Cold-responsive MtCBF4-MtJMJ13 positive feedback loop negatively regulates anthocyanin biosynthesis in Medicago truncatula

Legumes, being significant food sources for both humans and animals globally, experience several adverse conditions that considerably affect their crop yield. Medicago sp., an important forage crop, thrives under adverse conditions, such as drought, extreme temperatures (heat and cold), and salinity. Several studies have shed light on the regulatory pathways of cold/chilling stress tolerance in Medicago sp. in recent years. In the present study, we found that a cold-repressed transcription factor, MtCBF4, negatively regulates anthocyanin synthesis by directly repressing the expression of MtLAP1, a critical regulatory gene for anthocyanin synthesis in Medicago truncatula. It leads to the downregulation of anthocyanin biosynthesis genes, and reduced anthocyanin accumulation. Apart from the transcriptional cascade, MtCBF4 exerts its effect through histone H3K27 trimethylation. It directly binds to the promoter of MtJMJ13, an H3K27me3 demethylase, to activate its expression, resulting in decreased H3K27 trimethylation. Furthermore, MtJMJ13 demethylates the gene body of MtCBF4 to positively regulate its expression, forming a positive feedback loop that functions in a cold-responsive manner. MtJMJ13 also demethylates the gene body of MtMYB2, MtKFB1, and MtKFB2 genes, which are negative regulators of anthocyanin biosynthesis. Altogether, we have unveiled a new regulatory loop involving a transcriptional regulator and a histone demethylase, operating together to limit anthocyanin production. This regulatory module is repressed under cold stress, leading to increased anthocyanin production and enhanced cold stress tolerance in M. truncatula.