The GOLDEN2-LIKE (GLK) gene family, known for its role in chloroplast development, has recently been implicated in involvement of anthocyanin biosynthesis in kiwifruit (Actinidia spp.), but the underlying regulatory mechanism remains unclear. Here we report the characterization of a kiwifruit GLK homolog AcGLK2 in regulating anthocyanin accumulation. We found that expression of AcGLK2 is much higher exclusively in the red pigment-accumulated fruit tissue. Overexpression of AcGLK2 in Arabidopsis and kiwifruit significantly enhanced anthocyanin content, whereas its RNAi-mediated silencing compromised anthocyanin accumulation. RNA-Seq analysis revealed significant upregulation of many structural genes and transcription factors (TFs) associated with the flavonoid pathway in AcGLK2-overexpressing kiwifruit. ChIP-Seq analysis indicated that AcGLK2 directly binds to AcMYB5 and AcTRY. We showed AcMYB5, an R2R3-MYB TF, promotes anthocyanin accumulation by interacting with the bHLH protein AcbHLH42, while AcTRY, an R3-MYB protein, competitively inhibits the interaction between key MYBs and AcbHLH42, thereby repressing anthocyanin biosynthesis. Transgenic and molecular assays in tobacco, tomato, and kiwifruit demonstrated that AcGLK2 positively participates into regulation of anthocyanin accumulation through transcriptionally activating AcMYB5 expression and concomitantly suppressing AcTRY expression. This study reveals the dual regulatory mechanism of AcGLK2 in anthocyanin biosynthesis, broadening the understanding of GLK gene functions and providing a valuable genetic resource for molecular breeding of nutritional quality in kiwifruit and other crops.

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