Molecular characterization and subcellular localization of salt-inducible lipid transfer proteins in rice

Abstract

Rice (Oryza sativa L.) is a salt-sensitive species. Salt stress can cause injury to plant cellular membrane. Plant lipid transfer proteins (LTPs) are abundant lipid binding proteins that are important in membrane vesicle biogenesis and trafficking, however, the biological importance of LTPs on salt-stress response in rice remains unclear. Therefore, saltresponsive rice LTPs were identified and characterized in this study. Microarray analysis showed seven genes positively regulated by salinity, including five Ltp genes (LtpII.3, LtpII.5, LtpII.6, LtpV.1, and LtpV.2) and two Ltp-like (LtpL; LtpL1 and LtpL2) genes. Amino acid alignment revealed that all these Ltp and LtpL genes contained the N-terminal signal peptide. Apart from LtpL1, all salt-inducible Ltp genes have the conserved eight cysteine residue motifs backbone. Verification of gene expression to different stimuli in rice seedlings revealed that salt-regulated Ltp genes differentially responded to drought, cold, H2O2, abscisic acid (ABA) and CaCl2. Furthermore, the expression of Ltp and LtpL genes was tissue-specifically regulated by ABA-dependent and independent pathway. In silico analysis of an 1.5-kb 5’-upstream region of these genes showed regulatory cis-elements associated with ABA, calcium, and cold/drought responses. Three LtpII subfamily genes, including LtpII.3, LtpII.5, and LtpII.6, were strictly expressed in flowers and seeds, and LtpIII.1 mRNA strongly accumulated in stem tissue. Subcellular localization analysis of LTP-DsRed fusion proteins revealed that the five LTPs and two LTPLs localized at the endoplasmic reticulum. The results provide new clues to further understand the biological functions of Ltp genes.