Identification of Grape Laccase Genes and Their Potential Role in Secondary Metabolite Synthesis

Abstract

Laccase, a copper-containing oxidoreductase, has close links with secondary metabolite biosynthesis in plants. Its activity can affect the synthesis and accumulation of secondary metabolites, thereby influencing plant growth, development, and stress resistance. This study aims to identify the grape laccases (VviLAC) gene family members in grape (Vitis vinifera L.) and explore the transcriptional regulatory network in berry development. Here, 115 VviLACs were identified and divided into seven (Type I-VII) classes. These were distributed on 17 chromosomes and out of 47 VviLACs on chromosome 18, 34 (72.34%) were involved in tandem duplication events. VviLAC1, VviLAC2, VviLAC3, and VviLAC62 were highly expressed before fruit color development, while VviLAC4, VviLAC12, VviLAC16, VviLAC18, VviLAC20, VviLAC53, VviLAC60 and VviLAC105 were highly expressed after fruit color transformation. Notably, VviLAC105 showed a significant positive correlation with important metabolites including resveratrol, resveratrol dimer, and peonidin-3-glucoside. Analysis of the transcriptional regulatory network predicted that the 12 different transcription factors target VviLACs genes. Specifically, WRKY and ERF were identified as potential transcriptional regulatory factors for VviLAC105, while Dof and MYB were identified as potential transcriptional regulatory factors for VviLAC51. This study identifies and provides basic information on the grape LAC gene family members and, in combination with transcriptome and metabolome data, predicts the upstream transcriptional regulatory network of VviLACs.

Keywords: RNA-seq; berry development; gene family; grapevine; laccase; secondary metabolites.

Figure 1

Phylogenetic analysis of laccase proteins in grape and Arabidopsis thaliana. The seven feature branches of the series are highlighted in different colors. Numbers at nodes indicate branch support performed by the ultrafast bootstrap test. Members of the Arabidopsis protein family are shown in bold and star symbols.

Figure 2

Distribution of laccase genes on the grape chromosomes. The circles with different colors at the top represent different classifications of VviLACs, the rectangle represents the chromosome, the left shows the chromosome number, and the internal color represents gene density, red: dense, blue: sparse. The scale of chromosome length is measured in MB. “▬”segments represent tandem repeats (TR), while “—” are connected to fragmented replication (FR).

Figure 3

Phylogenetic relationships, the architecture of the conserved motifs and domains, exon–intron structures in grape laccase family members: (A) The phylogenetic tree was constructed based on the full-length sequences of grape laccase proteins using MEGA (version 7). (B) The conserved motifs of grape laccase proteins. Different colored rectangles indicate different motifs, which are numbered 1–10, and different colored bars indicate different domains, which are numbered 1–3. (C) Exon–intron structure of grape laccase genes. Green zones indicate coding sequence length (exon); black lines denote introns; and the number denotes the phases of the corresponding introns.

Figure 4

Synteny analysis of laccase genes between grape and six representative plant species: (A) Circle diagram of collinear relationships of grape (Vv), Arabidopsis thaliana (At), Solanum lycopersicum, (Sl) and Oryza sativa L. (Os) laccase genes. Different colored rectangles represent different species. (B) Circle diagram of collinear relationships of grape (Vv), Populus trichocarpa (Pt), Gossypium raimondii (Gr), and Prunus persica (Pp) laccase genes. (C) The number of homologous gene pairs between grape laccase gene and other species. (D) Relationship between the number of laccase genes homologous to grape and different species.

Figure 5

Expression heat map of VviLACs during grape berry development and qRT-PCR verification: (A) Expression of laccase gene during the development of grape berry sampled for three consecutive years. (B) qRT-PCR verification of laccase-related genes during the berry development stages of ‘Summer black’.

Figure 6

Correlation analysis between laccase gene expression and secondary metabolites. Line orange: positive correlation; Blue: Negative correlation. Circular nodes represent VvLACs, squares represent secondary metabolites, and node sizes are determined by network connectivity.

Figure 7

Transcription factors target the regulatory network of laccase genes. Different color circles represent different types of transcription factors, gray represents grape laccase family genes. The line coarseness is controlled by the predictive regulation threshold. The circle size is determined by the weight.