<scp>MsCDPK29</scp> ‐ <scp>MsbZIP14</scp> modulates <scp>ABA</scp> accumulation in response to saline‐alkali stress in alfalfa

Salinization of arable land is a major adversity factor affecting crop yields. Calcium-dependent protein kinases (CDPKs/CPKs) play crucial regulatory roles in multiple stress responses. However, their functions in alfalfa and the mechanisms of CDPKs in regulating saline-alkali stress are not well elaborated. In this study, we identified MsCDPK29 as a positive regulator of saline-alkali tolerance in alfalfa. Overexpression (OE) of MsCDPK29 in alfalfa displayed tolerance to saline-alkali stress, with higher antioxidant capacity, whereas electrolyte leakage, hydrogen peroxide (H₂O₂), superoxide anions (O₂ ^-), and malondialdehyde (MDA) contents were lower than wild-type (WT). Conversely, MsCDPK29 RNA interference (RNAi) alfalfa displayed opposite phenotypes. Additionally, MsbZIP14, a basic region-leucine zipper transcription factor, was identified as a MsCDPK29-interacting protein. OE of MsbZIP14 promotes abscisic acid (ABA) accumulation and enhances saline-alkali tolerance. Meanwhile, we showed that MsbZIP14 was phosphorylated by MsCDPK29 at threonine236 (T236) and serine237 (S237) sites, and the phosphorylation is required for the biological function of MsbZIP14 in regulating ABA biosynthesis and saline-alkali stress response. MsbZIP14 directly binds to the promoter of MsNCED3 and activates its expression. Moreover, MsCDPK29 activated MsbZIP14 to enhance the expression of MsNCED3. Collectively, this study uncovered the mechanism whereby MsCDPK29 positively regulates saline-alkali tolerance in alfalfa by phosphorylation of MsbZIP14 to activate MsNCED3 expression.