Summary: Flavonoids have a major contribution to the fruit quality in cultivated strawberries and are regulated by MYB, bHLH and WD40 transcriptional factors. We reported here the identification of the FaMYB5, an R2R3‐MYB transcription factor, which positively regulated the accumulation of anthocyanins and proanthocyanidins through the trans‐activation of the F3'H and LAR. The strawberry FaEGL3 and FaLWD1/FaLWD1‐like interact with the R2R3‐FaMYB5 to form an MYB‐bHLH‐WD40 complex (MBW), enhancing the regulatory efficiency. The R2R3‐FaMYB5 was constitutively expressed in various tissues and in fruits of different developmental stages, which was strikingly contrasting to the fruit‐specific expression patterns of FaMYB10. Meanwhile, R2R3‐FaMYB5 failed to promote a stable accumulation of anthocyanin glycosides in the mature fruits of the myb10 mutant, mainly due to the suppressed expression of TT19. The R2R3‐FaMYB5 was regulated by an antisense long noncoding RNA lncRNA‐myb5. Additionally, the R2R3‐FaMYB5 protein could interact with FaBT2 and was degraded through the ubiquitin/26 S proteasome pathway. Transcriptome and metabolome data showed that R2R3‐FaMYB5 enhanced the gene expression and the metabolite accumulation involved in the flavonoid, phenylpropanoid and lignin biosynthesis pathways. Collectively, we conclude that the FaMYB5 is an R2R3‐MYB activator involved in the composition of MBW, which positively regulates the biosynthesis of anthocyanin and proanthocyanidin. These findings provided new insights into the molecular mechanisms that regulate flavonoids in strawberry fruits.

Light conditions can cause quantitative and qualitative changes in anthocyanin. However, little is known about the underlying mechanism of light quality-regulated anthocyanin accumulation in fruits. In this study, light-emitting diodes (LEDs) were applied to explore the effect of red and blue light on strawberry coloration. The results showed contents of total anthocyanins (TA), pelargonidin 3-glucoside (Pg3G) and pelargonidin 3-malonylglucoside (Pg3MG) significantly increased after blue and red light treatment. Pg3G was the major anthocyanin component in strawberry fruits, accounting for more than 80% of TA, whereas Pg3MG accounted for a smaller proportion. Comparative transcriptome analysis was conducted using libraries from the treated strawberries. A total of 1402, 5034, and 3764 differentially-expressed genes (DEGs) were identified in three pairwise comparisons (red light versus white light, RL-VS-WL; blue light versus white light, BL-VS-WL; blue light versus red light, BL-VS-RL), respectively. Photoreceptors and light transduction components remained dynamic to up-regulate the expression of regulatory factors and structural genes related to anthocyanin biosynthesis under red and white light, whereas most genes had low expression levels that were not consistent with the highest total anthocyanin content under blue light. Therefore, the results indicated that light was an essential environmental factor for anthocyanin biosynthesis before the anthocyanin concentration reached saturation in strawberry fruits, and blue light could quickly stimulate the accumulation of anthocyanin in the fruit. In addition, red light might contribute to the synthesis of proanthocyanidins by inducing LAR and ANR.

Anthocyanins endowing strawberry fruit red color are regulated by the MYB-bHLH-WD40 complex. By analyzing the MYBs involved in the flavonoid biosynthesis in strawberry, we found that R2R3-FaMYB5 promoted the content of anthocyanin and proanthocyanidins in strawberry fruits. Yeast two-hybrid and BiFC assays confirmed that MBW complexes connected with flavonoid metabolism were FaMYB5/FaMYB10-FaEGL3 (bHLH)-FaLWD1/FaLWD1-like (WD40). Transient overexpression and qRT-PCR analysis revealed that disparate MBW models hold different patterns in the regulation of flavonoid biosynthesis in strawberry fruits. Compared with FaMYB10, FaMYB5 and its dominant complexes showed a more specific regulatory range on strawberry flavonoid biosynthetic pathway, while FaMYB10 was more extensive. In addition, the complexes involved in FaMYB5 facilitated PAs accumulation primarily through the LAR tributary while FaMYB10 mainly by the ANR branch. FaMYB9 and FaMYB11 tremendously elicited the accumulation of proanthocyanidins by up-regulating the expression levels of both LAR and ANR, and also affected anthocyanin metabolism by changing the ratio of Cy3G and Pg3G which were constituted as two major anthocyanin monomers in strawberries. Our study also illustrated that FaMYB5-FaEGL3-FaLWD1-like directly targeted the promoters of F3′H, LAR, and AHA10 thus committing to flavonoid accumulation. These results allow the specific members involved in the MBW complex to be deciphered and provide new insights into the regulatory mechanisms of anthocyanins and proanthocyanidins regulated by the MBW complex.

• Fructose and glucose were the predominant sugars in Chinese cherry fruits. • Malic acid was the most abundant organic acid in Chinese cherry fruits. • Increase in fructose and reduction in malic acid during domestication. • Rating criteria of flavor evaluation indicators was established.

WRKY transcription factors play a nonnegligible role in plant growth and development, but little is known about the involvement of WRKY transcription factors in the regulation of fruit ripening. In this study, FaWRKY71 was identified to be closely related to fruit maturation in octoploid strawberry. FaWRKY71 protein localized in the nucleus and responded to cold, salt, low phosphate, ABA, and light quality in strawberry seedlings. The temporal and spatial pattern expression analysis indicated that FaWRKY71 was expressed in all the detected tissues, especially in the full red fruits. In addition, FaWRKY71 gave rise to the accumulation of anthocyanin content by promoting the expression of structural genes FaF3’H, FaLAR, FaANR, and transport factors FaTT19 and FaTT12 in the flavonoid pathway, and softening the texture of strawberry via up-regulating the abundance of FaPG19 and FaPG21. Furthermore, FaWRKY71 was a positive regulator that mediated resistance against reactive oxygen species by enhancing the enzyme activities of SOD, POD, and CAT, reducing the amount of MDA. Altogether, this study provides new and comprehensive insight into the regulatory mechanisms facilitating fruit ripening in strawberry.