Anthocyanin Accumulation in Muscadine Berry Skins Is Influenced by the Expression of the MYB Transcription Factors, MybA1, and MYBCS1

Abstract

The skin color of grape berry is very important in the wine industry. The red color results from the synthesis and accumulation of anthocyanins, which is regulated by transcription factors belonging to the MYB family. The transcription factors that activate the anthocyanin biosynthetic genes have been isolated in model plants. However, the genetic basis of color variation is species-specific and its understanding is relevant in many crop species. This study reports the isolation of MybA1, and MYBCS-1 genes from muscadine grapes for the first time. They are designated as VrMybA1 (GenBank Accession No. KJ513437), and VrMYBCS1 (VrMYB5a) (GenBank Accession No. KJ513438). The findings in this study indicate that, the deduced VrMybA1 and VrMYBCS1 protein structures share extensive sequence similarity with previously characterized plant MYBs, while phylogenetic analysis confirms that they are members of the plant MYB super-family. The expressions of MybA1, and MYBCS1 (VrMYB5a) gene sequences were investigated by quantitative real-time PCR using in vitro cell cultures, and berry skin samples at different developmental stages. Results showed that MybA1, and MYBCS1 genes were up-regulated in the veràison and physiologically mature red berry skins during fruit development, as well as in in vitro red cell cultures. This study also found that in ripening berries, the transcription of VrMybA1, and VrMYBCS1 in the berry skin was positively correlated with anthocyanin accumulation. Therefore, the upregulation of VrMybA1, and VrMYBCS1 results in the accumulation and regulation of anthocyanin biosynthesis in berry development of muscadine grapes. This work greatly enhances the understanding of anthocyanin biosynthesis in muscadine grapes and will facilitate future genetic modification of the antioxidants in V. rotundifolia.

Keywords: MYB gene; MYBCS1; MybA1; anthocyanins; antioxidants; muscadine grapes; pigments.

Figure 1

The complete cDNA sequence and amino acid sequence of the protein encoded by MybA1 (GenBank accession number: KJ513437).

Figure 2

The complete cDNA sequence and amino acid sequence of the protein encoded by MYBCS1 (GenBank accession number: KJ513438).

Figure 3

Amino acid sequence alignment of MybA1 (A); and MYBCS1 (B) proteins from different plant species. Residues highlighted in blue represent identical and similar amino acids, respectively. The alignment was performed using multi-align software as defined in the Materials and Methods section.

Figure 4

The computational modeled three dimensional structure of muscadine MybA1 (A); and MYBCS-1 (B).

Figure 5

Comparison of the deduced amino acid sequences of R2R3-type MYB transcription factors from higher plants. (A) A phylogenetic tree for plant MybA1 transcription factors; (B) A phylogenetic tree for plant MYBCS1 transcription factors including the one isolated from muscadine grapes (V. rotundifolia) in the present study.

Figure 6

Plant samples used in the experiment (A) (Red cells); (B) (Mature berries); (C) (Veràison berries); and (D) (Green berries) and mRNA expression levels of VrMybA1 (I); and VrMYBCS-1 (II) genes in different developmental stages of muscadine grape berry skins. The values are expressed relative to a level of transcription (I) the veràison, which was set as 0.1. Values are the means of three replicates ± SE.

Figure 7

Analysis of anthocyanin accumulation in in vitro cell cultures (Callus), as well as in different development stages of muscadine berry skins (Mature, Veriason, and Green skins). Mean values with different small letters (a–d) are significantly different at (p = 0.01). The accumulation pattern of anthocyanins follows the pattern of expression of VrMybA1, and VrMYBCS-1.