Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (9): 1975-1986.doi: 10.3864/j.issn.0578-1752.2026.09.010

• HORTICULTURE • Previous Articles     Next Articles

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

MA ZongHuan(), NAN XinTong, CHEN LiZhen, DU YunBo, TANG Xing, LI WenFang, MAO Juan, CHEN BaiHong()   

  1. College of Horticulture, Gansu Agricultural University, Lanzhou 730070
  • Received:2025-12-29 Accepted:2026-02-23 Online:2026-05-01 Published:2026-05-06
  • Contact: CHEN BaiHong

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Abstract:

【Objective】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.【Method】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.【Result】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.【Conclusion】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.

Key words: grape (Vitis vinifera L.), MYB, light signals, anthocyanin accumulation, protein interaction

Fig. 1

Bioinformatics and tissue-specific expression analysis of VvMYB14 A: Multiple sequence alignment of MYB14 proteins from different species. Vv: Vitis vinifera, At: Arabidopsis thaliana, Md: Malus domestica, Zm: Zea mays, Ma: Musa acuminata, Sl: Solanum lycopersicum, Qr: Quercus robur, Zb: Zanthoxylum bungeanum, Fv: Fragaria vesca, Hs: Hibiscus syriacus, Dz: Durio zibethinus, Gs: Glycine soja, Ae: Actinidia eriantha, 1-Cs: Camellia sinensis, 2-Cs: Cannabis sativa, 3-Cs: Chaenomeles speciosa, Cq: Chenopodium quinoa, Cc: Capsicum chinense, Bv: Beta vulgaris; B: Phylogenetic tree of MYB14 homologous proteins from different species; C: Relative expression levels of the VvMYB14 gene in different tissues of grape. Different lowercase letters indicate significant differences between groups (P<0.05). The same as below"

Fig. 2

Analysis of VvMYB14 promoter activity A: Analysis of cis-regulatory elements in the promoter region of VvMYB14; B: Histological staining and relative expression analysis of GUS reporter gene in tobacco leaves; C: Assay for the transcriptional autoregulatory activity of VvMYB14"

Fig. 3

Analysis of the phenotype, anthocyanin content, and VvMYB14 expression in apple and grape skins after transient transformation of VvMYB14 A: Instantaneous transformation phenotype of apple peel; B: Content of anthocyanins in apple peel and relative expression level of VvMYB14 gene; C: Instantaneous transformation phenotype of grape peel; D: Content of anthocyanins in grape peel and relative expression level of VvMYB14. ****：P<0.0001"

Fig. 4

Analysis of phenotypes and anthocyanin-related gene expression in tobacco overexpressing VvMYB14 A: Phenotypic observation of tobacco plants overexpressing VvMYB14; B: Analysis of relative expression levels of genes related to the anthocyanin biosynthesis pathway"

Fig. 5

Analysis of anthocyanin content and expression of related genes in grapevine callus overexpressing VvMYB14 A: Phenotypes of different grape calli after L45D treatment; B: Anthocyanin content in different grape calli; C: Expression analysis of genes related to anthocyanin biosynthesis. L: Light treatment. *: P<0.05; **: P<0.01; ***: P<0.001"

Fig. 6

Analysis of VvMYB14 transcriptional autoactivation activity and verification of its interaction with VvGRP4 A: Detection of transcriptional activation activity of VvMYB14; B: Yeast two-hybrid experiment; C: Luciferase complementation assay"

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