Comprehensive Genome-Wide Exploration of C2H2 Zinc Finger Family in Grapevine (Vitis vinifera L.): Insights into the Roles in the Pollen Development Regulation

Abstract

Some C2H2 zinc-finger proteins (ZFP) transcription factors are involved in the development of pollen in plants. In grapevine (Vitis vinifera L.), it has been suggested that abnormalities in pollen development lead to the phenomenon called parthenocarpy that occurs in some varieties of this cultivar. At present, a network involving several transcription factors types has been revealed and key roles have been assigned to members of the C2H2 zinc-finger proteins (ZFP) family in model plants. However, particularities of the regulatory mechanisms controlling pollen formation in grapevine remain unknown. In order to gain insight into the participation of ZFPs in grapevine gametophyte development, we performed a genome-wide identification and characterization of genes encoding ZFP (VviZFP family). A total of 98 genes were identified and renamed based on the gene distribution into grapevine genome. The analysis performed indicate significant changes throughout VviZFP genes evolution explained by high heterogeneity in sequence, length, number of ZF and presence of another conserved domains. Moreover, segmental duplication participated in the gene family expansion in grapevine. The VviZFPs were classified based on domain and phylogenetic analysis into three sets and different groups. Heat-map demonstrated differential and tissue-specific expression patterns of these genes and k-means clustering allowed to identify a group of putative orthologs to some ZFPs related to pollen development. In transgenic plants carrying the promVviZFP13::GUS and promVviZFP68::GUS constructs, GUS signals were detectable in the anther and mature pollen grains. Expression profiling of selected VviZFP genes showed differential expression pattern during flower development and provides a basis for deepening in the understanding of VviZFPs role on grapevine reproductive development.

Keywords: C2H2 zinc-finger protein; gene expression profiling; genome-wide; grapevine (Vitis vinifera L.); pollen development; transcription factor.

Figure 1

Graphical view of the classification criteria used according to ZF domain distribution. ZF arrays are composed by tandem ZFs linked by zero to ten amino acid residues, with five residues as the most frequent linker length [1]. The VviZFPs in set A possess one ZF array and the VviZFPs in set B possess more than one array of tandem ZF. Both groups may present another’s dispersed ZFs besides the ZF arrays. The VviZFPs in set C possess one or more dispersed ZFs (The ZF position showed is illustrative). * ZF arrays in grapevine contain 2 up to 4 ZF domains. Created with BioRender.com.

Figure 2

Sequence logos for the ZF motifs of VviZFPs. The x-axis represents the sequence positions in ZF domains and the y-axis represents the information content measured in bits. The sequence logos were derived from the complete peptide sequence in the MEME suite software [38].

Figure 3

Analytical view of the set A in the VviZFP family. (A) Unrooted phylogenetic tree built by the neighbor-joining method using aligned full-length amino acid sequences. The bootstrap values lower than 50 were not shown. (B) Schematic diagrams for intron/exon structures of VviZFP genes in the grapevine genome. The blue boxes indicate the exons, the lines indicate introns and the UTRs are displayed by yellow boxes. (C) Schematic representation of conserved motifs within VviZFPs. Green boxes represent ZF domains and the other colored boxes indicate other conserved domains identified. Gene models, proteins and length of the motifs can be estimated using the scale at the bottom. Different groups are highlighted with colors. In green box VviZFP52 the only B-set gene. EAR motif, Ethylene-responsive element binding factor-associated amphiphilic repression motif; EXOIII, Exonuclease RNase T/DNA polymerase III (IPR013520); JmjC, Jumonji C (IPR003347); JmjN, Jumonji N (IPR003349).

Figure 4

Analytical view of the set C in the VviZFP family. (A) Unrooted phylogenetic tree built by the neighbor-joining method using aligned full-length amino acid sequences. The bootstrap values lower than 50 were not shown. (B) Schematic diagrams for intron/exon structures of VviZFP genes in the grapevine genome. The blue boxes indicate the exons, the lines indicate introns and the UTRs are displayed by yellow boxes. (C) Schematic representation of conserved motifs within VviZFPs. Green boxes represent ZF domains and the other colored boxes indicate other conserved domains identified. Gene models, proteins and length of the motifs can be estimated using the scale at the bottom. Different groups are highlighted with colors. EAR motif, Ethylene-responsive element binding factor-associated amphiphilic repression motif (PF07897).

Figure 5

Overview of the VviZFPs EAR-repressome. (A) Proportion and classification of sequences containing EAR-motifs within the VviZFP family. (B): Total number of predicted EAR motifs containing VviZFPs, and incidence of EAR-motif sites within this protein family. (C): Distribution of EAR-motif sites in VviZFPs. (D): Frequency of EAR-motif sites in the N-terminal, C-terminal, or middle region of VviZFPs (C, C terminal; M, middle; N, N terminal). (E): Incidence of EAR-motif types in VviZFPs.

Figure 6

Genomic distribution and tandem duplication of VviZFP genes over 19 grapevine chromosomes. The chromosome number is indicated at the base of each chromosome. Each number indicates the VviZFP gene name and the symbol shows the position in chromosomes. Blue, green and yellow symbol correspond to set A, B and C VviZFPs coding genes, respectively. Tandem repeated genes are enclosed in a square.

Figure 7

Circos plot of 41 members of VviZFP gene family and their paralogs along Vitis vinifera (assembly 12X.V2) genome. The genome is represented through its chromosomes as segments of the main circumference (Circos). The names and position of the 41 annotated VviZFP genes that have paralogs are labeled along the chromosomes. Inside the Circos, lines of the same color connecting two positions of the genome represent the paralog regions of each family member.

Figure 8

Differential expression of VviZFP genes over grapevine flower development. (A) Hierarchical clustering of the expression profiles of 85 VviZFP genes covering the flower development and male floral samples. (B) Details of samples used in the analysis. Developmental stages are abbreviated according to [48]. (C) Centroid graphs of VviZFP gene expression via k-means clustering. The patterns identified showed (1) downward and (2) upward expression along the flowering time. The (3) cluster did not show a distinguishable expression pattern. The number of genes in every cluster is indicated. (D–F) Expression profiles of VviZFP genes over V. vinifera cv. Carménère flower development. (D) Details of samples used in the analysis and the Eichhorn-Lorenz modified phenological stages (E–L system). The relative expression of genes selected of the (E) downward and (F) upward clusters are presented. The x-axis represents developmental stages of whole inflorescences. The bars represent the standard error of the mean (SEM) from four biological replicates. Significant differences (p, 0.05) of expression between samples are indicated by different letters (Tukey’s HSD test). Y, young flower; W, well developed flower; SF, start of flowering; F, flowering; P, pollen; S, stamen.

Figure 9

The gene expression patterns of VviZFP13 and VviZFP68 in flower tissues. Histochemical analysis of GUS activity in promVviZFP13::GUS and promVviZFP68::GUS transgenic N. benthamiana mature flowers. (A) mature anther; (B) Mature pollen grain; (C) Stygma and style; (D) whole flower; (E) flower open manually and (F) anthers close-up; (G) Mature pollen grains.