Genome-wide investigation of WRKY transcription factors in Tartary buckwheat (Fagopyrum tataricum) and their potential roles in regulating growth and development

Abstract

Background: The WRKY gene family plays important roles in plant biological functions and has been identified in many plant species. With the publication of the Tartary buckwheat genome, the evolutionary characteristics of the WRKY gene family can be systematically explored and the functions of Fagopyrum tataricum WRKY (FtWRKY) genes in the growth and development of this plant also can be predicted.

Methods: In this study, the FtWRKY genes were identified by the BLASTP method, and HMMER, SMART, Pfam and InterPro were used to determine whether the FtWRKY genes contained conserved domains. The phylogenetic trees including FtWRKY and WRKY genes in other plants were constructed by the neighbor-joining (NJ) and maximum likelihood (ML) methods. The intron and exon structures of the FtWRKY genes were analyzed by the gene structure display server, and the motif compositions were analyzed by MEME. Chromosome location information of FtWRKY genes was obtained with gff files and sequencing files, and visualized by Circos, and the collinear relationship was analyzed by Dual synteny plotter software. The expression levels of 26 FtWRKY genes from different groups in roots, leaves, flowers, stems and fruits at the green fruit, discoloration and initial maturity stage were measured through quantitative real-time polymerase chain reaction (qRT-PCR) analysis.

Results: A total of 76 FtWRKY genes identified from the Tartary buckwheat genome were divided into three groups. FtWRKY genes in the same group had similar gene structures and motif compositions. Despite the lack of tandem-duplicated gene pairs, there were 23 pairs of segmental-duplicated gene pairs. The synteny gene pairs of FtWRKY genes and Glycine max WRKY genes were the most. FtWRKY42 was highly expressed in roots and may perform similar functions as its homologous gene AtWRKY75, playing a role in lateral root and hairy root formation. FtWRKY9, FtWRKY42 and FtWRKY60 were highly expressed in fruits and may play an important role in fruit development.

Conclusion: We have identified several candidate FtWRKY genes that may perform critical functions in the development of Tartary buckwheat root and fruit, which need be verified through further research. Our study provides useful information on WRKY genes in regulating growth and development and establishes a foundation for screening WRKY genes to improve Tartary buckwheat quality.

Keywords: Development; Fruit; Tartary buckwheat genome; FtWRKY.

Figure 1. Schematic representation of the chromosomal distribution of tartary buckwheat WRKY genes.

The chromosome number is indicated to the left of each chromosome. (A–H) stands for eight chromosomes of tartary buckwheat.

Figure 2. Phylogenetic relationships, gene structure, architecture of conserved protein motifs and cis-acting elements analysis of the WRKY genes from tartary buckwheat.

(A) A phylogenetic tree based on the full-length sequences of tartary buckwheat WRKY proteins was constructed using Geneious R11 software. (B) Exon-intron structure of tartary buckwheat WRKY genes. Blue boxes indicate zinc finger structure; yellow boxes indicate coding regions; black lines indicate introns. The WRKY domains are indicated by red boxes. The numbers indicate the phases of corresponding introns. (C) Motif composition of tartary buckwheat WRKY proteins. The motifs (numbered 1–10) are displayed in differently colored boxes. The sequence information for each motif is provided in Table S2. The lengths of the proteins can be estimated using the scale at the bottom. (D) The cis-acting elements analysis of FtWRKY genes promoters. Blocks of different colors represent light responsiveness elements, low temperature responsiveness elements, salicylic acid responsiveness elements, abscisic acid responsiveness elements, MeJA responsiveness elements, auxin responsiveness elements, gibberellin responsiveness elements and defense, stress responsiveness elements and wound responsiveness elements.

Figure 3. Unrooted phylogenetic tree representing relationships among the WRKY genes of tartary buckwheat and A. thaliana use ML method.

The genes in tartary buckwheat are marked in red, while those in A. thaliana are marked in black. The different-colored arcs indicate different groups (or subgroups) of WRKY genes.

Figure 4. Schematic representations of the interchromosomal relationships of tartary buckwheat FtWRKY genes.

The colored lines indicate the synteny blocks in the tartary buckwheat genome.

Figure 5. Phylogenetic relationships and motif compositions of WRKY proteins from seven different plant species.

Outer layer: an unrooted phylogenetic tree constructed with the ML method. Inner layer: distribution of conserved motifs in WRKY proteins. The different-colored boxes represent the different motifs and their positions in the WRKY protein sequences.

Figure 6. Synteny analysis of the WRKY genes in tartary buckwheat and seven representative plant species.

The gray lines in the background indicate the collinear blocks within tartary buckwheat and other plant genomes; the red lines indicate the syntenic WRKY gene pairs. A–G represent the synteny relationship between WRKY genes in tartary buckwheat and that in Arabidopsis thaliana, Oryza sativa, Beta vulgaris, Glycine max, Solanum lycopersicum, Vitis vinifera and Helianthus annuus, respectively.

Figure 7. Tissue-specific gene expression of 26 tartary buckwheat WRKY genes and and the correlation between the gene expression patterns of FtWRKY genes.

The expression patterns of 26 FtWRKY genes in flower, leaf, root, stem and fruit tissues were examined by qPCR (A–Z). Error bars were obtained from three measurements. The small letter(s) above the bars indicate significant differences (α = 0.05, LSD) among the treatments. AA shows the correlation between the gene expression patterns of FtWRKY genes. Purple: positively correlated; blue: negatively correlated. The red numbers indicate significant correlation at the 0.05 level.

Figure 8. Gene expression of 21 tartary buckwheat WRKY genes during fruit development and the correlation between the gene expression of FtWRKY genes during fruit development.

Expression patterns of 21 FtWRKY genes at the green fruit stage, the discoloration stage and the initial maturity stage examined by qPCR (A–U). Error bars were obtained from three measurements. The small letter(s) above the bars indicate significant differences (α = 0.05, LSD) among the treatments. (V) shows the correlation between the gene expression of FtWRKY genes during fruit development. Yellow: positively correlated; Dark green: negatively correlated. The red numbers indicate significant correlation at the 0.05 level.