Genome-wide identification and characterization of WRKY gene family in Salix suchowensis

Abstract

WRKY proteins are the zinc finger transcription factors that were first identified in plants. They can specifically interact with the W-box, which can be found in the promoter region of a large number of plant target genes, to regulate the expressions of downstream target genes. They also participate in diverse physiological and growing processes in plants. Prior to this study, a plenty of WRKY genes have been identified and characterized in herbaceous species, but there is no large-scale study of WRKY genes in willow. With the whole genome sequencing of Salix suchowensis, we have the opportunity to conduct the genome-wide research for willow WRKY gene family. In this study, we identified 85 WRKY genes in the willow genome and renamed them from SsWRKY1 to SsWRKY85 on the basis of their specific distributions on chromosomes. Due to their diverse structural features, the 85 willow WRKY genes could be further classified into three main groups (group I-III), with five subgroups (IIa-IIe) in group II. With the multiple sequence alignment and the manual search, we found three variations of the WRKYGQK heptapeptide: WRKYGRK, WKKYGQK and WRKYGKK, and four variations of the normal zinc finger motif, which might execute some new biological functions. In addition, the SsWRKY genes from the same subgroup share the similar exon-intron structures and conserved motif domains. Further studies of SsWRKY genes revealed that segmental duplication events (SDs) played a more prominent role in the expansion of SsWRKY genes. Distinct expression profiles of SsWRKY genes with RNA sequencing data revealed that diverse expression patterns among five tissues, including tender roots, young leaves, vegetative buds, non-lignified stems and barks. With the analyses of WRKY gene family in willow, it is not only beneficial to complete the functional and annotation information of WRKY genes family in woody plants, but also provide important references to investigate the expansion and evolution of this gene family in flowering plants.

Keywords: Duplication; Evolution; Expression; Phylogenetic analysis; WRKY protein; Willow.

Figure 1. Comparison of the WRKY domain sequences from 85 SsWRKY genes.

The WRKY gene with the suffix -N and -C indicates the N-terminal and C-terminal WRKY domain of group I members, respectively. “-” has been inserted for the optimal alignment. Red indicates the highly conserved WRKYGQK heptapeptide, and the zinc finger motifs are highlighted in green. The position of a conserved intron is indicated by an arrowhead.

Figure 2. Chromosomal location of SsWRKY genes.

Red indicates group I, blue indicates group II and green indicates group III. Red lines indicate gene clusters. The chromosome numbers are given at the top of each chromosome and the left side of each chromosome is related to the approximate physical location of each WRKY gene. Only one unmapped SsWRKY gene is shown on ChrN.

Figure 3. Phylogenetic tree of WRKY domains from willow and Arabidopsis.

The phylogenetic tree was constructed using the neighbor-joining method in MEGA 6.0. The WRKY genes with the suffix ‘N’ and ‘C’ indicate the N-terminal and the C-terminal WRKY domains of group I, respectively. The different colors indicate different groups (I, II and III) or subgroups (IIa, b, c, d and e) of WRKY domains. Circles indicate WRKY genes from willow, and diamonds represent genes from Arabidopsis. The purple trapezoid region indicate a new subgroup belonging to IIc.

Figure 4. Genomic organization of SsWRKY genes.

(A) The phylogenetic tree built on the basis of full-length SsWRKY genes was depicted using the neighbor-joining method in MEGA 6.0. The short black lines indicate the existence of duplicated gene pairs; (B) The graphic exon–intron structure of SsWRKY genes is displayed using GSDS. Green indicates exons, and gray indicates introns. The introns phases 0, 1 and 2 are indicated by numbers 0, 1 and 2, respectively.

Figure 5. Expression profiles of the 85 SsWRKY genes in root, stem, bark, bud and leaf.

Color scale represents RPKM normalized log2 transformed counts and red indicates high expression, blue indicates low expression and white indicates the gene is not expressed in this tissue.