Leucine-Rich Repeat Receptor-Like Kinases Are Important Regulators of Cold Tolerance in Sainfoin

Background Sainfoin ( Onobrychis viciifolia ) is a perennial legume that combines high protein content with bloat-preventive tannin compounds, rendering it a high-quality forage species. Nevertheless, its inadequate cold hardiness limits geographic expansion, especially in northern pastoral areas, constraining its broader agricultural adoption. Results In a three-year field trial, cultivar ‘Lanhong No.1’ (LH) showed a 15.08% higher average winter survival rate (over two winters) and a 33.84% higher average forage yield (over three years) than the locally grown cultivar ‘Gansu’ (GS). After exposure to -6℃ for 1.5 h, LH seedlings exhibited an approximately 5-fold higher survival rate than GS, along with lower ion leakage and reduced accumulation of reactive oxygen species. To elucidate the molecular basis of the cold-tolerant phenotype of LH, transcriptome analysis identified 3,330 differentially expressed genes (DEGs) in LH compared to GS under cold stress. These DEGs were functionally enriched in stress response, hormone signaling, and metabolic regulation, including 44 upregulated cold response genes. Strikingly, numerous genes encoding leucine-rich repeat receptor-like kinases (LRR-RLKs) were significantly up-regulated in LH, some of which are not homologous to components in the established plant cold stress response network. Up-regulation of four of these LRR-RLK s was verified by qPCR. Heterologous expression of each of these four genes in yeast significantly improved growth under cold stress. Likewise, transient expression in tobacco leaves strongly induced the cold-responsive marker genes NbCBF s. Conclusions Collectively, our findings suggest that four LRR-RLK s are potential key components of cold signal perception and transduction in a cold-tolerant sainfoin cultivar. This provides a mechanistic basis for improving cold tolerance in forage legumes via molecular breeding.