Identification of WRKY Family Members and Characterization of the Low-Temperature-Stress-Responsive WRKY Genes in Luffa (Luffa cylindrica L.)

Abstract

The plant-specific WRKY transcription factor family members have diverse regulatory effects on the genes associated with many plant processes. Although the WRKY proteins in Arabidopsis thaliana and other species have been thoroughly investigated, there has been relatively little research on the WRKY family in Luffa cylindrica, which is one of the most widely grown vegetables in China. In this study, we performed a genome-wide analysis to identify L. cylindrica WRKY genes, which were subsequently classified and examined in terms of their gene structures, chromosomal locations, promoter cis-acting elements, and responses to abiotic stress. A total of 62 LcWRKY genes (471-2238 bp) were identified and divided into three phylogenetic groups (I, II, and III), with group II further divided into five subgroups (IIa, IIb, IIc, IId, and IIe) in accordance with the classification in other plants. The LcWRKY genes were unevenly distributed across 13 chromosomes. The gene structure analysis indicated that the LcWRKY genes contained 0-11 introns (average of 4.4). Moreover, 20 motifs were detected in the LcWRKY proteins with conserved motifs among the different phylogenetic groups. Two subgroup IIc members (LcWRKY16 and LcWRKY31) contained the WRKY sequence variant WRKYGKK. Additionally, nine cis-acting elements related to diverse responses to environmental stimuli were identified in the LcWRKY promoters. The subcellular localization analysis indicated that three LcWRKY proteins (LcWRKY43, LcWRKY7, and LcWRKY23) are localized in the nucleus. The tissue-specific LcWRKY expression profiles reflected the diversity in LcWRKY expression. The RNA-seq data revealed the effects of low-temperature stress on LcWRKY expression. The cold-induced changes in expression were verified via a qRT-PCR analysis of 24 differentially expressed WRKY genes. Both LcWRKY7 and LcWRKY12 were highly responsive to the low-temperature treatment (approximately 110-fold increase in expression). Furthermore, the LcWRKY8, LcWRKY12, and LcWRKY59 expression levels increased by more than 25-fold under cold conditions. Our findings will help clarify the evolution of the luffa WRKY family while also providing valuable insights for future studies on WRKY functions.

Keywords: Luffa cylindrica; WRKY transcription factors; abiotic stress; expression analysis.

Figure 1

Chromosomal distribution of the LcWRKY genes. The chromosomal position of each LcWRKY gene can be determined using the scale on the left.

Figure 2

Luffa cylindrica LcWRKY promoter elements and structures according to phylogenetic relationships. The phylogenetic tree was constructed on the basis of the full-length L. cylindrica WRKY protein sequences using MEGA (version 7.0). The proportional lengths of the WRKY genes are presented. The groups are differentiated by color. (a) The promoter elements were analyzed using the TBtools software (version 1.6). (b) The LcWRKY gene structures were examined using the Gene Structure Display Server 2.0 software.

Figure 3

Conserved motifs in L. cylindrica WRKY proteins according to phylogenetic relationships. (a) Unrooted phylogenetic tree constructed on the basis of full-length WRKY protein sequences using MEGA (version 7.0). (b) Distribution of conserved motifs among WRKY proteins. Different motifs are indicated by different colored blocks as indicated at the top of the figure.

Figure 4

Tissue-specific and low-temperature-stress-induced LcWRKY expression profiles. Transcriptome data were used to determine the LcWRKY expression profiles. The color scale represents the FPKM normalized log₁₀-transformed counts, with blue and red indicating low and high expression levels, respectively. (a) Tissue-specific LcWRKY expression profiles. (b) LcWRKY expression levels in the leaves at five time-points during a low-temperature treatment.

Figure 5

Quantitative real-time polymerase chain reaction analysis of the expression of selected LcWRKY genes associated with the L. cylindrica leaf response to low-temperature stress. The 18S rRNA gene was used as the internal control. Error bars represent the standard error of three biological replicates.

Figure 6

Regression analysis of the fold-change values determined on the basis of transcriptome sequencing (RNA-seq) and qRT-PCR data. For the RNA-seq analysis, the FPKM values at 2, 4, 8, and 12 h were compared with the FPKM value at 0 h to calculate the fold-change. For the qRT-PCR analysis, the expression levels at 2, 4, 8, and 12 h were normalized against the expression level at 0 h to calculate the fold-change. **, significant correlation (p < 0.01).

Figure 7

Subcellular localization of three LcWRKY proteins in the lower epidermal cells of Nicotiana benthamiana. The green fluorescence, visible light, and merged green fluorescence and visible light images are presented. 35S::GFP: Agrobacterium tumefaciens strain carrying the empty vector (pCAMBIA1300-GFP); 35S::LcWRKY::GFP: A. tumefaciens strain carrying a recombinant vector (pCAMBIA1300-LcWRKY7-GFP, pCAMBIA1300-LcWRKY23-GFP, or pCAMBIA1300-LcWRKY43-GFP). Scale bars = 50 µM.