Functional analysis of rice acyl-CoA-binding protein2 in seed development

Abstract

As rice seeds provide food for more than three billion people worldwide, the identification of rice genes that enhance grain size and composition is needed to satisfy the demands of a growing global population. Acyl-CoA-binding proteins (ACBPs) are a family of proteins that facilitate the binding of long-chain acyl-CoA esters at a conserved acyl-CoA-binding domain. ACBPs act to form intracellular acyl-CoA pools, transport acyl-CoA esters and regulate lipid metabolism. Earlier reports have indicated that Arabidopsis thaliana ACBPs are important in seed development but they do not seem to influence seed size. In Oryza sativa (rice), six ACBPs (OsACBPs) have also been identified but, unlike in Arabidopsis thaliana, they are not as well-characterized. Oryza sativa (rice) ACBP2 is demonstrated here to be not only important in seed development and germination, but also for enhancing grain size. OsACBP2 mRNA accumulated in embryos and endosperm of germinating seeds in qRT-PCR analysis, while β-glucuronidase (GUS) assays on OsACBP2pro::GUS rice transformants showed GUS expression in embryos as well as the scutellum and aleurone layer of germinating seeds. Deletion analysis of the OsACBP2 5'-flanking region revealed five copies of the seed-specific cis-element, Skn-I-like motif (-1486/-1482, -956/-952, -939/-935, -826/-822 and -766/-762), and the removal of any adversely affected expression in seeds. When OsACBP2 function was investigated using osacbp2 mutants and transgenic rice overexpressing OsACBP2 (OsACBP2-OE), osacbp2 was retarded during germination, while OsACBP2-OEs were enhanced in seedling growth, grain size and seed weight. Transmission electron microscopy detected the accumulation of oil and protein bodies in the OsACBP2-OE scutellum cells, while lipid profiling using GC-MS analysis of mature OsACBP2-OE seeds revealed gains in triacylglycerols and C18:1- and C18:2-fatty acids over the vector-transformed control. As dietary rice bran contains beneficial bioactive components and rice embryo globulins accumulated in OsACBP2-OEs, OsACBP2 is a promising candidate for enriching seed nutritional value. To further understand its role in lipid metabolism, the crystal structure of OsACBP2 was elucidated and its affinities toward acyl-CoA esters were examined using isothermal titration calorimetry. OsACBP2 structure resembles human liver ACBP, and consists of four helices: Helix 1 (H1) extents from residues 1 to 13; H2 from residues 23 to 27; H3 from residues 51 to 62; and H4 from residues 68-86. OsACBP2 was predicted to possess a deep (depth 8-9 Å) and narrow (9-10 Å) groove for acyl-CoA esters, which consist of the middle of Helix 1 (H1) to the middle of the H1-H2 loop (residues 7-19), the start to the middle of H2 (residues 23-33) and the start to the middle of H3 (residues 51-55). Isothermal titration calorimetry (ITC) experiments revealed that OsACBP2 preferred C18:2- and C18:3-CoAs, which offers an explanation for the accumulation in OsACBP2-OE embryos of C18:1- and C18:2-fatty acids over controls.