Actinidia arguta is a newly emerged, commercially cultivated Actinidia species. A. arguta has a beautiful appearance and is rich in anthocyanin, and is thus highly welcomed by consumers. However, the mechanism of anthocyanin regulation in A. arguta remains unclear. In this study, we assembled the nearly complete genome of the first red A. arguta cultivar, ‘Tianyuanhong’, with an N50 of 21 Mb. Comparative genome analysis revealed a role of the expansion/contraction of gene families in the species-specific trait formation of A. arguta. Through verification of transient overexpression and stable transformation, RNA-seq analysis revealed a key bHLH transcription factor, AaBEE1, which negatively regulates anthocyanin biosynthesis. DAP-seq analysis combined with Y1H, EMSA, Chip-qPCR and LUC suggested that AaBEE1 binds to the G-box of the AaLDOX promoter and suppresses its expression. Overall, we assembled the genome of A. arguta and clarified its AaBEE1-AaLDOX module-mediated molecular mechanism of anthocyanin regulation.

Red Actinidia arguta has recently become highly popular because of its red appearance resulting from anthocyanin accumulation, and has gradually become an important breeding direction. However, regulators involved in anthocyanin biosynthesis have not been fully characterized in A. arguta. Here, we demonstrated that a key R2R3-MYB transcription factor, AaMYB61-like, plays a crucial role in A. arguta anthocyanin biosynthesis. The RT-qPCR results revealed that transient overexpression of AaMYB61-like in A. arguta fruit at 90–100 DAFB significantly promoted anthocyanin biosynthesis, as did the gene expression levels of AaCHS, AaCHI, AaF3H, AaLDOX, and AaF3GT, whereas the result of VIGS revealed the opposite results in A. arguta fruit at 105–115 DAFB. A transcriptional activation assay indicated that AaMYB61-like exhibited transcriptional activation activity. Y1H and LUC assays revealed that AaMYB61-like activates the promoters of AaCHS, AaLDOX, and AaF3GT. In addition, AabHLH137 was found to be related to fruit color from the transcriptome data. We demonstrated that AaMYB61-like promotes anthocyanin biosynthesis by interacting with AabHLH137 via Y2H, BiFC, and Agrobacterium-mediated co-transformation. Our study not only reveals the functions of AaMYB61-like and AabHLH137 in anthocyanin regulation, but also broadly enriches color regulation theory, establishing a foundation for clarifying the molecular mechanism of fruit coloration in kiwifruit.

Actinidia arguta possesses different colors in the fruit skin and flesh, but the underlying mechanism has not yet been clarified. In this study, we conducted 36 samples RNA-seq to investigate the phenotypic expression of different fruit tissues (skin and flesh) in red and green A. arguta varieties during different coloring phases. GO and KEGG enrichment results of differentially expressed genes (DEGs) suggested that the red color of the skin and flesh was derived from anthocyanin transport and flesh softening, respectively. Weighted gene co-expression network analysis (WGCNA) revealed MEyellow and MEblack modules significantly correlated with skin and flesh coloration, and two genes, Glutathione S-transferases (AaGST) and β-galactosidases (AaBGAL), were identified as hub genes involved in different tissue-specific coloration. Transient overexpression in apples and kiwifruits confirmed the role of AaGST and AaBGAL in color formation. Our results preliminarily explore the mechanism of red color formation in different A. arguta fruit tissues and provide novel insights into red color formation.