VvMYB14 Regulates Anthocyanin Biosynthesis in Grape and Screening of Its Interacting Proteins

Anthocyanins are core secondary metabolites that determine the fruit color, flavor and antioxidant activity of grape (Vitis vinifera L.). Their biosynthesis is regulated at multiple levels by light signals, transcription factors and key genes in the metabolic pathway, which directly affects the commercial and nutritional value of grape fruits. To investigate the regulatory mechanism of MYB transcription factor VvMYB14 (NCBI ID: XM_002278747.2) in response to light signals during anthocyanin biosynthesis, so as to provide a theoretical basis for analyzing the interaction between light signals and secondary metabolism, as well as improving grape fruit quality.

Grape cultivars Pinot Noir and Red Globe, apple cultivar Ruixue, and Nicotiana benthamiana were used as experimental materials. Homologous sequences of VvMYB14 were retrieved by BLAST tool from the NCBI database. A phylogenetic tree was constructed using MEGA 5.0 software, and multiple amino acid sequence alignment was performed with DNAMAN. Experimental vectors including VvMYB14 overexpression vector and promoter-GUS fusion vector were constructed. Cis-acting elements in its promoter were predicted using the PlantCARE database, and promoter activity was verified by GUS staining and qRT-PCR assay in tobacco leaves. Combined with transient transformation in apple and grape peels, stable transformation in tobacco, and genetic transformation in grape calli, the function of VvMYB14 was explored through phenotypic observation and molecular detection. Yeast two-hybrid (Y2H) and luciferase complementation assay (LCA) were used to screen and verify the interacting proteins of VvMYB14.

VvMYB14 was most closely related to QrMYB14 of Quercus robur. Tissue-specific analysis showed that VvMYB14 was mainly expressed in grape leaves and fruits. Its promoter contained light-responsive elements (Box4, I-box), and light treatment significantly enhanced the promoter activity. Meanwhile, GUS expression levels were significantly higher than those of the control at three stages (G0, G12, G24). Functional verification indicated that VvMYB14 promoted anthocyanin biosynthesis in response to light: after transient expression in Ruixue apple and Red Globe grape peels, anthocyanin contents were 2.25-fold and 2.1-fold of the empty vector control, respectively. Transcription levels of key anthocyanin pathway genes CHS, F3′H, DFR and UFGT were significantly up-regulated in VvMYB14-overexpressing tobacco and grape calli. Interaction analysis confirmed that VvMYB14 specifically interacted with VvGRP4, a gibberellin-regulated protein.

VvMYB14 is a light-responsive transcription factor. It activates self-expression by recognizing light-responsive elements in the promoter, and then up-regulates the transcription of key genes in the anthocyanin biosynthetic pathway, ultimately promoting anthocyanin accumulation in grapes. In addition, its interacting protein VvGRP4 was identified.