Chalcone synthase 2 (BpCHS2), a structural gene, was activated by low temperature to promote anthocyanin synthesis in Broussonetia papyrifera to improve its cold tolerance

Broussonetia papyrifera is an important source of unconventional feed. However, freezing injuries in winter are a considerable threat to the popularization and application of B. papyrifera. Notably, the use of anthocyanins is a promising approach for enhancing plant stress resistance. However, B. papyrifera contains low levels of anthocyanins, and the anthocyanin synthesis process in this plant remains unclear. In this study, the one-month-old seedlings of B. papyrifera were grown at low and average temperatures. After 3 weeks, petioles and veins from different growth environments were harvested for transcriptome and anthocyanin-targeted metabolome analyses. The targeted metabolome analysis revealed that following cold treatment, the levels of cyanidin-3-O-galactoside, cyanidin-3-O-rutinoside, cyanidin-3-O-(6-O-p-coumaroyl)-glucoside and cyanidin chloride increased by 756.22, 306.87, 222.28, and 776.67 times, respectively. Transcriptome analysis revealed 17 pivotal anthocyanin-related differential expressing genes, among which chalcone synthase 2 (BpCHS2) was significantly upregulated at low temperatures. The combined transcriptome and metabolome disclosed an apparent positive correlation between BpCHS2 and cyanidin derivatives. Transgenic experiments demonstrated that overexpression of BpCHS2 in tobacco markedly increased the expression of anthocyanin-related genes, promoted anthocyanin accumulation, and enhanced the activities of peroxidase, superoxide dismutase, and catalase. These results suggest that the expression of BpCHS2 is markedly increased in B. papyrifera under low-temperature stress, improving anthocyanin accumulation and cold tolerance.