Whole Genome Analysis of Cyclin Dependent Kinase (CDK) Gene Family in Cotton and Functional Evaluation of the Role of CDKF4 Gene in Drought and Salt Stress Tolerance in Plants

Abstract

Cotton (Gossypium spp.) is the number one crop cultivated for fiber production and the cornerstone of the textile industry. Drought and salt stress are the major abiotic stresses, which can have a huge economic impact on cotton production; this has been aggravated with continued climate change, and compounded by pollution. Various survival strategies evolved by plants include the induction of various stress responsive genes, such as cyclin dependent kinases (CDKs). In this study, we performed a whole-genome identification and analysis of the CDK gene family in cotton. We identified 31, 12, and 15 CDK genes in G. hirsutum, G. arboreum, and G. raimondii respectively, and they were classified into 6 groups. CDK genes were distributed in 15, 10, and 9 linkage groups of AD, D, and A genomes, respectively. Evolutionary analysis revealed that segmental types of gene duplication were the primary force underlying CDK genes expansion. RNA sequence and RT-qPCR validation revealed that Gh_D12G2017 (CDKF4) was strongly induced by drought and salt stresses. The transient expression of Gh_D12G2017-GFP fusion protein in the protoplast showed that Gh_D12G2017 was localized in the nucleus. The transgenic Arabidopsis lines exhibited higher concentration levels of the antioxidant enzymes measured, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) concentrations under drought and salt stress conditions with very low levels of oxidants. Moreover, cell membrane stability (CMS), excised leaf water loss (ELWL), saturated leaf weight (SLW), and chlorophyll content measurements showed that the transgenic Arabidopsis lines were highly tolerant to either of the stress factors compared to their wild types. Moreover, the expression of the stress-related genes was also significantly up-regulated in Gh_D12G2017(CDKF4) transgenic Arabidopsis plants under drought and salt conditions. We infer that CDKF-4s and CDKG-2s might be the primary regulators of salt and drought responses in cotton.

Keywords: abiotic stress; cotton cyclin dependent kinase; gene expression; oxidant and antioxidant enzymes; plant growth and development; segmental duplication; transgenic arabidopsis lines.

Figure 1

Gene structure and motif compositions of cotton CDK genes. (A) Exon/intron structures of CDK genes in cotton, exons introns and up/down-stream were represented by yellow boxes, black lines, and blue boxes, respectively. (B) MEME software characterized conserved motifs of G. raimondii (D), G. hirsutum (AD), and G. arboreum (A). Different colors represent different motifs and each motif is represented by a box numbered at the bottom. The names of genes and combined P value are exhibited on the left side of the figure. Grey lines represent the non-conserved sequences; the length of protein can be estimated using the scale at the bottom.

Figure 2

Phylogenetic tree and motif identification among the CDK proteins. (A) The neighbor-join phylogenetic tree of Cotton, Rice and Arabidopsis CDK genes. The red circle: CDKs from rice, blue: CDKs from G. hirsutum, green: CDKs from G. raimondii, brown: CDKs from Arabidopsis and purple: CDKs from G. arboreum. (B) Alignment of CDK motif domain. Color shading indicates types of amino acid residues conserved. S: motif, K: motif, P: motif and D: motif.

Figure 3

Average number of the cis-promoters ABRELATERD1 (ACGTG), DRECRTCOREAT (G/ACCGAC), MYBCORE (TAACTG), LTRE1HVBLT49 (CCGAC) and others in promoter region of the three cotton genomes (A) Gossypium hirsutum; (B) Gossypium raimondii and (C) Gossypium arboreum, CDK genes from each CDK subfamilies. The promoter regions were analyzed in the 1 kb upstream promoter region of translation start site using the PLACE database.

Figure 4

RNA sequence profiling of the CDK gene expression under salt and drought stress. (I) Heat map displaying expression changes of differentially expressed CDK genes in upland cotton, G. hirsutum plants stressed for 1 h, 3 h, 6 h, and 12 h of salt stress compared with the control; (II) Venn diagram depicting the expression of the CDK genes in various tissues and (III): Heat map displaying expression changes of differentially expressed CDK genes in upland cotton, G. hirsutum plant stressed for 1 h, 3 h, 6 h and 12 h of drought compared with the control.

Figure 5

Transcriptome profiling of CDK gene expression through RT-qPCR under salt and drought stress. (A–C) Heat map displaying expression changes of differentially expressed CDK genes in G. hirsutum, G. raimondii, and G. arboreum respectively plants stressed for 3 h, 6 h, 12 h and 24 h of salt and drought stress compared with the control; (I, II and III) Venn diagram depicting the number of up regulated CDK genes in root and leaf tissues at 24 h of drought (PEG) and salt (NaCl) stress exposure.

Figure 6

RT-qPCR expression profile of the top five highly expressed CDK genes in RNA sequences as compared to control with expression value 1.0 in upland cotton under drought stress. Gh: Gossypium hirsutum, BC: BC₂F₁ generation, Gt: Gossypium tomentosum. 0, 7 and 14 are days of drought stress exposure. I: Expression on leaf tissue; II: Expression on stem tissue and III: Expression on root tissue.

Figure 7

Localization of Gh_D12G2017 (CDKF4) in onion epidermal cells. (A–C) Onion epidermal cells transformed with 35S::GFP. (D–F) Onion epidermal cells transformed with 35S: Gh_D12G2017_GFP (A,D) Light field with magnification of X400 to display morphology. (B,E) Dark field images for the detection of green fluorescent protein (GFP) fluorescence. (C,F) Superimposed light and dark field images.

Figure 8

The RT-qPCR analysis of the expression of the cloned gene (A) Total RNA isolated from various tissue of cotton plant under normal conditions; (B) Total RNA extracted from drought-stressed cotton seedlings (C) Total RNA extracted from salt-stressed cotton seedlings; (D) Polymerase chain reaction (PCR) analysis performed to check 1299 bp coding sequence (CDS) integration in transformed T₁ generation, number 1–10 transgenic Arabidopsis lines, 11 negative control (wild type) and 12 is the positive control (pWM101-Gh_D12G2017 (CDKF4) (E) The gene expression levels of the Gh_D12G2017 (CDKF4) of T₂ transgenic Arabidopsis lines analyzed through RT-qPCR, in three biological replicates. Different letters (a, b, and c) indicate significant differences between expression levels of the gene in different tissues of the Gh_D12G2017 (CDKF4) overexpressed Arabidopsis lines (ANOVA; p < 0.05).

Figure 9

Determination of physiological traits (A) Transgenic Arabidopsis lines and wild type under abiotic stress conditions (B). chlorophyll content determination (C) Quantitative determination of relative water content (RLWC) (D) Quantitative determination of excised leaf water loss ELWL (E) Quantitative determination of cell membrane stability (CMS) as ion leakage concentration in leaves of wild type and transgenic Arabidopsis lines (L4, L5 and L7) after 8-day post stress exposure. Each experiment was repeated three times. Bar indicates standard error (SE). Different letters indicate significant differences between wild type and transgenic Arabidopsis lines (ANOVA; p < 0.05). CK: normal conditions.

Figure 10

Determination of oxidant and antioxidant enzymes under drought and salt stress conditions (A) Quantitative determination of hydrogen peroxide (H₂O₂) concentration (B) Quantitative determination of malondialdehyde (MDA) concentration (C) Quantitative determination of POD concentration. (D) Quantitative determination of SOD concentration. (E) Quantitative determination of CAT in leaves of wild type and transgenic Arabidopsis lines (L4, L5 and L7) after 8-day post stress exposure. Each experiment was repeated three times. Bar indicates standard error (SE). Different letters indicate significant differences between wild type and the transgenic Arabidopsis lines (ANOVA; p < 0.05). CK: normal conditions.

Figure 11

Expression levels of abiotic stress-responsive genes (ABF4, CBL1 and RD29B) in transgenic Arabidopsis lines (L4, L5 and L7) and wild type Arabidopsis Atactin2 gene was used as the reference, each experiment was repeated three times, mean values with ± SD, (a,b) calculated by Student’s t-test with p < 0.05.