Mutation in rice protein kinase OsRRK1 confers drought tolerance via non-phosphorylating fructose-1,6-bisphosphate aldolase 6

Drought can severely restrict the productivity of rice, and thus gaining an understanding of its molecular basis is essential for developing viable stress-tolerant cultivars. In this study, to identify the key genetic regulators of drought tolerance, we screened a gamma-irradiated population of 150 rice M₁₀ core mutant lines and identified drought-insensitive TILLING line 5 (ditl5) carrying a frameshift mutation caused by a single-base guanine deletion in Rop-interacting receptor-like kinase 1 (OsRRK1). To characterize this line, we performed whole-genome re-sequencing, gene expression analysis, in vivo and in vitro protein–protein interaction assays, and protein kinase activity assays. Transgenic plants, including CRISPR/Cas9-mediated knockout, overexpression, and complementation lines, were generated to evaluate the function of OsRRK1 under drought conditions. The mutant form of OsRRK1, OsRRK1p.Gly100fs (frameshift at the 100th glycine) was found to be characterized by disruption of the phosphorylation of fructose-1,6-bisphosphate aldolase 6 (OsFBA6), resulting in upregulation of glycolytic pathway-related genes and an increase in the accumulation of soluble sugars. However, OsRRK1-knockout lines showed enhanced drought tolerance, overexpression lines were characterized by greater susceptibility, thereby confirming the regulatory role of the gene. Collectively, our findings provide evidence to indicate that OsRRK1 negatively regulates drought tolerance by modulating OsFBA6-dependent glycolysis via phosphorylation. These findings thus highlight the links between protein post-translational modifications and metabolic adaptation during periods of drought and provide a genetic resource for breeding drought-tolerant rice varieties.