MdMPK6a/b‐mediated phosphorylation of MdANN2 restricts cytosolic Ca ²⁺ influx and attenuates cold tolerance in apple

SUMMARY

Annexins (ANNs) are Ca ²⁺ ‐dependent phospholipid‐binding proteins that play important roles in plant responses to environmental stress. However, the apple ANN gene family and the regulatory mechanisms by which ANNs participate in cold‐stress responses remain poorly understood. In this study, 15 MdANN family members were identified in the apple genome and divided into three subfamilies. Among them, MdANN2 expression was strongly induced by low temperature. Functional analyses showed that MdANN2 overexpression markedly enhanced cold tolerance in apple, whereas virus‐induced gene silencing (VIGS) of MdANN2 compromised cold tolerance. Subcellular localization assays revealed that MdANN2 localized to the plasma membrane, cytoplasm, and nucleus. Non‐invasive micro‐test technology (NMT) further showed that MdANN2 is associated with Ca ²⁺ ‐permeable channel activity: cold stimulation significantly enhanced cytosolic Ca ²⁺ influx in MdANN2 ‐overexpressing plants, whereas exogenous application of the Ca ²⁺ channel blocker LaCl ₃ or the Ca ²⁺ chelator EGTA substantially inhibited this influx. The kinases MdMPK6a and MdMPK6b physically interacted with and phosphorylated MdANN2 both in vitro and in vivo . Silencing MdMPK6a/b significantly increased apple cold tolerance, whereas overexpression of MdMPK6a/b reduced apple cold tolerance. Moreover, transient overexpression of MdMPK6a/b in the MdANN2 ‐overexpression background attenuated MdANN2‐mediated cold tolerance and Ca ²⁺ influx. In addition, co‐localization assays showed that MdMPK6a/b decreased the accumulation of MdANN2 at the plasma membrane. Together, these findings demonstrate that MdANN2 positively regulates apple cold tolerance by promoting cold‐induced Ca ²⁺ influx, a process inhibited by MdMPK6a/b‐mediated phosphorylation. This study identifies a novel MdMPK6a/b‐MdANN2 module that governs Ca ²⁺ signaling in apple under cold stress, thereby providing promising molecular targets for improving cold tolerance in apple and other crop species.