Genome-wide characterization and analysis of bHLH transcription factors related to anthocyanin biosynthesis in spine grapes (Vitis davidii)

Abstract

As one of the largest transcription factor family, basic helix-loop-helix (bHLH) transcription factor family plays an important role in plant metabolism, physiology and growth. Berry color is one of the important factors that determine grape quality. However, the bHLH transcription factor family's function in anthocyanin synthesis of grape berry has not been studied systematically. We identified 115 bHLH transcription factors in grape genome and phylogenetic analysis indicated that bHLH family could be classified into 25 subfamilies. First, we screened six candidate genes by bioinformatics analysis and expression analysis. We found one of the candidate genes VdbHLH037 belonged to III (f) subfamily and interacted with genes related to anthocyanin synthesis through phylogenetic analysis and interaction network prediction. Therefore, we speculated that VdbHLH037 participated in the anthocyanin synthesis process. To confirm this, we transiently expressed VdbHLH037 in grape and Arabidopsis transformation. Compared with the control, transgenic materials can accumulate more anthocyanins. These results provide a good base to study the function of the VdbHLH family in anthocyanin synthesis of grape berry.

Figure 1

The distribution of 115 VdbHLH genes on the nineteen grape chromosomes. The centromeric positions are shown according to the location of each VdbHLH gene.

Figure 2

An un-rooted phylogenetic tree of the VdbHLH family was constructed through the neighbor-joining method. The phylogenetic tree was constructed online with Interactive Tree of Life (iTOL). The numbers are bootstrap values based on 1000 iterations. Only bootstrap values larger than 50% support are displayed.

Figure 3

The characterization and distribution of bHLH domains across all VdbHLH. (a) The sequence logo of the bHLH domain. The asterisks represent the 19 conserved amino acids mentioned previously by Atchley. The capital letters represent conserved amino acids that contain more than 50%. (b) The distribution of amino acids in the bHLH domain. The columns of different colors represent the percentage of amino acids at this site.

Figure 4

The analysis of paralogous bHLH genes and their orthologues in grape, Arabidopsis and tomato. (a) The analysis of paralogous and orthologues in grape (Chr1-Chr19) and Arabidopsis (A1-A5). The blue lines represent the paralogous bHLH genes in grape. The green lines represent the paralogous bHLH genes in Arabidopsis. The red lines represent their orthologous bHLH genes. (b) The analysis of paralogous and orthologues in grape (Chr1-Chr19) and tomato (A1-A12). The blue lines represent the paralogous bHLH genes in grape. The green lines represent the paralogous bHLH genes in tomato. The red lines represent their orthologous bHLH genes. The relationships of orthologous and paralogous bHLH genes among grape, Arabidopsis and tomato were drawn by Circos 0.69.6 (http://circos.ca/).

Figure 5

The heatmap of black and white spine grape berry bHLH genes at different developmental stages. Note: B1, B2 and B3 represent black spine grape berry of 40, 80 and 120 days after anthesis, respectively. W1, W2 and W3 represent white spine grape berry of 40, 80 and 120 days after anthesis, respectively.

Figure 6

Interaction network analysis for VdbHLH003, VdbHLH004, VdbHLH033, VdbHLH037, VdbHLH062 and VdbHLH097. Note: The predicted results are based on the orthologous gene in Arabidopsis. VdbHLH genes are shown in brackets.

Figure 7

Transient expression result of ‘Kyoho’ berries. (a) ‘Kyoho’ fruits agroinfiltrated with VC (vector control) and OE (overexpression) at 6 days after injection. (b) Hygromycin gene (resistance marker gene) of pHB vector in fruits screened by RT-PCR. (c) Color parameters (L*, a*, b*) of VC and OE berries. (d) The total anthocyanin content of VC and OE berries. RT-PCR were normalized to the expression of VlActin.

Figure 8

Functional analysis of VdbHLH037 in transgenic Arabidopsis. (a) The phenotype of four-week-old transgenic Arabidopsis with VC and OE. (b) The transcription level of VdbHLH037 in OE and VC transgenic Arabidopsis by qRT-PCR. (c) The total anthocyanin content of four-week-old transgenic Arabidopsis with VC and OE. (d) The transcription level of anthocyanin biosynthesis genes in transgenic Arabidopsis with VC and OE. qRT-PCR were normalized to the expression of AtUBQ3.