Ca2+/MtCML39-MtMYB6 module regulates MtCBF1 expression and cold tolerance in Medicago truncatula

Calcium is a universal second messenger regulating plant responses to environmental stresses, while the cold-responsive CBF pathway plays a key role in plant cold tolerance. However, it remains largely unknown what molecular module bridges the gap between Ca2+ signaling and the CBF pathway. An R2R3-type transcription factor, MtMYB6, is examined to regulate cold tolerance in M. truncatula negatively. It interacts with a calmodulin-like protein MtCML39 that upregulates cold tolerance with dependence on the presence of Ca2+. Based on the shared downstream CBFs and cold-responsive genes regulated by either MtMYB6 or MtCML39, MtCBF1 but not MtCBF2/3/4 is identified to be the direct target gene of MtMYB6 by binding to a MYB cis-element (TTGTTG) in the promoter of MtCBF1 for repressing its expression, while the binding is disrupted in the presence of Ca2+/MtCML39. In addition, genetic evidence confirms that MtMYB6 acts downstream of MtCML39, while physiological measurements indicate that the increased cold tolerance by overexpressing MtCML39 or by editing MtMYB6 is reversed in the absence of Ca2+. It is suggested that Ca2+/MtCML39 interacts with MtMYB6 under cold conditions to relieve its repression on MtCBF1 expression to activate MtCBF1 and its dependent cold response pathway for enhanced freezing tolerance.