Integrated metabolome and transcriptome analysis reveals the cause of anthocyanin biosynthesis deficiency in litchi aril.

Anthocyanins are health-promoting compounds with strong antioxidant properties that play important roles in disease prevention. Litchi chinensis Sonn. is a well-known and economically significant fruit due to its appealing appearance and nutritional value. The mature pericarp of litchi is rich in anthocyanins, whereas the aril (flesh) has an extremely low anthocyanin content. However, the mechanism of anthocyanin differential accumulation in litchi pericarp and aril remained unknown. Here, metabolome and transcriptome analysis were performed to unveil the cause of the deficiency of anthocyanin biosynthesis in litchi aril. Numerous anthocyanin biosynthesis-related metabolites and their derivatives were found in the aril, and the levels of rutin and (-)-epicatechin in the aril were comparable to those found in the pericarp, while anthocyanin levels were negligible. This suggests that the biosynthetic pathway from phenylalanine to cyanidin was present but that a block in cyanidin glycosylation could result in extremely low anthocyanin accumulation in the aril. Furthermore, 54 candidate genes were screened using weighted gene co-expression network analysis (WGCNA), and 9 genes (LcUFGT1, LcGST1, LcMYB1, LcSGR, LcCYP75B1, LcMATE, LcTPP, LcSWEET10, and LcERF61) might play a significant role in regulating anthocyanin biosynthesis. The dual-luciferase reporter (DLR) assay revealed that LcMYB1 strongly activated the promoters of LcUFGT1, LcGST4, and LcSWEET10. The results imply that LcMYB1 is the primary qualitative gene responsible for the deficiency of anthocyanin biosynthesis in litchi aril, which was confirmed by a transient transformation assay. Our findings shed light on the molecular mechanisms underlying tissue-specific anthocyanin accumulation and will help developing new red-fleshed litchi germplasm.

R3-MYB transcription factor LcMYBx from Litchi chinensis negatively regulates anthocyanin biosynthesis by ectopic expression in tobacco.

Anthocyanin accumulation is one of the remarkable physiological changes during fruit ripening. In plants, anthocyanin synthesis is regulated by MYB activators, but the MYB repressors has been recognized recently. Here, we isolated a repressor of anthocyanin synthesis, LcMYBx, from Litchi chinensis Sonn. LcMYBx encoded a typical R3-MYB protein and contained a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif for interacting with bHLH proteins. Overexpression of LcMYBx in tobacco suppressed anthocyanin accumulation resulting in faded petals from pale-pink to almost white. Gene expression analysis showed the strong down-regulation of endogenous anthocyanin structural and regulatory genes by LcMYBx overexpression. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that LcMYBx could interact with the transcription factors LcbHLH1 and LcbHLH3. Transient promoter activation assays showed that LcMYBx could inhibit the activation capacity of LcMYB1-LcbHLH3 complex for LcDFR gene. These results suggest that LcMYBx competed with LcMYB1 to LcbHLHs, thus preventing the activation of LcDFR by LcMYB1-LcbHLHs complex and negatively controlling anthocyanin biosynthesis.