The transmembrane domain of the rice small protein OsS1Fa1 is responsible for subcellular localization and drought tolerance

Abstract OsS1Fa1, a homologue of spinach S1Fa, is a small protein in rice that contains four distinct conserved motifs and participates in drought tolerance. However, the biological functions of these conserved motifs have not been characterized to date. Therefore, we investigated the roles of these conserved domains in the localization and cellular function of OsS1Fa1. We analysed the subcellular localization of OsS1Fa1 using confocal laser scanning microscopy (CLSM), following particle bombardment and bacterial infiltration. An E. coli in vivo reconstituted sumoylation assay was conducted to investigate sumoylation of OsS1Fa1. We characterized the function of the transmembrane domain of OsS1Fa1 in drought tolerance using transgenic Arabidopsis plants. Fluorescence analysis showed that OsS1Fa1 localized to the nuclear and cytoplasmic membranes. Mutation and cell fractionation analyses revealed that the membrane localization domain determined the subcellular localization of OsS1Fa1. The rice homologue OsS1Fa2 and Arabidopsis orthologs AtS1Fa1, AtS1Fa2, and AtS1Fa3 also exhibited similar localization patterns as OsS1Fa1. Sumoylation analysis demonstrated that OsS1Fa1 was conjugated with the small ubiquitin‐related modifier (SUMO). Transgenic analysis showed that overexpression of OsS1Fa1(TMm1), a mutant form of the transmembrane domain of OsS1Fa1, in Arabidopsis did not enhance drought stress tolerance, whereas OsS1Fa1 overexpression improved the drought tolerance of transgenic Arabidopsis . Our data indicate that rice and Arabidopsis S1Fa1 proteins localize in the nuclear and cytoplasmic membranes, and that transmembrane domain determines subcellular localization and plays an important role in drought stress tolerance.