Background

Casein kinase I (CKI) is a serine/threonine specific protein kinase, highly conserved in plant and animal species [1,2,3]. Compared to other protein kinases, CKI proteins contain four short conserved peptides: HIPXR, LPWQGLKA, EXSRRDD, and LLGPSLEDLF [4]. Due to the broad distribution of the CKI gene family members and their substrates, CKI genes have been found to participate in many biological activities. In yeast, CKI plays important roles in gene expression regulation, vesicle trafficking, cell morphogenesis, cell cycle, cell colonization, and DNA repair [5,6,7,8]. In mammals, CKI is involved in cell proliferation, cytokine production, and the transduction and regulation of a variety of signaling pathways related to apoptosis and tumor production and development, such as Wnt and Hedgehog [9,10,11].

The CKI family in some plants has been studied. The CKI family has been divided into two types, one type is canonical casein kinase 1, another type is plant-specific casein kinase 1 [12, 13]. In Arabidopsis, there are 17 members in CKI family, among which 13 members are canonical casein kinase 1, belonging to CKI-like (CKL) cluster, and 4 members are plant-specific CKI, belonging to Mut9p-LIKE KINASEs (MLKs) cluster [12]. In rice, there are 9 canonical casein kinase 1 and 6 plant specific casein kinase 1 [13]. Both two types of CKI family have important functions in the regulation of a variety of biological processes related to growth and development, as well as various responses to environmental stimuli [14]. In rice, abscisic acid (ABA) and brassinolide caused up-regulation of OsCKI1 (canonical casein kinase 1), while OsCKI1 deficiency caused shorter primary roots and fewer lateral and adventitious roots [2]. In Arabidopsis, the casein kinase I-like 2 AtCKL2 and AtCKL3, are necessary for ABA to regulate seed germination, root growth and gene expression [15,16,17], and overexpression of either AtCK1.3 or AtCK1.4 delayed flowering under long-day conditions in Arabidopsis [13]. In addition, MLK3, a plant-specific casein kinase 1, is critical for maintaining proper flowering time [18]. In cotton, GhCKI was speculated to regulate not only tapetal programmed cell death (PCD) and anther dehiscence [19], but also somatic embryogenesis by modulating auxin homeostasis [20]. Since the functions of most CKI genes in higher plants are largely unknown, their identification and characterization are particularly necessary for understanding their role and to potentially employ them as genetic resources for improving crop plant defense against biotic and abiotic stresses. In cotton, no genome-wide characterization of the CKI gene family has been reported so far. Besides, there was no systematic research to trace the origin and divergence of two types CKI genes. Fortunately, the recently published cotton species genomic information is a solid foundation for characterizing CKI genes at a genome-wide level. Here we have investigated several fundamental questions regarding the CKI gene family evolution: (i) the diversity of gene structure and domain architecture of cotton CKI family; (ii) the evolutionary expansion in cotton of CKI family; (iii) the origin and divergence speculation of CKI family; (iv) the expression profiles of the cotton CKI genes under different conditions. In summary, we retraced the evolution of the CKI genes to better understand their essential elements and thus be able to exploit this knowledge for plant growth and development.

Results

Identification and classification of the Casein Kinase I (CKI) in Gossypium

To extract CKI sequences in Gossypium, the Hidden Markov Model (HMM) was built with 17 Arabidopsis CKI protein, then the protein databases of five sequenced cotton species (G. raimondii, G. arboreum, and G. hirsutum acc. TM-1, G.barbadense, G.herbaceum) [21,22,23,24] were extracted with hmmserach. As a result, 31, 29, 27, 58 and 57 CKI members were identified in G. raimondii (D genome), G. arboretum (A genome), G.herbaceum (A genome), G. hirsutum (AD genome) and G.barbadense (AD genome), respectively (Table S1). To get a better understanding of the phylogenetic relationships between CKIs, an unrooted phylogenetic tree was generated using the CKI protein sequences from G. raimondii, G. arboretum, G.herbaceum, G. hirsutum acc. TM-1, G.barbadense. Clearly, the CKIs were classified into canonical CKI (named type I) and plant-specific CKI (named type II) in cotton (Fig. 1a). Mostly, a homolog of CKI genes can be found once in the diploid G. raimondii, once in G. arboretum, once in G.herbaceum, two copies in the tetraploid G. hirsutum acc. TM-1 and two copies in G.barbadense 3–79 (Fig. 1a), may indicating the CKI family in tetraploid is derived from A and D diploid.

Fig. 1
figure 1

Phylogenetic tree of the Casein Kinase I (CKI) in Gossypium and chromosomal distribution and gene duplication in Gossypium hirsutum. a The phylogenetic tree was constructed using 202 cotton CKI protein sequences from G.hirsutum (58), G.arboreum (30), G.raimondii (31), G.barbadense (57), G.herbaceum (28) with the maximum likelihood (ML) method in MEGA 6. The five different symbols represent the five cotton species: red for G.hirsutum, blue for G.barbadense, green for G.arboretum, purple for G.herbaceum, yellow for G.raimondii. The Gene ID of CKI genes from G. hirsutum, G. arboreum, G. raimondii, G.barbadense, G.herbaceum were listed in Table S1. b The scale is in megabases (Mb), the value on each chromosome represents chromosome length, and the paralogous GhCKIs were connected with a blue line

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Tetraploid cotton species G. hirsutum, the most widely cultivated cotton species in the world, are thought to have formed by a polyploidization event that occurred approximately 1–2 million years ago, which involved D and A genome species [25]. Consequently, G. hirsutum genome information was used to characterize the location of the CKI family genes on chromosome. GhCKI were almost evenly distributed on At and Dt subgenomes (Fig. 1b), so the duplication events may illuminate the mechanism about the expansion of GhCKI gene family. Then the GhCKI phylogenetic tree was independently constructed using the MEGA 6 software (Fig. 2a). All G. hirsutum CKI proteins fell into two distinct groups, which was consistent with the results of Fig. 1a. The type I CKI proteins were further divided into three subclasses: Group A, B, and C; the type II CKI proteins were classified into two subclasses: Group D and Group E.

Fig. 2
figure 2

Phylogenetic tree, protein motif and Exon/Intron organization of the Casein Kinase I (CKI) in G. hirsutum.a Phylogenetic tree of the Casein Kinase I (CKI) in G.hirsutum and b Exon/Intron organization of the CKI genes in G.hirsutum. Red boxes represent exons and black lines indicate introns. c Motif composition of GhCKI proteins. Conserved motifs in the GhCKI proteins are indicated by colored boxes

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Gene structure, conserved motifs and domains of CKIs in G. hirsutum

To get a better understanding of the diversification of type I and type II GhCKI genes in G. hirsutum, the exon/intron organization was analyzed. Most GhCKI genes within the same group showed very similar exon/intron distribution patterns in terms of exon length and intron number (Fig. 2b). For example, most type I GhCKI genes in groups A, B, and C had thirteen to fifteen exons of similar length, whereas members of type II GhCKI genes within group D and E contained more exons. Then, we further analyzed motifs with the program MEME (http://meme-suite.org/tools/meme) to obtain more insights into the diversity of motif compositions among GhCKI proteins. As shown in Fig. 2c, the conserved motifs 1–8 were identified. We clearly observed type I and type II GhCKI proteins have different motif compositions that the type I GhCKI proteins shared five similar motif compositions except Gh_A13G1711 and Gh_D13G2059, but most type II GhCKIs possess eight motifs, implying that GhCKI members within the same type may perform similar functions and that some motifs may play an important role. However, divergence was observed in group E of type II GhCKI, maybe indicating group E members have pluralistic function. Generally, the consistency of motif compositions of GhCKIs with the phylogenetic groups further supported the close evolutionary relationships among GhCKIs, as well as the reliability of our phylogenetic analysis (Fig. 2a). Thus, members belonging to the same group showed similar exon/intron organization and similar motif composition, indicating their functional similarities. And the diverse evolutionary patterns in exon numbers of the CKI genes may hint at their functional diversifications in gene expression. These results further supported the classification between type I and type II GhCKI genes.

The putative conserved domains of GhCKI proteins in G. hirsutum were investigated by using the online program Conserved Domain Search Service (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The type I GhCKI proteins were highly conserved within their kinase domains and N-terminal, but differed significantly in the length and primary structure of C-terminal domains (Figure S1a, Figure S2a). The gene structure of type I GhCKI proteins were consistent with previous reports [3, 4, 26]. The type II GhCKI proteins also presented conserved kinase domains. However, contrary to the type I GhCKI proteins, type II possessed a variable N-terminal and conserved C-terminal (Figure S1b and Figure S2b). In addition, several short sequences are absolutely conserved among the CKIs and not found in other kinases. To further confirm whether the conserved fragments are present in type I and II GhCKI proteins, sequence alignment was performed. The result showed type I GhCKI proteins possess the four short sequences LLGPSLEDLF, HIPXR, EXSRRDD, and LPWQGLKA (Figure S1a). Type II GhCKI proteins contained three of the four conserved sequences (LGPSL, SRRDD, and LPWQG) (Figure S1b). However, compared with the type I CKI proteins, two specific fragments LGKGGFGQV and HGDVKPEN were present in the type II GhCKI proteins only. Comprehensively, LGPSL, SRRDD, and LPWQG appeared in both type I and type II GhCKI proteins. Same result was got in other Gossypium (G. raimondii) (Figure S3).

CKI family expanded with tetraploid event in cotton

Four duplication events have been detected in cotton from the ancient angiosperms development period to the stage of tetraploid cotton species: an ancient whole genome duplication (WGD) very early in angiosperm evolution, one triplication event, the recent WGD event specific for cotton, and the tetraploid event [25, 27, 28]. Based the phylogenetic tree we constructed (Fig. 1a), GhCKI might expanded by hybridization of diploid A genome species and D genome species [29]. To further examine the evolutionary history of the GhCKI genes, we firstly carried out gene collinearity analysis between diploid genome (A genome: G.arboreum and D genome: G. raimondii) and tetraploid genome (AD genome: G.hirsutum) (Fig. 3a). The collinearity of chromosomal regions which contained CKI genes indicated that these genes were the products of WGD or segment duplication. Clearly, both most of genes of two types have a homologous gene in diploid genome and two homologous in tetraploid genome. For example, type I gene GrCKI3 (Gorai.005G050700) on D genome and GaCKI (Gar03G24920) on A genome expanded from one copy to two copies GhCKI3A (Gh_A02G0391) and GhCKI3D (Gh_D02G0444) on the AD genome (Fig. 3b). Type II gene GrCKI25 (Gorai.004G018000) on D genome and GaCKI (Gar08G01750) on A genome expanded from one copy to two copies GhCKI25A (Gh_A08G0110) and GhCKI25D (Gh_D08G0154) on the AD genome (Fig. 3c). These results revealed that both type I and type II CKI genes were replicated in cotton tetraploid event. In addition, we investigated the selection pressure of two types CKI genes by calculating the ratio of nonsynoymous substitutions per nonsynonymous site (Ka) to synonymous substitutions per synonymous site (Ks) (omega) for each homologous gene pair in three above Gossypium species (Table S2). Genes in G. hirsutum as reference. We found all CKI genes were under negative selection (Fig. 3d). The result showed that all CKI genes were highly conserved during the evolution of Gossypium, which show the importance of CKI genes for Gossypium.

Fig. 3
figure 3

Collinear analysis among G. hirsutum, G. arboreum, G. raimondii and the distribution of Ka/Ks values of two types CKI genes. a Collinear analysis of chromosome fragments containing 20 adjacent genes upstream and downstream of two types of CKI genes among G. hirsutum, G. arboreum, G. raimondii.b-c A case of collinearity for a type I paralogous gene pairs and type II paralogous gene pairs respectively. Red for type I, green for type II. d The Ka/Ks values of homologous gene pair between diploid (G. arboretum and G. raimondii) and tetraploid (G. hirsutum) of two types CKI genes. G. hirsutum genes as reference. The above was the result of type I CKI, the below was the result of type II CKI

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The origin and divergence speculation of two types CKI family

Cotton was diverged from an ancestor shared with Theobroma cacao at least 60 Myr ago, and individual grape chromosome segments resembling ancestral eudicot genome structure [27]. To further explore the evolutionary history of the two types CKI genes, we first studied the evolution between three dicotyledons: grape, cacao, cotton (Fig. 4a). As observed in cotton, there were two types of CKI syntenic relationships among the collinearity of grapes, cocoa, and cotton. Therefore, the divergence of two types CKI genes should be earlier than the formation of dicots. Interestingly, it can be observed that there were more type I CKIs than type II CKIs among the collinearity of grape to cacao (Fig. 4a). Maybe this was why there were more type I CKI genes than type II CKI genes in cotton. Then phylogenetic analysis between the G. raimondii and Arabidopsis (eudicots) and rice (monocot) were carried out (Figure S4). Clearly, two types CKI genes from eudicots (G. raimondii and Arabidopsis) and monocot (rice) were present in all subgroups (Figure S4). These results indicating that the divergence of two type CKI genes in plants precedes the divergence between monocots/eudicots.

Fig. 4
figure 4

Collinear analysis among grape, cacao, G. raimondii and phylogenetic tree and number of the Casein Kinase I (CKI) in eighteen species. a Collinear analysis of chromosome fragments containing 20 adjacent genes upstream and downstream of two types CKI genes among grape, cacao, G.raimondii.b Phylogenetic tree of the Casein Kinase I (CKI) in eighteen species. Blue dot represent algae of Protista, Black triangle represent plants of Plantae. c The number of two types CKI genes in eighteen species

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To better trace the origin of the two types CKI genes divergence, CKI genes from 18 species, including algae, mosses, ferns, gymnosperms, monocotyledons and dicotyledons, were found to construct phylogenetic trees (Table S1, Table S3). It can be clearly detected that CKI genes of all species were also divided into two types (Fig. 4b). And the extremely absorbing results were found when we counted the number of two type CKI genes respectively (Fig. 4c). Type I CKI genes were present in all species, but type II genes were lost in algae besides green algae. According to some reports, red algae and green algae diverged about 1,500 MYA ago [30], then other algae were produced by secondary endosymbiosis [31], so the result above may imply that type I genes arose at a very ancient time and type II CKI genes were newly generated upon the divergence of red alga and green algae. Besides, two types CKI homologs in plant including mosses, ferns, gymnosperms, monocot and dicot showed a tendency to increase from lower to higher plants, the result means that after the divergence of red algae and green algae, green algae produced the type II CKI genes in the subsequent evolutionary process, then two type of CKI genes expanded in the subsequent evolutionary process to form the two type of CKI family in various plant today.

Analysis of cis-acting elements of GhCKI and GhCKI expression profile in different tissues from G. hirsutum

Cis-acting elements play an important role in gene expression regulation in plant. We found many cis-acting elements in the promoters of GhCKI genes and identified four main types of cis-acting elements: light responsive element, abiotic stress responsive element, plant hormones responsive element, plant growth and development related element (Figure S5). This result indicates that CKI were involved in many biological processes through different cis-acting element. To further determine which tissue CKI mainly plays a role in cotton, the expression profiles in different organs/tissues (including roots, stems, leaves, petals, anthers, and 5 DPA [days post anthesis] ovules) were examined by quantitative RT-PCR (qRT-PCR) in G. hirsutum. Because of the high sequence similarity between A subgenome and D subgenome cDNAs and regulatory region of homologous genes, so we named the 58 putative G. hirsutum CKI genes as GhCKI1A/D to GhCKI31A/D. We designed one common primer pair for analyzing CKIA/D gene expression. After the specificity for each primer pair was verified, suitable qRT-PCR primer pairs for 44 (26 type I CKI genes and 18 type II CKI genes) of the 61 CKI genes (33 type I CKI genes and 28 type II CKI genes) were obtained (Table S4). As shown in Figure S6, the most of the type I and type II CKI genes exhibited different tissue expression. GhCKI2A/D, GhCKI3A/D, GhCKI8A/D, GhCKI10A/D, GhCKI11A/D, GhCKI14A/D, GhCKI15A/D, GhCKI18A/D, GhCKI20A/D, and GhCKI27A/D were constitutively expressed in every tested tissue, implying that these genes may play regulatory roles at multiple developmental stages. In addition, some genes were highly relatively expressed in leaf and anther, such as GhCKI4A/D, GhCKI14A/D, GhCKI19A/D, GhCKI20A/D, GhCKI27A/D, GhCKI28A/D, GhCKI29A/D, GhCKI30A/D. Some of these genes, such as GhCKI4A/D, GhCKI14A/D, GhCKI20A/D, and GhCKI27A/D, were preferentially expressed in leaves. Interestingly, these genes were also relatively conserved in evolution and may have earlier differentiation times (Fig. 3d, Table S2). These results provide additional insight into their roles during different growth and development processes in cotton.

Circadian rhythm and light signal regulation of GhCKI genes expression

After the divergence of green algae and red algae [30], green algae further evolved into higher plants that adapt to terrestrial life [32]. In this process, adaptation to biological processes such as light and circadian rhythm occured. Besides, analysis of cis-acting elements shows both types of GhCKI have many light-responsive components and plant growth and development related element including circadian rhythm (Figure S5). So CKI expression studies on circadian rhythms and light responses were performed. To determine whether the expression of cotton two types CKI genes is regulated by photoperiod (circadian clock), the transcription level of G. hirsutum CKI genes under different diurnal conditions were investigated. Thirty-six G. hirsutum CKI genes were expressed at a sufficient level to evaluate their circadian regulation (Fig. 5). Under short-day (SD) conditions, all the 36 CKI genes showed a high expression peak at 8:00, and the expression of GhCKI genes increased gradually in the dark and decreased in the light (Fig. 5). Under long-day (LD) conditions, twelve type I genes (GhCKI1A/D, GhCKI2A/D, GhCKI3A/D, GhCKI8A/D, GhCKI13A/D, and GhCKI14A/D) and ten type II genes (GhCKI19A/D, GhCKI20A/D, GhCKI27A/D, GhCKI28A/D, and GhCKI30A/D) showed a high expression peak at 12:00. However, the expression peaks of eight type I genes (GhCKI4A/D, GhCKI11A/D, GhCKI12A/D, and GhCKI15A/D) and six type II genes (GhCKI18A/D, GhCKI26A/D, and GhCKI31A/D) were at 16:00 under long-day (LD) conditions (Fig. 5), and GhCKI14D and GhCKI26D also were relatively conserved in evolution (Fig. 3d, Table S2). In addition, the expression of the 36 GhCKI genes was higher in the light than in the dark under LD conditions, was different with these genes under SD, LD seems to simulate an increase in the duration of sunlight during evolution from red algae to green algae. These results identified a set of clock-regulated GhCKI genes showing different phases of expression and provided additional insight to understand the underlying mechanisms of modulation in circadian rhythm.

Fig. 5
figure 5

Expression profiles of two types GhCKI genes under different diurnal conditions. a and b Expression profiles of the type I and type II CKI genes in G. hirsutum under different diurnal conditions, respectively. Diurnal changes in transcript levels of 36 G. hirsutum CKI genes under short-day (SD) and long-day (LD) conditions. SD, short day (8 h light); LD, long day (16 h light); Black boxes denote dark, while the solid red boxes show light. Values are means ± standard deviations. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01) by Student’s t-test. The GhUB7 (Gh_A11G096) was used as the reference gene to normalize the total amount of cDNA in each reaction

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To further investigate whether two types CKIs are involved in light signaling, the expression of G. hirsutum (Fig. 6) CKI genes and G. raimondii (Figure S7, Table S5) in cotyledon under light and dark conditions was examined by qRT-PCR. The expression of most CKI genes both in G. hirsutum and G. raimondii was up-regulated under light, except for GhCKI19A/D (Fig. 6), GrCKI6, GrCKI14, GrCKI25, and GrCKI26 (Figure S7), there was only a slight difference between the two types of CKI genes. The results indicated both type I and type II CKI might involve in plant light signal. However, intriguingly, genes expressed preferentially in leaves respond more strongly to light signals, such as GhCKI4A/D, GhCKI20A/D, GhCKI27A/D, implying the evolutionarily conserved CKI genes may play a more important role in the response of cotton to light signals.

Fig. 6
figure 6

Expression profiles of two types GhCKI genes under dark and light conditions. a and b Expression profiles of the type I GhCKI genes (26 genes) and type II GhCKI genes (18 genes) in upland cotton cotyledons under dark and light conditions, respectively. Error bars indicate ± standard deviations of triplicate experiments. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01) by Student’s t-test. The GhUB7 (Gh_A11G096) gene was used as the reference gene to normalize the total amount of cDNA in each reaction

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CKI genes respond to high temperature during cotton anther development process

CKI genes exhibited the highest expression in cotton anthers, except for GhCKI14A/D and GhCKI27A/D (Figure S6). CKI promoter also contain numerous abiotic stress responsive cis-acting element (Figure S5). Meanwhile, our previous studies showed that one member of the CKI gene family, GhCKI (Gh_A07G0121/GhCKI11A) was induced in H05 (the high temperature (HT)-sensitive line) anthers, but not in 84,021 (the HT-tolerant line) anthers under HT condition [19]. Genome-wide analyses of G. hirsutum CKI genes in response to HT during anthers development may lay a foundation for further understanding the mechanisms involved in HT tolerance or HT sensitivity. In the present study, a heat-map representing expression profiles was produced using the transcriptomic data which contained the three different anther development stages (TS, tetrad stage; TDS, tapetal degradation stage; ADS, anther dehiscence stage, the three most sensitive periods to high temperature) of 84,021 and H05 under HT and NT [33]. It was observed that 34 of 61 GhCKI genes (19 from type I and 15 from type II) were differently expressed in 84,021 and H05 under HT and NT, even after filtering with an absolute threshold log2 (fold-change) ≥ 1 (Fig. 7a). We found that most genes were up-regulated in H05 after HT exposure, such as type I genes (GhCKI10 and GhCKI11) and type II genes (GhCKI20 and GhCKI27) (Fig. 7a). To verify the result, the qRT-PCR experiments were also performed (Fig. 7b), consistently with the RNA-seq data, most CKI genes were up-regulated in H05 under HT condition. We also analyzed the HT induced differentially alternative spliced (AS) of CKI genes based on our RNA-seq data [33]. Several CKI genes have HT induced differentially AS event (Figure S8, Table S6), for instance, Gh_A10G0586 (GhCKI18A) have a retained intron event, Gh_A11G1211 (GhCKI8A) have a skipped exon event (Figure S8, Table S6). These results indicated that HT influenced the expression and alternative splice of GhCKI genes during anther development.

Fig. 7
figure 7

Expression profiles of two types G. hirsutum CKI genes in anthers at different development stages under NT and HT conditions. a-b Expression patterns of G. hirsutum CKI genes in 84,021 and H05 at different anther developmental stages under NT and HT conditions from RNAseq (a) and Quantitative RT-PCR experiment (b). 8 N and 8H refer to 84,021 (the HT-tolerant line) under NT and HT conditions, respectively; HN and HH refer to H05 (the HT-sensitive line) under NT and HT conditions, respectively; TS: tetrads stage; TDS: tapetal degradation stage; ADS, anther dehiscence stage; FPKM: fragments per kb of transcript per million fragments mapped. The RNAseq data of the expression profiles was cited from Min et al. [33]. Error bars indicate ± standard deviations of triplicate experiments. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01) by Student’s t-test. The GhUB7 (Gh_A11G096) was used as the reference gene to normalize the total amount of cDNA in each reaction

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Discussion

Structural characteristics of G. hirsutum CKI genes

In animals, certain characteristics of CKI that certainly influence its activity have been identified at the protein level: structure-related regulation, subcellular localization, interaction with other proteins, and post-translational modifications [34]. As a member of the superfamily of serine/threonine-specific kinases, the function of phosphorylation is a priority to focus on. In plant, some research found CKI family divided into two types, type I was canonical casein kinase 1, type II was plant-specific casein kinase 1 [12, 13]. However, systematically identification and characteristics of cotton CKI genes were not analyzed. In this study, we first systematically identified CKI genes in five sequenced cotton species (G. raimondii, G. arboreum, and G. hirsutum, G.barbadense, G.herbaceum) (Fig. 1a). Based on sequence comparison and phylogenetic analysis, cotton CKI genes were divided into two types similar to other reports, namely type I (canonical casein kinase 1) and type II (plant-specific casein kinase 1) CKI genes respectively (Fig. 1a). Motif compositions, and exon/intron distribution patterns in terms of exon number in G. hirsutum agreed with our hypothesis (Fig. 2).

The Ser/Thr kinase domain contains putative kinase catalytic loop, substrate recognition region and ATP-binding site. Regarding the functional characteristics of the N-terminal and conserved C-terminal regions, previous reports in mammals showed that CKI presents a β-sheets N-terminal lobe and mainly a α-helical C-terminal lobe, which are connected by a hinge region forming a catalytic cleft for substrate and ATP binding [35, 36]. Within the C-terminal region, a specific phosphate moiety binding motif has been identified allowing the recognition of phosphorylated protein substrates, which is believed to be involved in CKI regulatory interactions. These reports showed that the N-terminal and C-terminal lobes play an important role in substrate phosphorylation. In this study, type I CKI proteins resulted highly conserved within their kinase domains, but significantly differing in the length and primary structure of their C-terminal domains (Figure S1, Figure S3), which is consistent with previous reports [3, 4, 25, 33, 37]. Interestingly, contrary to type I, type II CKI proteins also presented conserved kinase domains and possessed variable N-terminal and conserved C-terminal regions (Figure S1, Figure S3). Based on the characteristic of plant CKI genes, we hypothesis the type I and type II CKI genes possess different functions on account of the difference in structures, especially regarding the function of phosphorylation. Functional studies will be needed in the future to further explore this difference.

Evolutionary expansion of the two types CKI gene family

The evolution of Gossypium was characterized by a history of multiple gene duplications at different stages as in the four duplication events (an ancient angiosperm WGD event, one triplication event, a specific and recent cotton WGD event, and the tetraploid event) [25, 28, 29]. First, we observed the expansion of the two types CKI genes in cotton (Fig. 3a), and found that the two types CKI genes were expanded with cotton tetraploid event. Most genes were one copy in diploid and became two copies in tetraploid. But the collinearity of three cotton species chromosomal regions were not contained all CKI genes. For example, Gh_D10G2569 (type I) and Gh_D10G0542 (type II) didn’t have homologous gene in synteny blocks between G. raimondii and G. hirsutum or G. arboretum and G. hirsutum (Fig. 3a). We speculated these genes were newly created copies during the cotton tetraploid event, or errors in genome assembly. In order to better trace the origin and divergence of the two types CKI genes, the genome of 18 species were collected to construct the phylogenetic tree. We found that CKI genes in all species were divided into two types, and red algae did not have type II gene (Fig. 4). But type II CKI genes were identified in green algae (Fig. 4), consistent with previous studies [38]. After red algae and green algae diverged about 1,500 MYA ago [30], other algae were produced by secondary endosymbiosis [31]. Then the green alga evolved into plant [32]. Therefore, we speculate that the two type CKI genes diverged about 1,500 MYA ago, then CKI genes have replicated and expanded differently in different species during the evolution of green algae to higher plants. Besides, the gene families which possess an ancient evolutionary history have important functions, such as MADS-box genes [39], Auxin response factors [40], or ASYMMETRIC LEAVES2-LIKE/ LOB-DOMAIN transcription factors [41]. Thus, we speculate that CKI genes may play important roles in plant development and be involved in very diverse biological roles to adapted to environmental stress.

Expression patterns of the GhCKI genes

To date, although the functions of only one CKI gene have been characterized in tetraploid cotton [19], no systematic functional analysis of expression patterns for different groups of the tetraploid cotton CKI gene family was done. In this study, we demonstrated that CKI genes displayed expression divergence in different tissues (Figure S6). For instance, GhCKI2A/D, GhCKI3A/D, GhCKI8A/D, GhCKI10A/D, GhCKI11A/D, GhCKI15A/D, and GhCKI18A/D were constitutively expressed in every tested tissue (such as roots), implying that these genes may play regulatory roles at multiple development stages. In Arabidopsis, AtCKL2 and AtCKL3 were required for ABA regulation of seed germination, root growth, and gene expression [16, 17]. In rice, OsCKI1 deficiency resulted in shorter primary roots and fewer lateral and adventitious roots [2]. Similar expression patterns suggest that these preferentially or specifically expressed G.hirsutum CKI genes might play important roles in root formation and development. Among these 61 identified CKI genes, GhCKI (namely GhCKI11A) is the only one that was speculated to regulate tapetal programmed cell death and anther dehiscence in cotton [19]. Under HT condition, AtCKL2 and AtCKL7 were expressed in the tapetum, in anther microspores at stages 9–12, and in anther pollen grains at stages 13–14, which imply AtCKL2 and AtCKL7 may be key regulators of tapetal development under HT [42]. Apart from GhCKI11A (formerly GhCKI), the GhCKI1A/D, GhCKI5A/D, GhCKI9A/D, GhCKI12A/D, GhCKI13A/D, GhCKI19A/D, GhCKI26A/D, GhCKI28A/D, GhCKI29A/D, GhCKI30A/D, and GhCKI31A/D genes were exclusively expressed in anther. This finding suggested that CKI genes were components of a complex transcriptional network regulating anther development.

Previous studies had showed that HT stress causes premature programmed cell of the tapetum, resulting in male sterility and catastrophic loss of crop production [30, 43,44,45]. However, the mechanism underlying successful male reproductive development under HT remains largely unknown. Except for the GhCKI11A (formerly GhCKI) gene, which was reported to regulate tapetum development under HT [20], no other CKI genes have demonstrated to participate in the regulation of anther development. In this study, the expression of type I and type II CKI genes were analyzed in cotton anther respond to HT, no differences between type I and type II CKI genes were found (Fig. 7). Thus, two types CKI genes both participated in regulating stamen development under HT and that will be employed in future works.

Circadian clocks are molecular timekeepers. Many plants use photoperiod (circadian clock) information to prepare for daily environmental changes and increase their fitness in changing environments [46]. The circadian clocks share similar network architecture of feedback loops that form by transcriptional and post-translational regulation among the clock components [47, 48]. Phosphorylation is a common post-translational modification and is an integral part of circadian regulation [49]. Another function of CKI is to take a series of biological process via phosphorylating various substrates [3]. In Arabidopsis, both CK1.3 and CK1.4 showed a high expression peak before dark under LD conditions, which indicated that the expression of CK1.3 and CK1.4 was strictly regulated by a circadian rhythm; overexpression of either CK1.3 or CK1.4 delayed flowering under LD conditions [13]. However, in cotton, it is not clear that the systematic function of the tetraploid cotton CKI gene family is involved in photoperiod (circadian clock). In our present study, the expression of CKI genes is circadian. Under long-day (LD) conditions, the expression in the light is higher than in the dark. Under short-day (SD) conditions, the expression of all the 36 CKI genes showed a high expression in dark. (Fig. 5). The results indicated that the expression of CKI was strictly regulated by a circadian rhythm. According to these evidences, we propose that at least some CKI genes are components of a diurnal rhythm complex network. The functions of these GhCKI genes in regulating the circadian clock on the diurnal rhythms will be further characterized in future works.

Plant reception of light signal and its subsequent reaction is important for both growth and development. In Arabidopsis, CK1 genes are involved in the light signaling pathway mainly through phosphorylation; casein kinase 1 proteins CK1.3 and CK1.4 phosphorylate CRYPTOCHROME 2 (CRY2) which is the blue-light receptor, to regulate blue light signaling [13]. Our results showed that the expression of most type I and type II CKI genes was upregulated under light conditions compared to dark (Fig. 6, Figure S7). We thus believe that both type I and type II CKI gene are involved in the response to light signals. But we do not know whether they participate in light signals by phosphorylating photoreceptors CRY2 or light signal components, including HY5, HF5, HFR1, COP1, and PIF1, which in Arabidopsis are phosphorylated by casein kinase 2 proteins [49]. We also do not know whether the pathways and capabilities of these two types of genes are the same.

We found cis-acting element of GhCKI involve light responsive element, abiotic stress responsive element, plant hormones responsive element, plant growth and development related element, so we carried two types of cotton CKI expression analysis about high temperature response, biological rhythm, and light response. The results of expression analysis indicated that GhCKI genes may involve in the above biological functions. Therefore, GhCKI maybe participate in these biological functions through different cis-elements. Although the expression patterns of type I and type II CKI genes were basically the same in our study, we believe that these two types of genes are functionally differentiated because of the diversity in structure, which requires further investigations in the future.

Conclusion

Our study offers a promising landscape to unravel the underlying structural characteristics and evolutionary expansion of cotton CKI genes and further elucidate their expression patterns in different tissues and various conditions; this is crucial to better understand their characteristics and to elucidate their precise functions in regulating various facets of plant growth and development.

Materials and methods

Database search and identification of CKI genes

The genome information of twenty-two species was retrieved from the website shown in Table S3. The CKI genes in A.thaliana were downloaded from TAIR (www.arabidopsis.org) database. The Hidden Markov Model (HMM) was built with 17 Arabidopsis CKI protein by using hmmbuild command of HMMER (v3.2.1) [50]. The protein databases of the remaining twenty-one species were searched with hmmsearch command (-E 1e-20) [50]. All possible CKI genes were identified in these twenty-one species. To further ensure the accuracy of each CKI gene, we identified the conserved kinase domains (domain id is: http://pfam.xfam.org/family/PF00069) present in possible CKI proteins using SMART (http://smart.embl-heidelberg.de/), Pfam (http://pfam.xfam.org). Only gene containing the conserved kinase domains considered as true CKI.

Chromosomal location, gene structure and phylogenetic analysis

The chromosomal location of GhCKIs were obtained from cotton genome database (https://www.cottongen.org/), then gene duplication events of GhCKIs were detected based on principle described in previous study [51], moreover the Circos-0.69 Software was used to visualize chromosomal location and gene duplication [52]. Sequence alignments were generated with CLUSTALX [53], and the alignments among CKIs were adjusted before the tree was constructed. The online Gene Structure Display Server 2.0 [54] (http://gsds.cbi.pku.edu.cn/) was used to identify the exon/intron organization. G.hirsutum CKI protein sequences were submitted to online MEME (Multiple EM for Motif Elicitation) program [55] (http://meme-suite.org/tools/meme) to identify conserved protein motifs. Phylogenetic trees were constructed by the maximum likelihood (ML) method, the Poisson correction model, complete deletion and bootstrap analysis performed with 2,000 replicates [56].

Collinearity analysis and the estimation of selection pressure of CKI genes

The chromosome location of CKI genes in each genome was obtained according to the GFF3 file. When homologous CKI genes were located in the same chromosome with no more than one intervening gene, these genes were defined as tandem duplication genes. We used the method as described in Maher et al. to identify large-scale duplication events [57]. If two genes were located in the same duplication block, the protein sequences on their flanks were highly similar at the amino acid level. Therefore, we located the CKI genes on the genome and used this location as the initial anchor site to obtain 20 protein coding genes upstream and downstream of each site respectively [56]. This chromosomal region containing 41 protein-coding genes were selected for collinearity analysis using the python version of MCScan [58].

The coding sequences were aligned and guided by alignments of protein sequences using the PAL2NAL software with the NOGAPS parameter [59]. The yn00 procedure in PAML package was used to calculate the ratio of nonsynonymous substitutions per nonsynonymous site (Ka) to synonymous substitutions per synonymous site (Ks) for each homologous gene pair [60]. According to the definition of Ka/Ks, values less than one represent negative or purifying selection, while values greater than one represent positive selection. The saturation effect was ruled out by removing gene pairs with Ks > 2.5.

Analysis of cis-acting elements in GhCKI promoter

The promoter sequences of the GhCKI genes (1,000 bp upstream of the initiation codon “ATG” were extracted from the cotton geome sequences (https://cottonfgd.org/), PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/search_CARE.html) was used to predict the cis-acting elements of the GhCKI promoter sequence [61], the light responsive element, abiotic stress responsive element, plant hormones responsive element, plant growth and development related element were analyzed.

High temperature-induced differential alternative spliced CKI gene detection

All putative high temperature-induced differential AS event were extacted from our RNA-seq data using rMATS [33, 62]. After weakly expressed AS events were filtered below 40% samples with total IJC + SJC ≥ 30, differentially AS events between NT and HT condition were identified using rMATS if the difference in the PSI of AS event between two conditions exceeds a stringent threshold (FDR ≤ 0.05, ΔPSI ≥ 10%). Those CKI genes containing AS event considered as high temperature-induced differential AS CKI Gene.

Plant materials, growth conditions and stress treatments

Four cotton accessions used in this experiment are provided by Huazhong Agricultural University. For the expression profiles in different organs/tissues, various samples of G. hirsutum cv. YZ1 were extracted from roots, stems, leaves, petal, anther, and ovules excised carefully from bolls five DPA. To analysis the expression patterns of Gossypium hirsutum CKI genes at different anther developmental stages under NT and HT conditions, two cotton (Gossypium hirsutum) lines with obvious differences in performance under HT were employed in this study: 84,021, which is tolerant to HT, and H05, which is sensitive to HT [33]. The plants were grown in a greenhouse at 28 °C to 35 °C/20 °C to 28 °C day/night as a normal condition. During HT treatment, the plants were cultivated at 35 °C to 39 °C/29 °C to 31 °C day/night in a greenhouse. When the plants were treated with HT for 7 d, buds of different lengths (6–7, 9–14, and more than 24 mm) were collected under HT and NT. The anthers were excised and immediately frozen in liquid nitrogen; they were then stored at -80 °C until use. The transcriptome profiles of CKI genes were isolated from the RNA-seq data [33]. In order to analyze the diurnal regulation of G. hirsutum CKIs gene expression, G. hirsutum cv. YZ1 was grown under short-day conditions (8 h light/16 h dark) and long-day conditions (16 h light/8 h dark), respectively. The leaves of four leaves period cotton plants were harvested. We also collected cotyledons under light and dark conditions, that from G. raimondii and G. hirsutum cv.YZ1 respectively.

qRT-PCR

Various plant samples were collected and immediately frozen in liquid nitrogen and stored at -80 °C. total RNA was isolated from the collected cotton tissues using previously published methods [63]. First-strand cDNA was generated from 3 μg total RNA using the M-MLV reverse transcriptase (Invitrogen). The cDNA was used as a template for qRT-PCR. The qRT-PCR reactions were performed using the 7500 Real-Time PCR System (Applied Biosystems). The primers used in this study were listed in Table S4, Table S5.