Calmodulin‐Like Protein MfCML50 Interacts With Carveol Dehydrogenase in <i>Medicago falcata</i> to Regulate Cold Tolerance Through Mediating ROS Homeostasis

Low temperature triggers Ca²⁺ signalling and reprogramming of gene expression and metabolism in plants. However, how the Ca²⁺ signal is transduced to the downstream metabolic pathways remains unknown. The involvement of a cold-induced calmodulin-like protein, MfCML50, from Medicago falcata in regulation of cold tolerance was examined in the present study. The interaction of MfCML50 with isopiperitenol/carveol dehydrogenase (MfCDH) was identified, which was dependent upon Ca²⁺ and led to activated activity of MfCDH. MfCDH catalysed the production of carvone from carveol, with K_m value of 10.25 μM. Overexpression of MfCML50 or MfCDH in Medicago truncatula led to enhanced cold tolerance with increased accumulation of carvone under cold conditions. The knockout mutation of the ortholog MtCML50 and MtCDH in M. truncatula led to reduced cold tolerance with decreased accumulation of carvone in the mtcml50, mtcdh, or mtcml50 mtcdh mutants under cold conditions, which could be recovered by expressing MfCML50 or MfCDH or exogenous application of carvone. The diphenyl-2-picrylhydrazyl (DPPH) assay showed that carvone exhibited strong antioxidant activity. Less ROS were accumulated in MfCML50 and MfCDH overexpressing lines, but more ROS were accumulated in mtcml50 and mtcdh mutants under cold conditions. The results suggested that the Ca²⁺ signal activated the MfCML50-MfCDH module regulates cold tolerance through promoted production of carvone to maintain ROS homeostasis.