Screening of CBL genes in pigeon pea with focus on the functional analysis of CBL4 in abiotic stress tolerance and flavonoid biosynthesis

Pigeon pea, the only woody legume in the world,is also a medicinal-food homogenous crop. At the same time, the calcineurin B-like (CBL) proteins that mediate a variety of abiotic stress responses are largely unknown. Here, we performed a bioinformatics analysis of the CBL family in pigeon pea, and initially verified the function of CcCBL4 in stress resistance and secondary metabolic synthesis. CBL family genes respond to 4 degrees C, 42 degrees C, NaCl, and drought stresses, and we observed the chlorophyll, relative water, proline and soluble sugar in CcCBL4-OE (over-expression) were significantly higher than CK (empty vectors) plants after stresses, and the integrity of the cell membrane was better. Seven secondary metabolites were detected, and we found that the p-coumaric acid in OE was 1.4-fold that in CK plants, but the apigenin and pterostilbene were reduced. And the expression of genes in these pathways was also confirmed changes. Altogether, our work revealed a potential regulation mechanism between CcCBL proteins, stress resistance and flavonoid metabolism, which is conducive to enhance the adaptability of pigeon pea to the environment.