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• Ethephon enhanced anthocyanin accumulation and coloration of litchi fruit. • LcERF1/22/25/37 are positive regulators but LcERF64 is a negative regulator in anthocyanin biosynthesis. • LcERFs regulated coloration by binding to the promoters of key anthocyanin biosynthesis genes. Anthocyanins are the most prevalent phytochemicals that color the fruits. Ethylene promotes fruit ripening and modulates anthocyanin biosynthesis in plants, however, the underlying molecular mechanism in litchi is unclear. Herein, the slow-reddening cultivar 'Feizixiao' (FXZ) and swift-reddening cultivar 'Guiwei' (GW) were selected to study the regulation of anthocyanin biosynthesis in litchi. Total anthocyanin contents were found to be higher in 'GW' and lower in 'FZX'. We then comprehensively investigated ethylene response factor (ERF) subfamily genes and their expression patterns in different tissues and fruit developmental stages. Using transcriptome data, eight LcERF genes were identified as putative key genes associated with litchi pericarp coloration and anthocyanin accumulation. To test whether these genes are expressed under ethephon treatment, we applied 100 and 200 µL L−1 ethephon 4 weeks before harvest. Ethephon treatment dramatically increased anthocyanin accumulation as well as the expression of targeted ERF genes. LcERF1/22/25/37 were significantly upregulated in response to ethephon treatment, while LcERF64 was significantly downregulated. Anthocyanin content and expression of anthocyanin-related genes were considerably reduced in LcERF1/22/25/37 -silenced fruits, but the opposite trend was observed in LcERF64 -silenced fruits. Yeast-one-hybrid and dual luciferase assays revealed that the tested ERFs bind to the promoters of key anthocyanin biosynthesis genes and alter their expression. These findings suggested that ethylene promotes anthocyanin accumulation and consequently fruit coloration by stimulating the positive anthocyanin biosynthesis regulators LcERF1/22/25/37 , or by suppressing the negative regulator LcERF64 , which regulates the expression LcDFR and LcMYB1 by directly binding to their promoters. [ABSTRACT FROM AUTHOR]

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. • Cool temperature hastens litchi chlorophyll loss and anthocyanin accumulation. • ROS mediate cool temperature enhanced coloration of litchi. • Cool temperatures activate LcDREB2C to enhance anthocyanin synthesis. • LcDREB2C targets LcMYB1 and key structural genes. [ABSTRACT FROM AUTHOR]