The cytosolic protein GRP1 facilitates abscisic acid- and darkness-induced stomatal closure in Salvia miltiorrhiza

By screening an expressed sequence tag (EST) library of Salvia miltiorrhiza, we detected an acidic protein, SmGRP1, with no significant similarities to the other sequences in public databases. SmGRP1 encodes a peptide of 151 amino acids, 33.77 % of which are glutamic acid residues, and the peptide was positive according to "stains-all" staining. Expression analysis revealed that SmGRP1 was expressed in all examined tissues of S. miltiorrhiza but was most highly expressed in the leaves and stems. Without a signal peptide, SmGRP1 localized to the cytoplasm in protoplasts in subcellular localization experiments. SmGRP1 expression was prominently enhanced by ABA and darkness treatments; the protein could also be induced by high temperature, NaCl, and dehydration treatments, while low temperature suppressed its expression. Furthermore, although there were no obvious phenotypic differences in SmGRP1 overexpression and SmGRP1 knockdown mutants compared with control plants under normal culture conditions, the stomata of the knockdown lines remained open when treated with ABA, darkness, NO, and H2O2. In addition, the water loss rate of the knockdown mutants was faster than that of the control lines and overexpression mutants when exposed to air. These observations indicate that SmGRP1 is a novel acidic protein with potential calcium-binding capability and is involved in stomatal movement and stress resistance.