ROS- and CBF- mediated pathways are involved in chlorophyll degradation and anthocyanin accumulation enhanced by cool temperatures in ripening litchi fruits

Cool temperatures improve the color of certain fruit crops by promoting chlorophyll degradation and anthocyanin accumulation. However, the molecular mechanism that underpins improved coloring in response to chilling is elusive. In consistent with the significant enhanced chlorophyll degradation in response to chilling, key genes associated with chlorophyll degradation such as LcSGR, LcNYC, LcPPH and LcPAO, were either unaltered or down-regulated. Conversely, a violaxanthin de-epoxidase gene (VDE) and a serine/threonine-protein kinase (TAO1-B), both of which are involved in the inhibition of reactive oxygen species (ROS) production, were found to be substantially downregulated in response to chilling. Under cool temperature treatment, there was an outbreak of superoxide anion in the pericarp, and treatment with a free radical inducer (20 mg L−1 methylviologen ) resulted in enhanced chlorophyll degradation. These findings suggested that the effect of cool temperatures in chlorophyll degradation may be associated with ROS damage rather than transcription. In addition, enhanced anthocyanin accumulation was associated with activation of anthocyanin-related genes under cool temperature. LcDREB2C, a chilling-induced CBF/DREB transcription factor, was identified as a key gene regulating anthocyanin accumulation at cool temperatures. LcDREB2C overexpression promoted anthocyanin accumulation, whereas silencing it displayed the opposite effect, coinciding with the significant changes in the expression of key anthocyanin-related genes. LcDREB2C was found to directly bind to the promoters of LcMYB1, LcCHI, and LcF3H in Y1H, dual luciferase, and EMSA assays. These findings establish the link between cool temperature-promoted anthocyanin biosynthesis and CBF pathway.