Regulatory SNP in OsHKT2;1 governs OsRR22 interaction and salt tolerance in rice

Rice growth and productivity are severely impaired under salt stress caused by an excess of dissolved salts, mainly the sodium and chloride ions, in soil and irrigation water. An excessive influx of sodium ions into the plant shoot and root tissues disrupts its homeostasis, leading to cellular dysfunction. Sodium ion (Na⁺) homeostasis in rice is regulated by members of the HIGH-AFFINITY K⁺ TRANSPORTER (HKT) family, among them OsHKT2;1 is known to influx Na⁺ into the plant system under salt stress, thereby affecting its homeostasis. In this study, the crucial role of a regulatory single nucleotide polymorphism (rSNP) in the promoter motif of OsHKT2;1 in governing its interaction with B-Type RESPONSE REGULATOR 22 (OsRR22) was analyzed between the salt-susceptible cultivar (ASD16) and the salt-tolerant landrace (Pokkali). The ASD16 exhibits significant upregulation of OsHKT2;1 compared to Pokkali, and the differential expression is ascribed to an rSNP (5'- AAAT(A/T)TTCC - 3') in the regulatory motif of OsHKT2;1, viz., ASD16 harboring 'A' and Pokkali 'T', respectively. Molecular docking and MD simulation reveal that salt-susceptible rice ASD16 forms a more stable OsRR22-pOsHKT2;1 transcriptional complex compared to Pokkali; consequently, the upregulation of OsHKT2;1 in ASD16 under salt stress. CRISPR-Cas9 mediated Oshkt2;1 knock-out mutant of ASD16 shows improved seedling salt tolerance and exhibits low Na⁺ concentrations in shoot and root tissues under stress. The designated rSNP is therefore to play a significant role in regulating OsHKT2;1 expression and Na⁺ influx, resulting in a genotype-specific salt stress response function in rice, thus paving the way for crop improvement.