Genome-wide comparative and evolutionary analysis of Calmodulin-binding Transcription Activator (CAMTA) family in Gossypium species

Abstract

The CAMTA gene family is crucial in managing both biotic and abiotic stresses in plants. Our comprehensive analysis of this gene family in cotton resulted in the identification of 6, 7 and 9 CAMTAs in three sequenced cotton species, i.e., Gossypium arboreum, Gossypium raimondii, and Gossypium hirsutum, respectively. All cotton CAMTAs were localized in the nucleus and possessed calmodulin-binding domain (CaMBD) as identified computationally. Phylogenetically four significant groups of cotton CAMTAs were identified out of which, Group II CAMTAs experienced higher evolutionary pressure, leading to a faster evolution in diploid cotton. The expansion of cotton CAMTAs in the genome was mainly due to segmental duplication. Purifying selection played a significant role in the evolution of cotton CAMTAs. Expression profiles of GhCAMTAs revealed that GhCAMTA2A.2 and GhCAMTA7A express profoundly in different stages of cotton fiber development. Positive correlation between expression of these two CAMTAs and fiber strength confirmed their functional relevance in fiber development. The promoter region of co-expressing genes network of GhCAMTA2A.2 and GhCAMTA7A showed a higher frequency of occurrence of CAMTA binding motifs. Our present study thus contributes to broad probing into the structure and probable function of CAMTA genes in Gossypium species.

Figure 1

Schematic representation of functional domains of GaCAMTAs, GrCAMTAs and GhCAMTAs. Bioinformatics analysis of functional conserved domains were performed by Pfam database (http://pfam.janelia.org/). NLS and CaMBD were specifically searched in Motif scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan) and Calmodulin Target Database (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/) respectively. The domain structure of GaCAMTAs, GrCAMTAs and GhCAMTAs were drawn using Illustrator for Biological sequences software (http://ibs.biocuckoo.org/).

Figure 2

Conservation of CaMBD in all cotton CAMTA proteins. (a) Functionally proved motif in Arabidopsis CAMTAs. (b) Alignment of conserved CaMBD of cotton CAMTAs with 6 AtCAMTAs. (c) Sequence logo of the CaMBD of 22 putative cotton CAMTAs and 6 AtCAMTAs. In the square brackets “[]” are the amino acids allowed in this position of the motif; “X” represents any amino acid and the round brackets “()” indicate the number of amino acids. (d) Amphipathic α-helix structure in the predicted CaMBD of GhCAMTA2A.2 amino acid residues (Val⁹³²-Leu⁹⁴⁹). Circles, diamonds, triangles and pentagons represent hydrophilic residues, hydrophobic residues, potentially negatively charged and potentially positively charged residues respectively.

Figure 3

Phylogenetic relationships of cotton CAMTAs from Arabidopsis and other plant species. The unrooted phylogenetic ML tree was constructed using MEGA 5.2 software with 1000 bootstrap value. The numbers beside the branches indicate the bootstrap values that support the adjacent nodes. Different colors of dots represented the different species (Brown, Bryophytes; Sky blue, Lycopodiophytes; Red, Monocots; Blue, dicots; Pink, Gymnosperms). Cotton CAMTAs and AtCAMTAs represented by pink and sky blue color, respectively.

Figure 4

Chromosomal location and gene duplication status of CAMTA genes on Gossypium genomes. Physical maps show the position of CAMTA genes on A, D and AD genome separately. The paralogous CAMTA genes (segmental duplicated genes) connected with blue line. The orthologous relationship of CAMTA genes represented by various colored lines (Orange, A vs. D; Grey, A_T vs. D_T; Green, D_T vs. D; Pink, A_T vs. A). Horizontal gray line represents the location of each CAMTA genes. The chromosome number is indicated at the top of each chromosome. Upper and lower panels show the distribution of CAMTA genes in A, D and AD (A_T and D_T subgenomes) genomes, respectively. The scale is in mega bases (Mb).

Figure 5

Phylogenetic tree, gene structure and conserved protein motifs analysis of CAMTAs in G. arboreum, G. raimondii and G. hirsutum. (a) Phylogenetic tree of G. arboreum, G. raimondii and G. hirsutum CAMTAs constructed with ML method by using 1000 bootstrap values. Different colors of dots represented the different species of Gossypium (Blue, G. arboreum; Red, G. raimondii; Green, G. hirsutum). Subfamily-I, -II, -III, -IV, -V and -VI colored in pink, brown, yellow, red, orange, green, and purple respectively. (b) Schematic diagram for the exon/intron organization of cotton CAMTA genes. The green boxes and black lines indicate the exons and introns, respectively. (c) The conserved protein motifs in the cotton CAMTAs were identified using MEME tool. Each motif is indicated with a specific color.

Figure 6

Expression profiles of six GhCAMTAs present in Affymetrix cotton chip at different fiber developmental stages and Co-expression network analysis of GhCAMTA2A.2 and GhCAMTA7A. (a) Variation in expression of 6 GhCAMTAs in different fiber developmental stages were visualized by box plot. Each GhCAMTAs in box plot were represented with different colors. The vertical axis represents expression values, while the horizontal axis corresponds to the different cotton fiber developmental stages. The central line for each box plot indicated median. The top and bottom edges of the box indicated the 25th and 75th percentiles. Gene co-expression network was constructed using cytoscape software. (b) PCoEGs and (c) NCoEGs with GhCAMTA2A.2. (d) PCoEGs and (e) NCoEGs with GhCAMTA7A at different fiber developmental stages. Circles (nodes) represent transcripts and lines (edges) represent significant transcriptional interaction between GhCAMTAs and transcripts. MapMan based functional classification of PCoEGs and NCoEGs (f) Cell wall (g) ROS (h) Stress (i) Redox (j) Transcriptional regulation (k) Hormone metabolism. BINs coloured in green and red are significantly positively and negatively regulated groups respectively. Expression values in log₂ scale indicated by the scale bar in the middle.

Figure 7

Expression and promoter region analysis of PCoEGs and NCoEGs with GhCAMTA2A.2 and GhCAMTA7A. Box plot showing variation in expression level of (a) PCoEGs and (b) NCoEGs with GhCAMTA2A.2 (c) PCoEGs and (d) NCoEGs with GhCAMTA7A at different fiber developmental stages. Graph (e) and (f) showing the frequency of CAMTA recognition motif (MCGCGB/MCGTGT) in randomly selected transcripts, PCoEGs and NCoEGs with GhCAMTA2A.2 and GhCAMTA7A, respectively. The asterisks represent significant differences (Fisher’s exact test, P < 0.05).

Figure 8

Correlation analysis in 100 genotypes of G. hirsutum at 0 DPA. Different fiber quality traits values plotted against the expression values (1/dCt) of highly (GhCAMTA2A.2 and GhCAMTA7A) and least expressing CAMTA (GhCAMTA3D.1). The r and p represent the Pearson correlation coefficient and p-value, respectively. Red box represents significant positive correlation of GhCAMTA2A.2 and GhCAMTA7A with fiber strength.