Overexpression of rice acyl-CoA-binding protein confers salinity tolerance in transgenic plants

Abstract

Plants are continuously affected by the adverse effects from the ongoing climate change, which alter the abiotic conditions required for plant growth. In rice (Oryza sativa), a gene family encoding six members of acyl-CoA-binding proteins (ACBPs) co-exist designated as OsACBP1 to OsACBP6. These proteins have been reported to play pivotal roles in plant lipid metabolism and in biotic and abiotic stress responses. Based on its structure, rice OsACBP4, belongs to the same Class II ACBPs as AtACBP2 in Arabidopsis, containing a conserved acyl-CoA-binding domain that facilitates binding activities to various acyl-CoA esters, and ankyrin repeats that mediate protein-protein interactions. In this study, the distinct roles of rice OsACBP4 were studied in transgenic rice and Arabidopsis. Electrophoretic mobility shift assays identified four salinity-responsive elements in the rice OsACBP4 5'-flanking region which bind salt-treated rice nuclear proteins and provide a molecular basis for salt-inducible regulation of OsACBP4 expression, indicating a potential role in plant abiotic stress tolerance. High OsACBP4 expression in rice leaves and roots was confirmed by histochemical staining and quantitative β-glucuronidase (GUS) analysis of OsACBP4pro::GUS transgenic plants. When salt treatments were performed on rice, in the wild-type (WT), the vector-transformed control (VC), rice OsACBP4-overexpressors (OEs), OsACBP4-RNAi lines and AtACBP2-OEs, the OsACBP4-OEs conferred greatest protection. In addition, rice AtACBP2-OEs were more tolerant than the controls as well, but less tolerant than OsACBP4-OEs, whereas OsACBP4-RNAi lines were more sensitive than the controls. In the experiments on transgenic Arabidopsis, OsACBP4-OEs were more salt-tolerant than AtACBP2-OEs. RNA-sequencing of transgenic rice OsACBP4-OEs revealed significant upregulation of phospholipase-encoding genes involved in salt stress tolerance and genes encoding acyl-CoA synthetase (E.C. 6.2.1.3) in the fatty acid biosynthesis pathway, and which were all further confirmed by quantitative real-time polymerase chain reactions. Taken together, these results suggested that the salinity tolerance mechanism in rice OsACBP4-OEs may involve the phospholipid signaling pathway. Gas chromatography mass spectrometry (GC/MS) revealed increased levels of palmitic, stearic, linoleic and linolenic fatty acids, but no oleic acid in transgenic rice OsACBP4-OEs over the controls. Fatty acid content in OsACBP4-RNAi lines was lower than that of the controls. Inductively coupled plasma optical emission spectrometry (ICP-OE) ion content analysis revealed fewer toxic ions accumulated in stress-tolerant transgenic rice OsACBP4-OEs, followed by AtACBP2-OEs and then the controls, whereas the sensitive OsACBP4-RNAi lines accumulated the most. Antioxidant enzymes such as superoxide-dismutase (SOD), peroxidase (POD) and catalase (CAT) show higher enzyme activity in salt-tolerant transgenic rice OsACBP4-OEs when compared to the controls, whereas the activity decreased in the OsACBP4-RNAi lines. In the isothermal titration calorimetry experiments (ITC), His(6)-OsACBP4 had binding affinity for palmitoyl-CoA, stearoyl-CoA, linoleoyl-CoA and linolenoyl-CoA. Uniquely to the rice ACBPs, His(6)-OsACBP4 had ability to bind oleoyl-CoA. It is envisaged that the overexpression of OsACBP4 achieves protection against salinity stress, through its abilities to bind LC-CoAs and to interact with yet unidentified protein partners.