Decoding drought resilience: a comprehensive exploration of the cotton Eceriferum (CER) gene family and its role in stress adaptation

Abstract

Background: The cuticular wax serves as a primary barrier that protects plants from environmental stresses. The Eceriferum (CER) gene family is associated with wax production and stress resistance.

Results: In a genome-wide identification study, a total of 52 members of the CER family were discovered in four Gossypium species: G. arboreum, G. barbadense, G. raimondii, and G. hirsutum. There were variations in the physicochemical characteristics of the Gossypium CER (GCER) proteins. Evolutionary analysis classified the identified GCERs into five groups, with purifying selection emerging as the primary evolutionary force. Gene structure analysis revealed that the number of conserved motifs ranged from 1 to 15, and the number of exons varied from 3 to 13. Closely related GCERs exhibited similar conserved motifs and gene structures. Analyses of chromosomal positions, selection pressure, and collinearity revealed numerous fragment duplications in the GCER genes. Additionally, nine putative ghr-miRNAs targeting seven G. hirsutum CER (GhCER) genes were identified. Among them, three miRNAs, including ghr-miR394, ghr-miR414d, and ghr-miR414f, targeted GhCER09A, representing the most targeted gene. The prediction of transcription factors (TFs) and the visualization of the regulatory TF network revealed interactions with GhCER genes involving ERF, MYB, Dof, bHLH, and bZIP. Analysis of cis-regulatory elements suggests potential associations between the CER gene family of cotton and responses to abiotic stress, light, and other biological processes. Enrichment analysis demonstrated a robust correlation between GhCER genes and pathways associated with cutin biosynthesis, fatty acid biosynthesis, wax production, and stress response. Localization analysis showed that most GCER proteins are localized in the plasma membrane. Transcriptome and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) expression assessments demonstrated that several GhCER genes, including GhCER15D, GhCER04A, GhCER06A, and GhCER12D, exhibited elevated expression levels in response to water deficiency stress compared to control conditions. The functional identification through virus-induced gene silencing (VIGS) highlighted the pivotal role of the GhCER04A gene in enhancing drought resistance by promoting increased tissue water retention.

Conclusions: This investigation not only provides valuable evidence but also offers novel insights that contribute to a deeper understanding of the roles of GhCER genes in cotton, their role in adaptation to drought and other abiotic stress and their potential applications for cotton improvement.

Keywords: Biosynthesis of cuticle wax; Eceriferum (CER); Gene ontology; Stress conditions; Synteny; micro-RNA.

Fig. 1

The phylogenetic analysis of CER genes among Arabidopsis and four cotton species. The phylogenetic tree was constructed using the program MEGA11.0. (The likelihood) algorithm, bootstrap value = 1000). The prefixes Ga, Gr, Gh and Gb represent G. arboretum, G. raimondii, G. hirsutum and G. barbadense, respectively

Fig. 2

Chromosomal locations and gene duplication events of CER genes. The corresponding chromosome numbers are given to the left of each bar. CER gene pairs resulting from segmental and tandem duplications are connected by lines. Distribution of CER genes on chromosomes of G. hirsutum (A), G. barbadense (B), G. arboreum (C), G. raimondii (D)

Fig. 3

Multiple synteny analysis of cotton CER genes in cotton. Multiple synteny analysis was used to reveal the orthologous relationship of CER genes between (A) G. hirsutum and its ancestors G. arboreum, G. raimondii and (B) G. barbadense and its ancestors G. arboreum, G. raimondii. The chromosomes of the different cotton species are represented by different colours. (C) Collinearity analysis of the CER genes of G. hirsutum. and (D) collinearity analysis of the CER genes of G. barbadense. A01 to A13 represent chromosomes of the A subgenome, while D01 to D13 represent chromosomes of the D subgenome

Fig. 4

Phylogenetic relationships and structural characteristics of CER genes. (A) Phylogenetic analysis using the likelihood method for the CER family in four cotton species. (B) Patterns of predicted motifs within CER proteins. (C) Gene structures of the CER genes. Pink symbols represent untranslated 5′ and 3′ regions, blue symbols represent coding sequences (CDS), and lines indicate introns

Fig. 5

A. Analysis of cis-regulatory elements on CER genes. B. Total number of cis-elements and CER genes involved in four categories. Percentage (%) ratio of numerous cis-elements from each category is shown in bar graphs; B. plant growth and development responsive, (C) phytohormone responsive, (D) light responsive, (E) stress responsive

Fig. 6

A. Shows the predicted regulatory protein-protein interaction network of CER. The network was created using the online software STRING. The proteins were represented at the network nodes with 3D structures of the proteins in the nodes, and the line colors indicate different data sources. B and C represent different clusters

Fig. 7

Putative transcription factor regulatory network analysis of CER genes. (A) Top TFs involved in the regulation of GhCER genes. (B) Green hexagonal nodes represent transcription factors; orange hexagonal nodes represent GhCER genes, and node size represents the degree of interaction between nodes based on the degree value

Fig. 8

Predicted miRNA targeting CER genes. (A) Network representation of predicted miRNA-targeted CER genes. (B) Schematic representation of CER genes targeted by miRNAs

Fig. 9

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of CER genes (A) Highly enriched GO terms in CER genes. (B) Highly enriched KEGG pathways in CER genes

Fig. 10

Expression profile of 16 GhCER genes in upland cotton. (A) A heat map showing the expression profile of GhCER genes in different tissues of cotton. (B) Expression pattern analysis of GhCER genes in response to abiotic stress. FPKM values were log2 transformed and the heatmap was constructed using TBTools software. The red colour shows the highest and the blue colour shows the lowest expression levels in the expression bar

Fig. 11

Relative expressions of GhCER genes. qRT-PCR analysis was performed to observe the relative expression patterns of GhCER genes at 3 h, 6 h and 12 h under control and drought stress conditions. Vertical bars represent mean ± SD (n = 3). * and ** indicate significance at p ≤ 0.05 and p ≤ 0.01, respectively

Fig. 12

Silencing of the GhCER04A. A gene reduced the tolerance of cotton plants to drought stress. A Plant with albino phenotype (TRV: GhCLA1, positive control). B Phenotype of the negative control (TRV: null) and the transgenic plants with silenced GhCER04A gene (TRV:GhCER04A) under drought conditions for 48 h. C Relative expression of GhCER04A in the control plants (TRV: null) and the silenced plants (TRV:GhCER04A). The GhUBQ7 gene was used as an internal control. D-F Physiological indicators were measured on plants grown under control and drought conditions. D proline content; E malondialdehyde (MDA) content; (F) water loss rate; error bars indicate the standard deviation from three independent experiments