Combining molecular docking and experimental approaches to uncover how Ca2+/PpCaM1-PpCAMTA3 regulated PpLCD2 expression and hydrogen sulfide production contributing to chilling tolerance in peach fruit

Abstract

Hydrogen sulfide (H2S) and calcium ions (Ca2+) are implicated in various processes in fruit response to cold. L-cysteine desulfhydrase (LCD) produces H2S using L-cysteine as a substrate, but the exact role of Ca2+ signaling in LCD-mediated H2S synthesis to induce chilling tolerance in peach fruit remains unclear. Here, transcriptome analysis revealed that most differentially expressed genes associated with Ca2+ signaling after H2S treatment function in calcium ion binding and calmodulin (CaM) binding. CaM-binding transcription activator (CAMTA) is a class of CaM-binding proteins and CaM-CAMTA may be a master regulator of fruit response to cold. Further investigation implied that PpCAMTA3 positively regulated PpLCD2 transcription by binding to the CG-box motif via multiple interactions. Three PpCaM genes were identified, in which PpCaM1 interaction with PpCAMTA3 probably occurred through hydrogen bonding with the third IQ domain. Each dumbbell-shaped structure of PpCaM1 contained two EF-hand domains that bound two calcium ions, and the interaction of PpCaM1 and PpCAMTA3 was Ca2+-dependent, enhancing PpCAMTA3-mediated PpLCD2 transcriptional activation. Exogenous H2S further increased intracellular Ca2+ concentrations and PpCaM expression, which resulted in a higher PpLCD2 expression and H2S synthesis, contributing to chilling tolerance in peach fruit. These findings indicated that the Ca2+/PpCaM1-PpCAMTA3 module regulated H2S production in peach fruit under cold stress, thus shedding light on the mechanisms of peach fruit response to cold stress.