ClRbx1 as a component of the SCF complex in the regulation of self-incompatibility and salt stress response in 'Xiangshui' lemon.

RING box Protein 1 (Rbx1) is a RING-domain protein that serves as the catalytic core of the SKP1-Cullin1-F-box (SCF) ubiquitin ligase complex. It is implicated in the ubiquitination process during plant self-incompatibility, growth, and abiotic stress responses. Two Arabidopsis Rbx1 homologous genes, designated ClRbx1-1 and ClRbx1-2, were identified in the genome of 'Xiangshui' lemon. Both ClRbx1 genes are highly expressed in pollen, furthermore, their expression was generally upregulated under self-pollination compared to cross-pollination conditions. Additionally, under simulated salt stress, the expression of ClRbx1 in leaves increased overall. The result of yeast two-hybrid showed that ClRbx1-1 could interact with several proteins associated with self-incompatibility and stress-responses, including Cullin1-2, S1-RNase, S3-RNase, Ubc12, AP1M2, and Peroxidase25. Yeast three-hybrid assays further demonstrated that ClRbx1-1 interacts with both Cullin1-2 and SKP1-6/SKP1-14 to assemble a canonical SCF complex. Notably, Ubc12 is highly expressed in pollen and its expression level is higher under self-pollination than under cross-pollination. This suggests that ClRbx1-1, cooperating with Ubc12, might function to regulate the self-incompatibility response via the RUB (related to ubiquitin) modification pathway. Overexpression of ClRbx1 in Arabidopsis resulted in dwarfism, and significantly improved salt stress tolerance. Overexpression of ClRbx1 in 'Xiangshui' lemon, achieved via Agrobacterium rhizogenes mediated hairy root transformation, led to a significant increase in salt stress tolerance. In summary, this study collectively elucidates the critical functions of ClRbx1 in self-incompatibility and salt stress response in 'Xiangshui' lemon, laying a solid foundation for further dissection of the molecular mechanisms by which the SCF complex mediates self-incompatibility and abiotic stress in plants.