Brassica rapa receptor-like cytoplasmic kinase BrRLCK1 negatively regulates freezing tolerance in transgenic Arabidopsis via the CBF pathway

Some winter rapeseed (Brassica rapa) varieties can endure extremely low temperatures (-20°C to -32°C). However, because of a lack of mutant resources, the molecular mechanisms underlying cold tolerance in B. rapa remain unclear. In this study, we identified a low-temperature-sensitive mutant receptor-like cytoplasmic kinase (RLCK), BrRLCK1, using the B. rapa--Arabidopsis (Arabidopsis thaliana) full-length cDNA-overexpressing gene hunting system mutant library. BrRLCK1, localized to the plasma membrane and retained its localization under low temperatures. Phylogenetic analysis showed that BrRLCK1 is highly conserved across six widely cultivated Brassica species, but exhibits complexity due to genome hybridization and polyploidization. Notably, β-glucuronidase activity and qRT-PCR analysis showed that B. rapa BrRLCK1 and its homologous gene BrRLCK2 were mainly expressed in the main root, shoot, and leaves, with their expression being activated by low temperatures. Transgenic Arabodipsis expressing BrRLCK1 and BrRLCK2 reduced freezing tolerance and promoted root elongation. These combined results indicated that low temperatures can activate the expression of BrRLCK1 and BrRLCK2, negatively regulating freezing tolerance via the C-repeat-binding factor (CBF) pathway.