Genome-Wide Analyses of CCHC Family Genes and Their Expression Profiles under Drought Stress in Rose (Rosa chinensis)

Abstract

CCHC-type zinc finger proteins (CCHC-ZFPs), ubiquitous across plant species, are integral to their growth, development, hormonal regulation, and stress adaptation. Roses (Rosa sp.), as one of the most significant and extensively cultivated ornamentals, account for more than 30% of the global cut-flower market. Despite its significance, the CCHC gene family in roses (Rosa sp.) remains unexplored. This investigation identified and categorized 41 CCHC gene members located on seven chromosomes of rose into 14 subfamilies through motif distribution and phylogenetic analyses involving ten additional plant species, including Ginkgo biloba, Ostreococcus lucimarinus, Arabidopsis thaliana, and others. This study revealed that dispersed duplication likely plays a crucial role in the diversification of the CCHC genes, with the Ka/Ks ratio suggesting a history of strong purifying selection. Promoter analysis highlighted a rich presence of cis-acting regulatory elements linked to both abiotic and biotic stress responses. Differential expression analysis under drought conditions grouped the 41 CCHC gene members into five distinct clusters, with those in group 4 exhibiting pronounced regulation in roots and leaves under severe drought. Furthermore, virus-induced gene silencing (VIGS) of the RcCCHC25 member from group 4 compromised drought resilience in rose foliage. This comprehensive analysis lays the groundwork for further investigations into the functional dynamics of the CCHC gene family in rose physiology and stress responses.

Keywords: VIGS; abiotic stress; cut flowers; gene family analysis; zinc finger proteins.

Figure 1

Chromosomal distribution of CCHC gene family in roses. This diagram illustrates the localization of CCHC genes on rose chromosomes, depicted as green rectangles. The chromosome numbers are indicated above each rectangle, while the scale on the left shows the length of such chromosome in megabases (Mb). Positions of CCHC genes are precisely marked along the chromosomes.

Figure 2

Phylogenetic analysis, gene structures, and protein-conserved motifs of the rose CCHC gene family. (A) Phylogenetic tree of the CCHC gene family in rose, with branches color-coded to differentiate subfamilies. Subfamily names are annotated to the right of corresponding gene names. (B) Gene structure analysis of the rose CCHC gene family. Among them, yellow boxes represent exons, untranslated regions (UTRs) are indicated by green boxes, and introns are indicated by gray lines. (C) Distribution of conserved protein motifs within the rose CCHC gene family, with each motif depicted by a differently colored box. The length of each line corresponds to the protein length. Motif designations are listed in the lower right-hand corner of the figure.

Figure 3

Gene duplication modes within the rose CCHC gene family. The gray lines represent all homologous gene pairs identified in roses. Lines colored in red, green, blue, and black denote gene pairs resulting from disperse duplication (DSD), retrotransposed duplication (TRD), whole-genome duplication (WGD), and proximal duplication (PD), respectively. The outermost circle represents the seven chromosomes of rose, with the position of CCHC genes marked accordingly. The middle circle presents histograms of gene density and the innermost circle features a heatmap indicating gene density across the rose chromosomes.

Figure 4

Phylogenetic tree of CCHC genes across three species. The colors of the outer ring denote distinct subfamilies, and subfamily names are listed on the right. Preceding each gene name, colored circles identify the species, with corresponding scientific names delineated on the right side of the figure.

Figure 5

Collinearity analysis of CCHC genes between rose and other representative species. (A) Collinearity of CCHC genes between rose and four other representative species. Each rectangle represents a chromosome. Gray lines map all homologous gene pairs between rose and the representative species, highlighting the comprehensive genetic relationships. Blue lines specifically indicate the collinear CCHC gene pairs, emphasizing genes with shared evolutionary paths. The names of the species involved are listed on the left side of the panel. (B) Evolution tree of rose and nine other species, excluding Ostreococcus lucimarinus. The tree visualizes the evolutionary relationships and divergence among the species, based on the analysis of their CCHC genes, illustrating both close and distinct genetic affiliations.

Figure 6

Prediction of cis-acting elements in promoters of rose CCHC genes. The number of each type of element is represented through varying color gradients, offering a visual representation of their prevalence. These cis-acting elements are classified into three functional categories: abiotic and biotic stress, phytohormone responsive, and plant growth and development. The histogram on the right side of the figure uses yellow, green, and blue rectangles to represent the number of cis-acting elements for the three types mentioned above. The length of each rectangle correlates with the quantity of each type of cis-acting element found, providing a quick visual assessment of their distribution and potential regulatory significance in rose CCHC genes.

Figure 7

Expression profile of the CCHC gene family in rose under drought stress. (A) ND (no drought), MD (mild drought), and SD (severe drought) indicate the levels of drought stress experienced, while L (leaves) and R (roots) represent the tissue types examined. The genes are organized into five groups according to their expression trends, with each group labeled beneath the dendrogram. (B–G) Rt-qPCR analysis of six RcCCHC-ZFP family members in rose leaves at five periods of drought. Error bars indicate SE (n = 3). Student’s t-test was used for all statistical analyses (** p <0.01, *** p < 0.001, **** p <0.0001, ns means not significant).

Figure 8

Protein–protein interaction network of the CCHC gene family in rose. The dashed lines represent interactions between proteins. Each green circle symbolizes a protein, with the circle’s size reflecting the number of interactions that protein has within the network.

Figure 9

Functional analysis of RcCCHC25 gene. (A) Plant phenotypes after dehydration (Dehyd) of TRV (TRV-empty vector) and TRV-RcCCHC25. (B) TRV and TRV-RcCCHC25 phenotypes in leaves following dehydration (Dehyd) and subsequent rehydration (Rehyd). Bar = 1 cm. (C) comparative expression levels of RcCCHC25 in both TRV and TRV-RcCCH25 leaves. (D) Ion leakage rate. (E) Wilting ratio of the leaf area in TRV and TRV-RcCCHC25 leaves. Error bars indicate SE (n = 3). Student’s t-test was used for all statistical analyses (** p < 0.01, *** p < 0.001, ns means not significant).