PpCML30-PpWRKY11 module regulated PpOAT expression and proline accumulation in H2S-induced cold tolerance in peach fruit

Proline accumulation in peach fruit against chilling injury (CI) was promoted after hydrogen sulfide (H 2 S) treatment, whereas the underlying mechanisms remain to be deciphered. Here, H 2 S application up-regulated the proline synthesis-related genes, and further investigation implied that an ornithine δ-aminotransferase ( OAT )-encoding gene ( PpOAT ) served as a critical gene for H 2 S-induced proline accumulation in peaches. We identified 38 calmodulin-like proteins ( PpCMLs ) genes and screened for H 2 S-inducible PpCMLs and PpWRKYs . PpWRKY11, a group Ⅱ WRKY protein, has a single WRKY DNA-binding domain (DBD) responsible for W-box recognition. The Mantel test suggested that PpWRKY11 expression was strongly correlated with both proline accumulation and CI symptoms in peaches. PpCML30 interacted with PpWRKY11 in the nucleus to form the PpCML30-PpWRKY11 complex. Albeit PpWRKY11 individuals might not activate PpOAT transcription directly, upon co-expression with PpCML30, according to the dual-luciferase reporter (DLR) assay and fluorescent signaling detection. Molecular docking indicates that the globular structure of PpCML30 forms hydrogen bonds with the WRKYGQK motif of PpWRKY11 DBD, perhaps enhancing the DNA-binding ability of PpWRKY11 for the W-box. OAT activity and proline content were higher in tobacco leaves that transiently co-expressed PpCML30 and PpWRKY11 than in leaves expressing PpCML30 or PpWRKY11 alone. Our findings uncover the PpCML30-PpWRKY11 module that orchestrates the regulation of PpOAT expression and proline synthesis in H 2 S-mitigated CI in peach fruit. • Identification of CML family genes in peach. • H 2 S treatment affects the expression of PpCMLs and PpWRKYs during fruit CI. • The PpCML30-PpWRKY11 complex promotes PpOAT transcription and proline production. • The PpCML30-PpWRKY11 promotes proline biosynthesis for peach fruit cold tolerance.