OsClo5 functions as a transcriptional co-repressor by interacting with OsDi19-5 to negatively affect salt stress tolerance in rice seedlings

Caleosins constitute a small protein family with one calcium-binding EF-hand motif. They are involved in the regulation of development and response to abiotic stress in plants. Nevertheless, how they impact salt stress tolerance in rice is largely unknown. Thereby, biochemical and molecular genetic experiments were carried out, and the results revealed that OsClo5 was able to bind calcium and phospholipids in vitro and localized in the nucleus and endoplasmic reticulum in rice protoplasts. At the germination and early seedlings stages, overexpression transgenic lines and T-DNA mutant lines exhibited reduced and increased tolerance to salt stress, respectively, compared with the wild-type. Yeast two-hybrid, bimolecular fluorescence complementation and in vitro pull-down assays demonstrated that the EF-hand motif of OsClo5 was essential for the interactions with itself and OsDi19-5. Yeast one-hybrid, electrophoretic migration shift and dual-luciferase reporter assays identified OsDi19-5 as a transcriptional repressor via the TACART cis-element in the promoters of two salt stress-related target genes, OsUSP and OsMST. In addition, OsClo5 enhanced the inhibitory effect of OsDi19-5 in the tobacco transient system, which was confirmed by qRT-PCR analysis in rice seedlings under salt stress. The collective results deepen the understanding of the molecular mechanism underlying the roles of caleosin in the salt stress response. These findings will also inform efforts to improve salt tolerance of rice.