Genome-Wide Characterization of WRKY Gene Family in Camellia chekiangoleosa Identifies Potential Regulatory Components in Pigment Biosynthesis Pathways

Abstract

The WRKY gene family is essential for controlling a variety of plant physiological functions, yet the involvement of specific WRKY members in pigment biosynthesis and accumulation in Camellia chekiangoleosa remains unexplored, particularly in anthocyanins and carotenoids, which play crucial roles in the pigmentation of C. chekiangoleosa. This study systematically identified 87 WRKY genes across 15 chromosomes in C. chekiangoleosa through bioinformatic approaches. Further structural and phylogenetic analyses of these TFs enabled their classification into six different subgroups. WRKY family expansion was shown to be mostly driven by tandem duplication. W-box elements, which can be binding sites for WRKY transcription factors, were present in a number of biosynthetic genes in the pigment production pathway. Yeast one-hybrid assay confirmed that five WRKY transcription factors (CchWRKY15/24/33/47/76) directly bind to the promoter regions of two key biosynthetic genes, CchPSY1 and Cch4CL1. Intriguingly, among the five WRKYs tested, the expression levels of CchWRKY15, CchWRKY33, and CchWRKY47 showed the strongest positive associations with flavonoid accumulation (p < 0.05, Pearson correlation analysis).These findings provide novel insights into the evolutionary patterns, transcriptional regulation, and functional characteristics of CchWRKYs, while elucidating their possible regulatory mechanisms in the fruit coloration of C. chekiangoleosa.

Keywords: Camellia chekiangoleosa; WRKY gene family; fruit coloration; pigment synthesis; transcriptional regulation.

Figure 1

Chromosomal localization and gene clusters of CchWRKYs. Four colors are used to distinguish the groups corresponding to the genes. Red font represents Group I, blue font represents Group II, green font represents Group III, and gray font represents Group IV. The tandemly repeated genes are within the black boxes.

Figure 2

Phylogenetic tree of WRKY genes from Camellia chekiangoleosa and Arabidopsis thaliana. The N-terminal and C-terminal WRKY domains of Group I are represented by the groups with the letters N and C. WRKY groups I, II, III, and IV, as well as their subgroups IIa/b, IIc, and IId/e, are represented by distinct colors.

Figure 3

Represents motif (a) and conserved domain (b) distribution analysis of CchWRKYs.

Figure 4

Gene structure analysis of CchWRKY. The coding sequence is shown by the yellow portion, introns are shown by the black short lines, the untranslated area is shown by the blue portion, and the numbers on black short lines indicate intron phases.

Figure 5

Collinearity analysis of CchWRKYs. (a) CchWRKY homologous gene pairs in whole genome. Black lines link the homologous gene pairs of CchWRKY, whereas gray lines depict the background whole genome. (b) CchWRKYs interspecies collinearity study within Populus trichocarpa and A. Thaliana genomes. Blue lines indicate collinear genes of the WRKY family, while gray lines indicate collinear genes in general. Numerical labels denote chromosome numbers.

Figure 6

Examination of cis-acting components in CchWRKY promoters.

Figure 7

A heatmap showing the CchWRKY expression level in fruit samples at various phases of development. Blue denotes low expression, whereas red denotes strong expression. The six sample periods are indicated by S1–S6. Group I is represented by red, Group IIa/b by green, Group IIc by light blue, Group IId/e by dark blue, Group III by purple, and Group IV by pink color coding.

Figure 8

(a) Comparison of temporal trends in three pigment contents during different developmental stages. (b) Correlation between CchWRKY15 expression levels and flavonoid content during different developmental stages. (c) Correlation between CchWRKY33 expression levels and flavonoid content during different developmental stages. (d) Correlation between CchWRKY47 expression levels and flavonoid content during different developmental stages.

Figure 9

Yeast one-hybrid assay. (a) Positions of W-boxes in the promoter sequences of CchPSY1 and Cch4CL1; (b) schematic diagrams of the structures of prey and bait expression vectors; and (c) direct binding of CchWRKYs and CchPSY1 and Cch4CL1 in the yeast system. The interactions between pGADT7-53 and pAbAi-53 and pAbAi-PSY1/4CL1 serve as positive and negative controls, respectively.