Over-expression of SgRVE1 from fine-stem stylo (Stylosanthes guianensis var. intermedia) up-regulates CBF gene expression and enhances cold tolerance in Arabidopsis

Stylosanthes (stylo) species are commercially important forage and pasture legumes in tropical and subtropical regions, but their vulnerability to chilling and frost remains a challenge. The REVEILLE (RVE) proteins, known as circadian clock components, have been implicated in abiotic stress responses in several plant species. Using RNA-seq analysis, we identified SgRVE1 as a cold-responsive gene in stylos. Further quantitative Real-time PCR analysis confirmed that SgRVE1 was rapidly induced by low temperature in different tissues, including roots, leaves, stems, and flowers. Multiple sequence alignment revealed that SgRVE1 shares a SHAQKFF-class MYB domain with the CCA1 clade of the RVE family. Phylogenetic analysis demonstrated that SgRVE1 clusters within the CCA1 clade and is closely related to AtRVE1. SgRVE1 also shows high sequence identities with three Arachis RVE1 orthologs, ranging from 95 to 96%. Overexpressing SgRVE1 in Arabidopsis thaliana activated AtCBF1,2,3 gene transcription, promoted osmoprotectant accumulation and enhanced cold tolerance. A yeast one-hybrid assay suggests that SgRVE1 binds to two Evening Element (EE) motifs, EE-like (EEL: AATATCT) and Short EE (SEE: AAATATCT), which has been identified in AtCBF1,2,3 promoter sequences. This study provides the first evidence of a RVE gene in stylos and reveals its role in the transcriptional regulation of CBF genes and the cold-responsive mechanism.