Identification of the MIOX Gene Family
A total of 81 MIOX genes were identified from nine species, including 75 MIOXs in eight Gossypium genomes and 6 MIOXs in the Gossypioides kirkii genome. Except for one MIOX from G. herbaceum has significantly more amino acids (896 aa) and two MIOXs from G. barbadense has significantly less amino acids (101 aa and 113 aa), the others are relatively conservative (Additional file 1: Table S1). Furthermore, we found that all of the four diploid species contained 6 MIOX genes, the number of MIOX genes in tetraploid cotton is roughly twice that of diploid cotton, but the three wild tetraploid cottons (G. tomentosum, G. mustelinum and G. darwinii) missed a MIOX gene.
For convenience, we renamed the MIOX gene family. Gh, Gb, Ga, Gher, Gr, Gd, Gt, Gm, and Gkk were used as prefixes before the names of MIOX genes from G. hirsutum, G. barbadense, G. arboreum, G. herbaceum, G. raimondii, G. darwinii, G. tomentosum, G. mustelinum and Gossypioides kirkii respectively. we assigned the names to MIOX genes as (GhMIOX01- GhMIOX12) for G. hirsutum, (GbMIOX01 - GbMIOX12) for G. barbadense and so on.
Phylogenetic Analysis of the MIOX Gene Family
To reveal the evolutionary relationship of identified MIOX proteins, the amino acid sequences of 75 proteins from Gossypium species, 6 proteins from Gossypioides kirkii and 4 proteins from Arabidopsis thaliana were used to construct a phylogenetic tree (Fig. 1, Additional file 2: Figure S1). According to the result, 81 MIOX proteins form 8 Gossypium species and Gossypioides kirkii were divided into six groups of A-F, the number of MIOX genes was very stable in each evolutionary branch, diploid species containing 6 MIOXs had one distribution in each branch, while the other five tetraploid cottons containing 11 or 12 MIOX genes had one or two distribution in each branch. All of these genes showed one-to-one homology relationship among different genomes or subgenomes. Compared to the A~F groups, the group G~I only contained A. thaliana MIOX proteins.
Gene Structure and Protein Motifs of the MIOX Gene Family
To explore the structural diversity of MIOX genes, the intron-exon organization of each MIOX gene was analyzed (Fig. 2). The number of introns ranged from 3 to 21, most (52/81) MIOX genes contained 9 or 10 introns. In the same group, the intron numbers exhibited comparable exon-intron structure and intron numbers, while many Gossypioideskirkii MIOX genes were different from cotton, implying important evidence for the phylogenetic relationship among members of the MIOX gene family.
Furthermore, we investigated the conserved motifs in MIOX proteins to understand the diversity of motif compositions (Fig. 2). A total of 13 motifs, named Motif 1~Motif 13, were identified in MIOX proteins. The number of motifs varied from 3 to 12 in each MIOX protein and most MIOX proteins within the same group exhibited similar motif composition and arrangement, which indicates that the members of MIOX gene family that clustered in the same group may have similar biological functions. Motif 5 was found in 78 MIOX proteins, Motif 8 was completely missing only in group B, and Motif 13 was specific to group C. The gene structure and motif composition of the MIOX members from each group that obtained from phylogenetic analysis were similar, which indicates the classification was reliable.
Chromosomal Location and Gene Duplications of the MIOX Gene Family
To more intuitively understand the distribution of MIOX family genes on the chromosomes, we performed a chromosomal localization analysis. The result shows that MIOX genes were mapped onto 5/10 different chromosomes of diploid/tetraploid cotton. Each chromosome contained only one MIOX gene usually, while some chromosome contained 2 MIOX genes (Fig. 3). In addition, the distribution of Gossypioides kirkii MIOX genes showed similar to Gossypium.
Whole genome duplication, segmental duplication and tandem duplication provides major forces that drive the expansion of gene families. The number of MIOX genes in tetraploid Gossypium species was twice as much as that in diploid Gossypium species which indicates the expansion of MIOX gene family during polyploidization. We searched the segmental duplication using MCscanX within each genome, and identified 133 collinear genes pairs among the 72 MIOX genes. Of the 75 MIOX genes in eight Gossypium genomes, only 3 were located outside of the duplicated blocks, while 96% (72 of 75) were located in duplicated regions. In addition, 40, 38 duplication gene-pairs were found between diploid G. arboretum A-genome and tetraploid G. hirsutum, G. barbadense A-subgenome respectively. 18, 17 duplication gene-pairs were found between diploid G. raimondii D-genome and tetraploid G. hirsutum, G. barbadense D-subgenome respectively (Fig. 4). According to Holub’s description, a chromosomal region within 200 kb containing two or more genes was defined as a tandem duplication event. Our results indicated that the MIOX genes of the nine species has no tandem duplication.
Cis-elements in the Promoter of MIOX Genes
Cis-elements in promoters play vital roles in regulating the expression of genes. To gain more insight into the functions of MIOX genes, the cis-regulatory elements were scanned in the 2000 bp upstream of the transcription start sites of cotton MIOX genes (Fig. 5, Additional file 3: Table S2, Additional file 4: Figure S2). The results showed that there were many kinds of response elements, such as light responsive element, defense and stress responsive element involved in drought and low temperature, and hormone responsive element associated with salicylic acid, abscisic acid, gibberellin and MeJA. All of the cotton MIOX genes contained more than one light responsive element, however only 29.33% (22/75) of the cotton MIOX genes contained auxins responsive element.
The cis-elements of MIOX genes in the same phylogenetic group was similar. In addition, within the same group, half of the cis-elements of MIOX genes in tetraploid Gossypium species are similar to the diploid A genome species (G. herbaceum and G. arboretum), and half are similar to the D genome species (G. raimondii). These results further indicated the expansion of MIOX gene family during polyploidization.
Tissue-Specific Expression Profiles of MIOX Genes
To study the tissue-specific expression patterns of the MIOX genes, we analyzed the expression profiles of the MIOX genes in different tissues. As shown in Fig. 6, MIOX genes showed different expression levels in different tissues. In G. hirsutum, the expression of GhMIOX02 and GhMIOX08 were higher in stem, leaf, torus, pistil, bract and speal, while their expression was lower in root. The expression of GhMIOX04 and GhMIOX10 was higher in root, stem, leaf, torus and speal, while their expression was lower in pistil and bract. In G. barbadense, GbMIOX02, GbMIOX08, GbMIOX04 and GbMIOX10 had similar expression profiles with G. hirsutum. In addition, GhMIOX03 of G. hirsutum showed higher transcription level only in root, however, GbMIOX03 of G. barbadense showed higher transcription levels in root, stem and pistil.
The expression levels of MIOX genes at ovule and fiber developmental stages were also investigated (Fig. 6). At most stages of development, the expression of GhMIOX08 from G. hirsutum, GbMIOX08 from G. barbadense were higher than that of the other genes; Several genes were expressed at high levels during specific developmental stages; for instance, GhMIOX03, GhMIOX09, GhMIOX10 from G. hirsutum and GbMIOX03, GbMIOX09, GbMIOX10 from G. barbadense. In contrast, the RNA transcript levels of GhMIOX05, GhMIOX11 from G. hirsutum and GbMIOX05, GbMIOX11 from G. barbadense were low at all stages and all tissues. These findings indicated the MIOX genes play differential roles in tissue development.
Stress-Induced Expression Patterns of MIOX Genes
The expression patterns of MIOX genes were further analyzed in G. hirsutum and G. barbadense exposed to different durations of cold, heat, salt, and drought stresses for different times by RNA-seq data downloaded from the public database (Fig. 7). Based on the clustering analysis, MIOX genes in group C and D general responded to all of these stress, but the genes in other groups were not significantly responded. In addition, it was observed that MIOX genes exhibited variations in expression in response to one or more stresses. In the case of heat and salt treatment, the expression of GhMIOX02 and GhMIOX08 showed up-regulation in the early time points (1h~3h), then down-regulation (3h~6h), and then another up-regulation (6h~24h). While GhMIOX03 was up-regulated in the early time points (1h~6h), but down regulated in the late time points (12h~24h) under code stress. Moreover, GbMIOX02 showed continuously down-regulation at all of the time points under salt stress. These results indicate that 3~6 hour is an important time point for MIOX genes respond to abiotic stress.