OsACBP4 crystals appear in the form of dimer. The osacbp mutants were also identified and characterized. All the osacbp mutants except for osacbp4 had a significant reduced growth rate after germination, suggesting that the OsACBP genes are involved in the early development of rice.