Characterization of WRKY Gene Family in Whole-Genome and Exploration of Flowering Improvement Genes in Chrysanthemum lavandulifolium

Abstract

Chrysanthemum is a well-known ornamental plant with numerous uses. WRKY is a large family of transcription factors known for a variety of functions ranging from stress resistance to plant growth and development. Due to the limited research on the WRKY family in chrysanthemums, we examined them for the first time in Chrysanthemum lavandulifolium. A total of 138 ClWRKY genes were identified, which were classified into three groups. Group III in C. lavandulifolium contains 53 members, which is larger than group III of Arabidopsis. The number of introns varied from one to nine in the ClWRKY gene family. The "WRKYGQK" motif is conserved in 118 members, while other members showed slight variations. AuR and GRE responsive cis-acting elements were located in the promoter region of WRKY members, which are important for plant development and flowering induction. In addition, the W box was present in most genes; the recognition site for the WRKY gene may play a role in autoregulation and cross-regulation. The expression of the most variable 19 genes in terms of different parameters was observed at different stages. Among them, 10 genes were selected due to the presence of CpG islands, while nine genes were selected based on their close association with important Arabidopsis genes related to floral traits. ClWRKY36 and ClWRKY45 exhibit differential expression at flowering stages in the capitulum, while methylation is detected in three genes, including ClWRKY31, ClWRKY100, and ClWRKY129. Our results provide a basis for further exploration of WRKY members to find their functions in plant growth and development, especially in flowering traits.

Keywords: CpG islands; WRKY transcription factors; cis-acting elements; flowering traits; methylation; motifs; ornamental.

FIGURE 1

A screenshot of the MEGA X version 10.2.6, showing variation in the conserved domain (WRKYGQK) of ClWRKY proteins, and the variation of the conserved domain is encircled in different colors.

FIGURE 2

A phylogenetic tree of 138 ClWRKY proteins of Chrysanthemum lavandulifolium (blue) and 74 AtWRKY proteins of Arabidopsis thaliana (red) on the basis of amino acid sequences. The tree was constructed in MEGA X. The domains were clustered into three groups, namely, group I, group II, and group III. The group II was divided into five subgroups (a–e).

FIGURE 3

The intron-exon structure of 138 ClWRKY members according to the phylogenetic relationship. A phylogenetic tree was constructed with the ClWRKY protein sequences using MEGA X. The Gene Structure Display Server (GSDS) software was used to determine the intron-exon structure. Green bars represent exons in the gene. Different colors of WRKY members represent different groups in the ClWRKY family.

FIGURE 4

Schematic diagram of conserved motifs in ClWRKY proteins according to the phylogenetic relationship. Different color ClWRKY members indicate different groups. The conserved motifs were predicted using the TBtools software.

FIGURE 5

ClWRKY31, ClWRKY100, and ClWRKY129 Methylation-Specific Polymerase Chain Reaction (MSP) results (A–C). An MSP was conducted to determine the methylation status of selected genes. The PCR product length of the genes was as follows: ClWRKY31 = 156 bp, ClWRKY100 = 135 bp, and ClWRKY129 = 131 bp. “M” represents a methylated PCR product, and “U” represents an unmethylated PCR product. (D) The genomic DNA results for all samples. These results were verified through gel (1.5%) electrophoresis. The abbreviations of different growth stages and tissues are as follows: YR, young stage root; YS, young stage stem; YL, young stage leaf; AR, adult stage root; AS, adult stage stem; AL, adult stage leaf; FR, flowering stage root; FS, flowering stage stem; FL, flowering stage leaf; LB, little buds; MB, median buds; FF, first flower at just opening time.

FIGURE 6

Cis-acting element analysis in the promoter region of the ClWRKY family. The PlantCARE software was used to search the cis-acting elements. Abbreviation of important cis-acting elements are as follows: EE, endosperm expression; ARE, auxin-responsive elements; GR, gibberellin responsiveness; LR, light responsiveness; REEAI, regulatory element essential for anaerobic induction; AuR, elements involved in auxin responsiveness; SSR, seed-specific regulation; MeJAR, methyl jasmonate responsiveness; ERE, ethylene-responsive elements; GRE, gibberellin responsive elements; ESNE, endosperm specific negative expression; LR2, elements involved in light response; PAP2L, plant-AP2 such as AuR2, part of auxin responsiveness; and W-box, recognition site of WRKY TFs.

FIGURE 7

Expression analysis of the selected ClWRKY genes in different tissues (capitulum, leaf, stem, and root) at different stages (young, adult, and flowering). Expression of 19 genes: 10 genes were selected due to the presence of CpG island, nine genes were selected based on their close association with important Arabidopsis genes found in the phylogenetic tree built between C. lavandulifolium and A. thaliana. Abbreviations of different growth stages and tissues are as follows: YSR, young stage root; YSS, young stage stem; YSL, young stage leaf; ASR, adult stage root; ASS, adult stage stem; ASL, adult stage leaf; FSR, flowering stage root; FSS, flowering stage stem; FSL, flowering stage leaf; FSLB, Flowering stage little buds; FSMB, Flowering stage buds; FSF, Flowering stage first flower.