Research Article

Genome-wide identification and expression analysis of CIPK genes in diploid cottons

Published: November 03, 2016
Genet. Mol. Res. 15(4): gmr15048852 DOI: 10.4238/gmr15048852

Abstract

Calcineurin B-like protein-interacting protein kinase (CIPK) plays a key regulatory role in the growth, development, and stress resistance of plants by combining with phosphatase B subunit-like protein. In the present study, CIPK genes were identified in the whole genomes of diploid cottons and their sequences were subjected to bioinformatic analyses. The results demonstrated that the CIPK gene family was unevenly distributed in two diploid cotton genomes. Forty-one CIPKs were identified in the D genome, mainly located on chromosomes 9 and 10, whereas thirty-nine CIPKs were identified in the A genome, mainly located on chromosomes 8 and 11. Based on the gene structures, CIPKs in cotton could be classified into two types: one that is intron-rich and the other that has few introns. Phylogenetic analysis revealed that the CIPK gene family members in cotton had close evolutionary relationships with those of the dicotyledonous plants, such as Arabidopsis thaliana and poplar. The analysis of transcriptome sequence data demonstrated that there were differences in gene expression in different tissues, indicating that the expression of the CIPKs in cotton had spatio-temporal specificity. The expression analysis of CIPKs under abiotic stresses (drought, salt, and low temperature) in different tissues at trefoil stage demonstrated that these stresses induced the expression of CIPKs.

 

INTRODUCTION

Plants have evolved many complex signaling pathways, such as the mitogen activated protein kinase (MAPK) signaling pathway and the calcium (Ca2+) signal transduction pathway, to adjust to the continuous stimulation from the external environment. Research has demonstrated that Ca2+ signal transduction pathway participates to a great extent in many processes involved in the growth and development of plants, such as in the growth of root hairs and in guarding the movement of cells (Assmann and Wang, 2001). Moreover, plants are also affected by various abiotic stresses, such as drought, cold, salinity, hormones, and light, as well as biotic stresses, especially those induced by pathogenic bacteria (Sanders et al., 2002). Calcineurin B-like protein-interacting protein kinase (CIPK) is regulated by calcineurin B-like protein (CBL), and is one of the important components of Ca2+ signal transduction. The proteins encoded by CIPK genes are a family of Ca2+-dependent serine/threonine kinases; these kinases have a conserved SNF kinase domain and an NAF (Asn-Ala-Phe) amino acid domain and belong to the third class of SNF1-related protein kinase 3 (SnRK3) (Harper, 2001). These two CIPK domains are required for the kinase to function. The structure of NAF is a unique domain of CIPK; it is the binding site for interaction of CBL-CIPK and plays an important role in this interaction. Extensive research has proven that the CBL-CIPK signal system plays a key role in the growth and development of plants and in their response to various stresses (Weinl and Kudla, 2009); therefore, research on the identification, structure analysis, and function of CIPK genes in cotton is significant in investigating the role of CBL-CIPK interaction in the growth and development of cotton plants and in the resistance of plants to stress.

Twenty-six CIPKs have been identified in Arabidopsis thaliana. There are 30, 32, 27, and 43 CIPKs in rice, Sorghum, poplar, and maize, respectively. Different members of the CIPK gene family respond to different stimuli in specific plant tissues and at particular developmental stages (Halfter et al., 2000). The salt overly sensitive (SOS) signaling pathway is a classical CBL-CIPK pathway involved in the signal transduction of salt stress and has been proven to be conserved, at least in poplar (Tang et al., 2010). Many other CIPKs are also involved in the salt-stress response; the mechanisms used by these might be different from that of the SOS pathway. A study done on SiCIPK24 of tomato plants revealed that the main role of this gene in conferring salt stress resistance was in the transport and accumulation of more Na+ in the plant stems (Huertas et al., 2012). Overexpression of AtCIPK16 in A. thaliana and barley (Hordeum vulgare L.) resulted in very strong resistance of the transgenic plants to salt stress (Roy et al., 2013). Heterologous overexpression of Brassica napus L. BnCIPK6 in A. thaliana could improve the resistance of the transgenic plants to salt stress (Chen et al., 2012). In rice, upregulation of OsCIPK15 expression enhanced the resistance of plants to salt stress (Xiang et al., 2007). In 2006, Xu et al. (2006) cloned and identified AtCIPK23 from the low potassium-sensitive mutants using map-based cloning method and discovered that AtCIPK23 could directly interact with and activate the K+ channel, AKT1, in the cell membrane. Pandey et al. (2007) found that the sensitivity of the CIPK9 mutant to low-potassium stress was enhanced and that AtCIPK3 might regulate the expression of the cold-resistance gene RD29A by regulating the transcription factor genes of CBF/DREB1 (Huang et al., 2011). Overexpression of OsCIPK3 and OsCIPK12 in rice significantly increased the resistance of plants to cold and drought stress. Increasing evidence demonstrates that CIPK genes are widely involved in the abscisic acid (ABA) signaling pathway (Chae et al., 2007), absorption of mineral nutrients, and resistance of plants to diseases and pests (Schwachtje et al., 2006).

Cotton is an important economic crop that is cultivated for oil and fiber; the cultivated species include diploid and tetraploid cotton plants, which are models for the study of plant polyploidization, cell elongation, and cell wall biosynthesis (Paterson et al., 2012). As a glycophyte, cotton displays stronger drought and salt resistance than other crops (Iqbal et al., 2011). Few studies have been conducted on the CIPKs in cotton and these have been mainly focused on cloning, identification, and functional analysis of a single CIPK (He et al., 2013). In 2012, sequencing of the “D genome” of diploid cotton Gossypium raimondii was completed (Wang et al., 2012) and by 2013, sequencing of the “A genome” of Asian cotton Shixiya I (Gossypium arboreum L.) was also completed (Li et al., 2014). The completion of the whole genome sequencing of diploid cotton made the first comprehensive analysis and comparison of the CIPK gene family members possible. Previous researchers have cloned GhCIPK6 from Gossypium hirsutum L., and sequence alignment showed that this gene had homology with AtCIPK6 of A. thaliana. In addition, tissue-specific expression analysis illustrated that the gene was expressed in different tissues, such as the stylus and anthers, with the expression being induced by drought, salt, and ABA (He et al., 2013). This suggests that CIPKs play a positive regulatory role in the response to salt and drought stress. It can, therefore, be concluded that CIPKs are related to the processes involved in stress, adversity, and in the growth and development of cotton.

In the present study, genome-wide identification and bioinformatic analysis of CIPKs was conducted to explore the homologous CIPKs, and to analyze their distribution in the genomes as well as their gene structure. Analyses of the expression pattern of CIPKs in different tissues of cotton during different growth stages were also conducted to lay a foundation for further research on the CBL-CIPK signaling network in cotton. The results of this study would be of great importance for further research on the growth and development of cotton as well as in improving their resistance to stress.

MATERIAL AND METHODS

Recognition and identification of CIPK family genes in cotton

To predict the members of the CIPK gene family in cotton, BLAST analysis was performed using the protein sequences of the CIPK gene families of A. thaliana, rice, and maize as the query sequences and the sequence data of the D and A genomes of the diploid cottons, G. raimondii and G. arboreum L., respectively; the E-value was set at 0.0001. Further confirmation was done using the screened protein sequences present in the protein database (http://www.expasy.ch/prosite/). The protein sequences of the N-terminal SNF kinase and C-terminal NAF domains were selected as the subjects for further analysis. NAF is the specific domain of CIPK. The selection method used was as described earlier by Albrecht et al. (2001).

Phylogenetic analysis of CIPKs

The protein sequences of A. thaliana CIPKs were downloaded from the A. thaliana genome database (http://www.arabidopsis.org/), those of rice were retrieved from the rice genome database (http://rice.plantbiology.msu.edu/) and National Center of Biotechnology Information (NCBI; Oryza sativa subsp japonica), and those of maize were downloaded from the maize genome database (http://www.maizegdb.org/gbrowse/maize) and NCBI. The protein sequences of the CIPKs in Populus trichocarpa were downloaded from the genome database of P. trichocarpa (http://www.ncbi.nlm.nih.gov/). According to the naming conventions reported in the literature, CIPKs of A. thaliana, rice, maize, and P. trichocarpa were named as AraCIPK, OsCIPK, ZmCIPK, and PtCIPK, respectively. Multiple sequence alignments were performed on the sequences of the proteins encoded by all the CIPKs using ClustalW2. The DNAMAN software (http://www.lynnon.com/) was used for sequence alignment. The neighbor-joining method was used for the construction of the phylogenetic tree and the MEGA software (http://www.megasoftware.net/) was used for inferring the phylogenetic tree.

Prediction of the isoelectric point (pI), molecular weight, and subcellular localization of proteins encoded by CIPK family genes

The theoretical pI and molecular weight of CIPK were calculated using the ExpASy protein server (http://web.expasy.org/compute_pi/). The average isotopic weight of one molecule of protein was expressed in daltons (Da). The subcellular localization of each CIPK was analyzed using the CELLO v2.5 server.

Chromosome location and gene structure analysis

The sequences of CIPK cDNAs were obtained from the D genome of G. raimondii and the A genome of G. arboreum L., respectively. The complete D genome of G. raimondii and A genome of G. arboreum L. were compared using the cDNA sequence as a query to determine the chromosomal location of each CIPK; the CIPK cDNA and the corresponding genomic DNA sequences were compared to identify the exon/intron structure of the gene (http://gsds.cbi.pku.edu.cn/).

Transcriptome expression analysis of CIPKs

The expression of CIPKs was analyzed in three tissues, namely mature leaves, 0-DPA (day post-anthesis) ovules, and 3.0-DPA ovules of G. raimondii and G. arboreum L. The transcriptome sequencing data were obtained from the NCBI Sequence Read Archive (SRA); the registration numbers of the G. raimondii samples were SRX111367, SRX111365, and SRX111366, and the original sequence number of G. arboreum L. was SRA150181. To evaluate the expression levels of CIPKs, the sequence reading aligned on the sequence of CIPK was converted to RPKM (Mortazavi et al., 2008) and the equation used for the evaluation was as follows:

RPKM = 106C/(NL/103)

where C refers to the reading length aligned uniquely on the transcript, N is the total reading length aligned uniquely on one specific sample, and L is the base of the transcript.

Expression analysis of CIPKs under different abiotic stress conditions

The seedlings of diploid cottons, G. raimondii and G. arboreum L. cv. Shixiya 1, were planted using the sand culture method. The true leaves, stems (hypocotyls), and roots of the seedlings were harvested and minced after abiotic stress treatments (low temperature: 4.0°C, 24 h, salt stress: 150 mM NaCl, 24 h, drought: relative water content in sand was reduced to approximately 5.0%) at the trefoil stage. Subsequently, the samples were quickly frozen in liquid nitrogen and stored at -80°C for RNA isolation. Untreated controls samples were also obtained. Total RNA was extracted from the collected tissues (Carra et al., 2007), and its concentration and purity was determined by Nanodrop2000 nucleic acid analyzer (Thermo, America). The RNA was reverse transcribed into cDNA using PrimeScript RT reagent kit with gDNA eraser (TaKaRa, China). Primer Premier 5.0 (PREMIER Biosoft) was used to design fluorescent quantitative primers (Table S1). Quantitative real time PCR (qRT-PCR) was performed using actin gene (GenBank accession No. AY305733) as a reference. The PCR programs were set as follows: 94°C for 30 s, followed by 40 cycles of 94°C for 5.0 s, 55°C for 34 s, and 72°C for 34 s. The assays were designed with three biological and three technical replicates. Relative quantitative analysis of the target genes was performed using the 2-ΔΔCt method.

RESULTS

Identification of CIPK gene family members in the complete genome of cotton

The CBL-CIPK complex significantly affects the expression of target proteins, thereby, influencing various metabolic activities of plants. BLAST analysis was conducted using the CIPK sequences from A. thaliana, rice, and maize as the query sequences and the A and D genomes of cotton as the reference genomes. The results revealed that there were 39 CIPKs in the A genome (Table 1). On the basis of their order of presence on the chromosomes (from chromosome 1 to 13), the genes were named as GaCIPK1-GaCIPK39, with the first two letters indicating the corresponding species. There were 41 CIPKs in the D genome, which were similarly named as GrCIPK1-GrCIPK41. As demonstrated in Table 1, there were significant differences in the number of amino acid residues (AAs) present in the various CIPKs, which ranged from 196 to 1396. Most of the CIPKs contained between 412 and 480 AAs. GrCIPK37 was the smallest with 196 AAs because it lacked the NAF structure. The pIs ranged from 5.58 to 9.42. Except for GrCIPK2, GrCIPK15, GrCIPK22, GrCIPK30, and GaCIPK16, these five genes were slightly acidic, and the other 75 CIPKs were alkaline, similar to CIPKs of rice, Sorghum, and A. thaliana. Rice contains 30 CIPKs; except for OsCIPK1 and OsCIPK8, which were acidic, the remaining CIPKs were reported to be alkaline (Xiang et al., 2007). Of the 31 CIPKs reported in Sorghum, the proteins of all, except SbCIPK9, were reported to be alkaline (Li et al., 2010). The dicotyledonous A. thaliana contains 25 CIPKs. AtCIPK1, AtCIPK5, and AtCIPK16 were reported to be acidic whereas the others were alkaline (Li et al., 2010). The data indicate that the ancestors of CIPKs in plants were alkaline, whereas the acidic CIPKs evolved later. The molecular weight of CIPKs ranged from 21.92 to 151.57 kDa, with the majority of proteins being in the range from 41.59 to 58.75 kDa. GrCIPK37 had the minimum molecular weight of 21.92 kDa. The molecular weights of GaCIPK36, GaCIPK16, and GrCIPK30 were 81.96, 151.06, and 151.57 kDa, respectively. Two CIPKs, on each of the D (GrCIPK4 and GrCIPK6) and A (GaCIPK14 and GaCIPK17) genomes, were predicted to be localized to the mitochondria. In contrast, the proteins of five CIPKs on the D genome (GrCIPK8, GrCIPK19, GrCIPK30, GrCIPK33, and GrCIPK38) and four CIPKs on the A genome (GaCIPK16, GaCIPK21, GaCIPK28 and GaCIPK36) were predicted to localize to the nucleus. The remaining CIPKs were localized in the cytoplasm, suggesting that the subcellular localization of the proteins of CIPK gene family members in cotton was diverse. It is, therefore, suggested that their corresponding functions should also have diversity, and hence, their subcellular location needs further experimental verification. Previous studies reported that PsCIPK was localized in cytosol and outer membrane in leguminous plants, which was confirmed by immunofluorescence and confocal microscopy (Mahajan et al., 2002). AtCIPK1 could be located in the cell membrane, cytoplasm, and nucleus.

Basic characteristics of CIPK genes in cotton genome.

Gene name Chr Accession No. Introns CDS (bp) AA pI Mw (kDa) Predicted subcellular localization
GrCIPK1 Chr1 Cotton_D_gene_10028535 13 1335 444 8.43 50.63 Cytoplasmic
GrCIPK2 Chr1 Cotton_D_gene_10028390 12 1128 375 5.68 43.07 Cytoplasmic
GrCIPK3 Chr1 Cotton_D_gene_10016226 0 1326 441 9.42 49.68 Cytoplasmic
GrCIPK4 Chr1 Cotton_D_gene_10015278 0 1305 434 9.28 48.79 Mitochondrial
GrCIPK5 Chr1 Cotton_D_gene_10015330 0 1347 448 8.95 50.98 Cytoplasmic
GrCIPK6 Chr3 Cotton_D_gene_10012157 0 1224 407 9.21 46.02 Mitochondrial
GrCIPK7 Chr3 Cotton_D_gene_10012160 0 1326 441 8.29 50.26 Cytoplasmic
GrCIPK8 Chr4 Cotton_D_gene_10038532 13 1338 445 6.58 49.53 Nuclear
GrCIPK9 Chr5 Cotton_D_gene_10002592 0 1293 430 6.65 49.29 Cytoplasmic
GrCIPK10 Chr5 Cotton_D_gene_10002594 0 1353 450 8.87 50.9 Cytoplasmic
GrCIPK11 Chr5 Cotton_D_gene_10005264 0 1389 462 9.19 51.2 Cytoplasmic
GrCIPK12 Chr6 Cotton_D_gene_10012417 11 1341 446 6.73 49.92 Cytoplasmic
GrCIPK13 Chr6 Cotton_D_gene_10020650 1 1374 457 8.52 52.23 Cytoplasmic
GrCIPK14 Chr6 Cotton_D_gene_10020679 13 1455 484 9.01 54.34 Cytoplasmic
GrCIPK15 Chr6 Cotton_D_gene_10015865 10 1185 394 5.85 45.19 Cytoplasmic
GrCIPK16 Chr6 Cotton_D_gene_10021141 0 1377 458 8.44 50.81 Cytoplasmic
GrCIPK17 Chr7 Cotton_D_gene_10035560 0 1308 435 8.93 49.88 Cytoplasmic
GrCIPK18 Chr7 Cotton_D_gene_10035729 13 1368 455 8.99 51.47 Cytoplasmic
GrCIPK19 Chr8 Cotton_D_gene_10015925 11 1245 414 8.91 47.28 Nuclear
GrCIPK20 Chr9 Cotton_D_gene_10037080 0 1353 450 8.95 51.19 Cytoplasmic
GrCIPK21 Chr9 Cotton_D_gene_10037140 0 1296 431 9.16 48.67 Cytoplasmic
GrCIPK22 Chr9 Cotton_D_gene_10019034 12 1131 376 5.73 42.99 Cytoplasmic
GrCIPK23 Chr9 Cotton_D_gene_10007126 0 1347 448 9.22 50.63 Cytoplasmic
GrCIPK24 Chr9 Cotton_D_gene_10033669 0 1437 478 8.54 53.36 Cytoplasmic
GrCIPK25 Chr9 Cotton_D_gene_10001863 13 1350 449 8.37 51.13 Cytoplasmic
GrCIPK26 Chr10 Cotton_D_gene_10039487 0 1296 431 9.12 48.49 Cytoplasmic
GrCIPK27 Chr10 Cotton_D_gene_10039556 0 1350 449 8.67 51.08 Cytoplasmic
GrCIPK28 Chr10 Cotton_D_gene_10040782 0 1323 440 9.31 50.06 Cytoplasmic
GrCIPK29 Chr10 Cotton_D_gene_10040781 0 1449 482 6.75 53.66 Cytoplasmic
GrCIPK30 Chr10 Cotton_D_gene_10040693 19 4191 1396 5.63 151.57 Nuclear
GrCIPK31 Chr10 Cotton_D_gene_10005466 0 1263 420 9.18 47.18 Cytoplasmic
GrCIPK32 Chr10 Cotton_D_gene_10000482 0 1389 462 8.68 52.38 Cytoplasmic
GrCIPK33 Chr11 Cotton_D_gene_10031476 0 1437 478 8.55 53.45 Nuclear
GrCIPK34 Chr11 Cotton_D_gene_10031564 13 1353 450 9.13 50.49 Cytoplasmic
GrCIPK35 Chr11 Cotton_D_gene_10036335 0 1284 427 8.46 49.03 Cytoplasmic
GrCIPK36 Chr11 Cotton_D_gene_10018680 0 1320 439 9.38 49.66 Cytoplasmic
GrCIPK37 Chr13 Cotton_D_gene_10026726 0 591 196 8.94 21.92 Cytoplasmic
GrCIPK38 Chr13 Cotton_D_gene_10022266 11 1371 456 8.53 51.21 Nuclear
GrCIPK39 scaffold84 Cotton_D_gene_10031666 0 1323 440 8.85 48.8 Cytoplasmic
GrCIPK40 scaffold131 Cotton_D_gene_10011736 13 1350 449 6.59 50.96 Cytoplasmic
GrCIPK41 scaffold163 Cotton_D_gene_10017661 12 1275 424 8.83 47.93 Cytoplasmic
GaCIPK1 CA_chr1 Cotton_A_07248 0 1239 412 9.14 46.46 Cytoplasmic
GaCIPK2 CA_chr1 Cotton_A_19207 0 1341 446 8.78 50.71 Cytoplasmic
GaCIPK3 CA_chr1 Cotton_A_13289 13 1320 439 6.86 50.27 Cytoplasmic
GaCIPK4 CA_chr4 Cotton_A_01247 1 1218 405 9.15 45.8 Cytoplasmic
GaCIPK5 CA_chr4 Cotton_A_01175 0 1353 450 8.95 51.25 Cytoplasmic
GaCIPK6 CA_chr4 Cotton_A_32557 0 1377 458 8.44 50.78 Cytoplasmic
GaCIPK7 CA_chr4 Cotton_A_15642 13 1155 384 6.35 43.67 Cytoplasmic
GaCIPK8 CA_chr5 Cotton_A_04333 1 1554 518 8.38 58.75 Cytoplasmic
GaCIPK9 CA_chr5 Cotton_A_04331 0 1293 430 6.45 49.2 Cytoplasmic
GaCIPK10 CA_chr5 Cotton_A_08366 0 1377 458 9.03 50.77 Cytoplasmic
GaCIPK11 CA_chr6 Cotton_A_37254 0 1431 476 8.26 53.08 Cytoplasmic
GaCIPK12 CA_chr6 Cotton_A_25899 10 1098 365 9 41.59 Cytoplasmic
GaCIPK13 CA_chr7 Cotton_A_05019 0 1389 462 8.68 52.42 Cytoplasmic
GaCIPK14 CA_chr7 Cotton_A_04154 0 1224 407 9.21 46.07 Mitochondrial
GaCIPK15 CA_chr7 Cotton_A_04158 0 1326 441 8.63 50.41 Cytoplasmic
GaCIPK16 CA_chr7 Cotton_A_25709 19 4179 1392 5.58 151.06 Nuclear
GaCIPK17 CA_chr8 Cotton_A_17423 13 1335 445 8.98 50.43 Mitochondrial
GaCIPK18 CA_chr8 Cotton_A_38153 0 1440 479 7.09 53.45 Cytoplasmic
GaCIPK19 CA_chr8 Cotton_A_39763 0 1323 440 9.35 50.06 Cytoplasmic
GaCIPK20 CA_chr8 Cotton_A_15608 0 1308 435 9.26 48.91 Cytoplasmic
GaCIPK21 CA_chr8 Cotton_A_15585 11 1368 456 8.36 51.31 Nuclear
GaCIPK22 CA_chr8 Cotton_A_32831 0 1296 431 9.12 48.55 Cytoplasmic
GaCIPK23 CA_chr8 Cotton_A_36065 14 1371 456 9.09 51.94 Cytoplasmic
GaCIPK24 CA_chr8 Cotton_A_40954 0 1314 437 8.58 49.37 Cytoplasmic
GaCIPK25 CA_chr9 Cotton_A_32356 0 1275 424 8.91 48.66 Cytoplasmic
GaCIPK26 CA_chr9 Cotton_A_02928 13 1302 434 8.98 48.73 Cytoplasmic
GaCIPK27 CA_chr9 Cotton_A_23852 0 1320 439 9.39 49.62 Cytoplasmic
GaCIPK28 CA_chr9 Cotton_A_17529 0 1437 478 8.2 53.43 Nuclear
GaCIPK29 CA_chr10 Cotton_A_11022 13 1323 440 6.49 50.26 Cytoplasmic
GaCIPK30 CA_chr10 Cotton_A_19508 13 1350 449 8.56 51.23 Cytoplasmic
GaCIPK31 CA_chr10 Cotton_A_04065 0 1326 441 9.41 49.73 Cytoplasmic
GaCIPK32 CA_chr10 Cotton_A_17083 0 1347 448 9.27 50.73 Cytoplasmic
GaCIPK33 CA_chr11 Cotton_A_12475 0 1308 435 9 49.78 Cytoplasmic
GaCIPK34 CA_chr11 Cotton_A_16517 11 1317 439 6.15 49.15 Cytoplasmic
GaCIPK35 CA_chr11 Cotton_A_34885 13 1350 449 7.15 51.01 Cytoplasmic
GaCIPK36 CA_chr11 Cotton_A_23687 16 2193 730 8.45 81.96 Nuclear
GaCIPK37 CA_chr11 Cotton_A_01750 13 1329 442 6.91 50.66 Cytoplasmic
GaCIPK38 CA_chr12 Cotton_A_30062 1 1374 457 8.52 52.37 Cytoplasmic
GaCIPK39 CA_chr13 Cotton_A_09063 0 1296 431 9.14 48.63 Cytoplasmic

AA = amino acid amount; PI = isoelectric point; Mw = molecular weight, kilodalton (kDa) is used as unit.

Functional domain analysis of the CIPK family members in cotton

Functional domain analysis of the identified CIPK family members was conducted (http://prosite.expasy.org/) and the results revealed that all the protein sequences of CIPKs in the D and A genomes contained the protein kinase domain (PS50011). Except for GrCIPK37 in the D genome, all CIPKs contained the NAF domain (PS50816). Although GrCIPK37 had no NAF domain, Wang et al. (2012) annotated it as a CIPK; therefore, we also listed it as a CIPK family member in the present study and analyzed it with the other members. Proteins of 41 CIPKs in the D genome and 37 CIPKs (except for GaCIPK6 and GaCIPK12) in the A genome contained the serine/threonine protein kinase active site (PS00108). Except for GrCIPK2, GrCIPK7, and GrCIPK22 in the D genome and GaCIPK7, GaCIPK12, and GaCIPK15 in the A genome, all CIPKs encoded proteins with a protein kinase ATP binding domain (PROTEIN_KINASE_ATP, PS00107). All the CIPKs in the D and A genomes, except for GaCIPK12, contained a proton acceptance locus (ACT_SITE). There were some differences in the distribution of CIPKs and a number of CIPK family members contained five domains; it is speculated that these five domains have different roles in CIPK function. In addition, protein encoded by GrCIPK12 in the D genome and GaCIPK34 in the A genome contained the ribosomal protein L29 marker (RIBOSOMAL_L29, PS00579) and GrCIPK24 contained an EF-hand type calcium-binding domain (EF_HAND_1, PS00018). In contrast, the zinc finger ring type domain (ZF_RING_2, PS50089) appeared in GaCIPK36 but was not found in other CIPK family members. It is speculated that these genes experienced directional evolution in structure resulting from changes in the environment. These structures might have special functional properties, which would need further experimental verification.

Distribution of CIPK gene family members in the complete genome of cotton

The information about the position of genes on chromosomes provides important evidence for the study of evolution and the function of a gene family. Combined with the chromosome information of the cotton A and D genomes and the locations of the CIPKs on the chromosomes, the distribution map of CIPKs on the chromosomes was prepared (Figure 1). As shown in Figure 1A, 38 of the 41 CIPKs were mapped to 11 chromosomes of the D genome of cotton. In comparison, distribution of the remaining three CIPKs was as follows: GrCIPK39, GrCIPK40, and GrCIPK41 were located on scaffold84, scaffold131, and scaffold163, respectively. None of the three genes was located on the corresponding chromosomes. Chromosomes 2 and 12 had no CIPKs; conversely, chromosome 10 had the highest number of CIPKs (seven), followed by chromosome 9 with six CIPKs. Chromosomes 1 and 6 had five CIPKs and chromosomes 4 and 8 had one. As shown in Figure 1B, all CIPKs in the A genome of cotton were located on the chromosomes and were found on the 11 chromosomes; however, neither chromosome 2 nor chromosome 3 contained any CIPK. Chromosome 8 contained the maximum number (eight) of CIPKs, followed by chromosome 11 with five genes. In contrast, chromosomes 12 and 13 contained only one CIPK. The remaining chromosomes contained 2-4 CIPKs each. In the D genome, GrCIPK6 and GrCIPK7 were distributed on chromosome 3 in the form of a cluster. GrCIPK9 and GrCIPK10 were located on chromosome 5 in a close-linking mode. These two gene clusters contained two gene pairs, GrCIPK6/GrCIPK9 and GrCIPK7/GrCIPK10, which had fewer intron types and similar functions. Furthermore, GaCIPK8 and GaCIPK9 were closely linked on chromosome 5 on the A genome, and GaCIPK15 and GaCIPK14 were distributed on chromosome 7 in a cluster, both of which contained two gene pairs, GaCIPK8/GaCIPK15 and GaCIPK9/GaCIPK14, with fewer intron types and similar functions. This distribution pattern might have been caused by the substitution and insertion in chromosomes. A gene family is created by the random amplification of genes, resulting in the formation of gene clusters. The scattered distribution of the members of a gene family on many chromosomes is most likely caused by partial-fragment replication of chromosome regions (Schauser et al., 2005). Compared to other eukaryotes, plants have a higher gene duplication rate (Wei et al., 2014). The results of previous studies showed that gene duplication and separation of the latter stages are the two main objectives of evolution (Chothia et al., 2003), resulting in the diversity of gene family members. Wang et al. (2012) demonstrated that in G. raimondii, complete genome replications occurred at least two times. The scattered distribution mode of the CIPK genes in the complete chromosome might reflect a series of complete genomes, chromosomes, and large-fragment duplication events with typical characteristics of the D and A chromosome genomes. Duplication of genes leads to diversity of gene functions, which plays a very important role driving the evolution of new features, organ differentiation, and better adaptation to changes in environments (Flagel and Wendel, 2009). Phylogenetic analysis reveals that G. arboreum L. and G. raimondii were derived from a common ancestor dating about 5.0 mya, that the genomes of these two species exhibited a high degree of collinearity at the chromosome level, and that the number of genes and sequences were very similar (Li et al., 2014). For example, there were 13 CIPKs on chromosomes 9 and 10 of the D genome, and 13 CIPKs on chromosomes 8 and 11 of the A genome, which might also be the result of gene duplication or partial segment duplication over the long evolutionary history of the cotton genome.

Chromosome distribution of the CIPK genes in the cotton genome. A. CIPK genes in Gossypium raimondii; B. CIPK genes in Gossypium arboreum L.

Genetic structural analysis of the CIPK gene family members in cotton

Gene structure analysis is important in the research on gene evolution. According to the number of introns, the CIPK gene family members of the D and A genomes of cotton were divided into two categories (as shown in Figure 2), one with more than 10 introns and the other with fewer than three introns. In contrast, the category containing less than three introns had 26 members in the D genome, accounting for 63.41% of the members. Except for GrCIPK13, which contained two exons, all the 25 members contained only one exon. The category with more than 10 introns comprised 15 members in the D genome. GrCIPK15 contained only 11 exons and GrCIPK30 contained 20 exons. The rest of the CIPK members had between 12 and 14 exons each. The situation in the A genome was similar to that in the D genome. The category with less than three introns comprised 25 members, accounting for 64.10% of the members, of which GaCIPK4, GaCIPK8, and GaCIPK38 contained two exons and the remaining 22 members contained one exon. The category with more than 10 introns contained 14 members and more than 10 exons. GaCIPK12 had only 11 exons. In contrast, GaCIPK16 had the highest number (20) of exons. It is speculated that the number of exons of each CIPK member in the D and A genomes of cotton was similar during the evolutionary process of CIPK gene structure, which was relatively conserved, suggesting that the functions of these genes might also be consistent. The results of previous studies showed that the insertion of small fragments of DNA could change the function of the gene, and that the gene could disappear by natural selection (Long et al., 1995). CIPK members, GrCIPK30 and GaCIPK16, of the cotton D and A genomes, respectively, had the highest number (up to 20) of exons, with large differences in the exon length, suggesting that there were significant changes in the structures or functions of these two genes in the evolutionary process of cotton. In addition, this indicates that there were relatively stable exon-intron pairs in either the D or A genome during the evolutionary process of cotton. Compared to the introns, the exons were more vulnerable to selective pressures from the external environment. The structures of the exons in many replication-type gene families were generally conserved; therefore, the differences in the structures of exons and introns caused by insertion-deletion events could be used to predict the evolutionary history of gene families (Lecharny et al., 2003).

Intron-exon structure analysis of the CIPK gene family in cotton. Group I and Group II, CIPK genes in Gossypium raimondii; Group III and Group IV, CIPK genes in Gossypium arboreum L.

Gene pair analysis of the CIPK gene family

The results of cluster analysis conducted on the D genome revealed that 41 members of CIPK in the D genome could be divided into 2 categories, one with no or very few introns and the other with several introns (Figure 3A). Similar results were also obtained with the A genome (Figure 3B). In addition, there were 13 pairs of homologous genes in the D and A genomes (Table S2).

Phylogenetic analysis of the CIPK proteins in the D and A genomes in cotton. A. Phylogenetic trees of the CIPK proteins in D genome. B. Phylogenetic trees of the CIPK proteins in A genome. C. Phylogenetic trees of the CIPK proteins in D and A genomes.

The cluster analysis of the D and A genomes (Figure 3C) also illustrated that 36 CIPKs in the D genome were paired with 36 CIPKs the A genome (Table S2). The formation of such gene pairs might have been caused by duplication events. The sequence consistency of proteins encoded by the CIPK gene pairs of the D genome was from 65.53% (GrCIPK7/GrCIPK10) to 92.2% (GrCIPK25/GrCIPK40), with an average of 82.51% consensus in the sequences. By comparison, the proteins encoded by the CIPK gene pairs of the A genome had a sequence consistency from 65.53% (GaCIPK8/GaCIPK15) to 92.71% (GaCIPK3/GaCIPK29), with an average consensus of 82.79%. In contrast, the consensus in the sequences of corresponding proteins encoded by CIPK family gene pairs of the D and A genomes ranged from 89.05% (GrCIPK14/GaCIPK36) to 99.73% (GrCIPK2/GaCIPK3), with an average consensus of 97.47%. Of these pairs, seven showed >99.00% consensus; these pairs were as follows: GrCIPK32/GaCIPK13, GrCIPK26/GaCIPK22, GrCIPK37/GaCIPK39, GrCIPK6/GaCIPK14, GrCIPK34/GaCIPK26, GrCIPK40/GaCIPK35, and GrCIPK2/GaCIPK3. These results indicate that the interspecies genetic relationship between the CIPK gene family members of cotton is stronger than the intraspecies relationship. They also suggest that the common ancestor of G. arboreum L. and G. raimondii formed the CIPK gene family during the evolutionary process. After the differentiation of the two species about 5.0 mya from the same progenitor, the CIPK gene family experienced linear synchronous evolution in the two species. These comprehensive analyses demonstrate that a gene duplication event had a significant effect on the amplification of the CIPK gene family members in cotton, and that the amplification of the CIPK members might be the result of a high-stress environment. Cotton is chronically affected by an adverse environment during the growth period, which promotes the widespread amplification of genes with similar functions. Moreover, the newly formed CIPK genes must have evolved with the corresponding CBL genes. These functional limitations might have helped in the evolution of the current CBL-CIPK signaling network (Kolukisaoglu et al., 2004).

Phylogenetic analysis of the CIPK gene families of cotton and four other species

To test the evolutionary relationship between the CIPK gene family members in cotton and those in Arabidopsis, rice, maize, and poplar, the amino acid sequences of all CIPK members from these species were compared and a phylogenetic tree was constructed (see Material and Methods). The analysis of the phylogenetic tree revealed that CIPK of the D and A genomes of cotton were mostly clustered together (Figure 4). The relationship between G. raimondii and G. arboreum L. was closer than between the other four species. In addition, many members of the CIPK gene family of cotton and poplar clustered together, suggesting that these two species were evolutionarily close; this was followed by the evolutionary closeness of cotton with Arabidopsis (Table S3). These genes are similar in structure and it is predicted that their functions might also be similar. There were more family members in cotton than in Arabidopsis, rice, and poplar plants, suggesting a specific linear amplification of the gene family in cotton. Whether these additional members of the gene family have additional functions, as well, or whether they are only produced because functional redundancy requires further experimental verification.

Phylogenetic analysis of the CIPK proteins in cotton, Arabidopsis, rice, maize, and poplar. The species used for the construction of the phylogenetic tree are Gr, Gossypium raimondii; Ga, Gossypium arboreum L.; At, Arabidopsis; Os, rice; Zm, maize; and Pt, poplar.

CIPK expression analysis based on the transcriptome sequencing

Transcription analysis has been used for identifying the protein-coding genes during the annotation of the D and A genomes (Wang et al., 2012; Li et al., 2014). In this study, the sequence read lengths of the matched CIPK genes were converted to RPKM values and used to predict the gene expression levels. The data were retrieved from NCBI and at least 20 nucleotide sequences were selected and used in this study.

The abundance of transcripts in the cotton leaves at the beginning of flowering stage, and in 0 and 3.0 DPA ovules was detected (Figure 5). The results showed that about 9.76% of the CIPK genes (GrCIPK12, GrCIPK18, GrCIPK30, and GrCIPK31) in the D genome were expressed in the leaves (Figure 5A). Of these, the expression of GrCIPK18 and GrCIPK31 in the leaves was much higher than that of GrCIPK12 and GrCIPK30, indicating that these two genes play an important role in leaf development and that GrCIPK18 and PtCIPK13 are homologous. Nine CIPK genes (21.95%) were expressed in the 0-DPA ovules and eleven (26.83%) were expressed in the 3.0-DPA ovules. Compared to the expression in the 0-DPA ovules, the expression of GrCIPK4, GrCIPK7, GrCIPK8, GrCIPK30, GrCIPK31, GrCIPK36, and GrCIPK40 in the 3.0-DPA ovules was increased by 1.19-, 1.09-, 1.02-, 3.02-, 3.87-, 2.82-, and 1.78-times, respectively, indicating that these genes play a major role in fiber elongation. GrCIPK12, GrCIPK18, GrCIPK23, and GrCIPK26 were all downregulated with different extent in 3.0-DPA ovules compared with that in 0-DPA ovules, indicating that these genes play important roles in the initiation of fiber formation in cotton. Of the CIPK genes in the A genome, approximately 41.03% (sixteen) were expressed in the leaves, with five genes being highly expressed in the leaves of G. arboreum L. (Figure 5B). GaCIPK8, GaCIPK14, GaCIPK22, GaCIPK33, GaCIPK39, GaCIPK8, and OsCIPK2 were observed to be homologous. In contrast, 35.90% (fourteen) of the CIPK genes were expressed in the 0-DPA ovules, with five genes, GaCIPK4, GaCIPK8, GaCIPK14, GaCIPK33, and GaCIPK39, being highly expressed. One-third (13) of the CIPK genes were expressed in the 3.0-DPA ovules in G. arboreum L., and two of these, namely GaCIPK8 and GaCIPK39, were highly expressed. Compared to the expression in 0-DPA ovules, only GaCIPK2 was significantly upregulated (1.02-times) in the 3-DPA ovules, indicating that this gene plays a main role in fiber elongation. Compared to the expression in the 0-DPA ovules, the expression of three CIPK genes, namely GaCIPK7, GaCIPK14, and GaCIPK32, was downregulated in the 3.0-DPA ovules by 1.95-, 1.54-, and 1.27-times, respectively, indicating that these three genes play important roles in the initiation of cotton fiber. The expression of another nine CIPK genes (GaCIPK4, GaCIPK8, GaCIPK10, GaCIPK15, GaCIPK20, GaCIPK28, GaCIPK31, GaCIPK33, and GaCIPK39) in the 0- and 3.0-DPA ovules was similar, suggesting that they all play important roles in the initiation and development of fiber. GaCIPK22 and GaCIPK25 were expressed only in the leaves, and GaCIPK4 was expressed only in the 0-DPAovules, indicating that the expression of these three genes was tissue specific. GaCIPK22 and GrCIPK26 belong to homologous gene pairs, and GaCIPK22 was abundantly expressed only in the leaves. The expression of GrCIPK26 in the 0-DPA ovules was high, suggesting that homologous genes might also have functional diversity.

Transcript abundance analysis of the CIPK genes in mature leaves, 0-DPA and 3.0-DPA ovules in Gossypium raimondii and Gossypium arboreum L. The transcript expressions were calculated using the RPKM method (Mortazavi et al., 2008).

Expression analysis of the CIPK genes in diploid cottons under different stress conditions

To investigate the anti-adversity function of CIPK genes in cotton, their expression patterns were studied in different tissues of diploid cottons exposed to different stress conditions (low temperature, drought, and salt stress). The results showed that 80 CIPK genes were expressed in the roots, stems, and leaves of diploid cottons, but the expression levels of most of the genes were different in the roots, stems, and leaves after exposure to abiotic stresses (Figure 6).

Expression of the CIPK genes in different tissues of diploid cotton under different abiotic stress conditions. Gene expression analysis of three different tissues of diploid cotton species was launched under different stresses with qRT-PCR method. Red color represented genes upregulated and the expression folds were greater than or equal to 5; Pale red color represented the genes upregulated with expression folds was greater than 1 and less than 5; black color represented the genes with expression amount was almost unchanged; green color represents genes downregulated and the expression folds were less than 1.

Sixteen, 28, and 14 CIPK genes were upregulated in the roots, stems, and leaves of G. raimondii, respectively and 3, 11, and 22 genes were downregulated, respectively, after 4°C treatment. In the roots, stems, and leaves of G. arboreum L. 29, 15, and 22 CIPK genes were upregulated and 10, 19, and 16 genes were downregulated, respectively, after 4°C treatment. However, the expression levels of CIPK genes varied. Moreover, similar expression patterns of CIPK genes were observed under both drought and salt stress. In different tissues, the number of CIPK genes up- or downregulated was different, showing tissue specificity. The results also showed that 11 CIPK genes in G. raimondii and G. arboreum L. were upregulated in the leaves, stems, and roots simultaneously after low temperature treatment (Table S4). In addition, 12 and 15 CIPK genes were simultaneously upregulated in the leaves, stems, and roots in both the diploid cotton species after drought and salt treatment, respectively. These results suggest that these genes could be induced by low temperature, drought, and salinity, and thus, play an important role in the resistance of cotton to the imposed stress. Moreover, differences in the expression of CIPK genes under different stress conditions reveal specificity in the expression.

Analysis of anti-adversity function of CIPK genes in cotton showed that 9 (GrCIPK20/21/23/31, GaCIPK15/19/21/22/35), 14 (GrCIPK6/7/21/22/31/36/37, GaCIPK3/5/6/7/10/14/15), and 7 (GrCIPK22/34/39 GaCIPK32/33/34/36) genes showed upregulation in leaves, stems, and roots, respectively, after low temperature, drought, and salt stress, indicating that these genes could respond to various adversities. Therefore, we speculate that these CIPK genes could respond to many other stresses, and therefore, play a vital role in stress resistance.

DISCUSSION

In recent years, with the development of genome research, comparative genomics has been extensively used for research on gene families, which have attracted increased attention in many species. Several gene families, such as the TIFY (He et al., 2015) and MAPKKK (Yin et al., 2013) gene families, have been identified in cotton. As a pioneer crop in saline soils, the adaptability of cotton is widespread. CIPK genes are important for stress resistance. The previous transcriptome sequencing experiments demonstrated that some CIPK genes of cotton were associated with resistance to cold, salt, and high temperatures. Considering the fact that little research has been conducted on the CIPK gene family in cotton, in the present study, we identified CIPK genes present in the complete genomes (the D and A genomes) of diploid cotton. The CIPK gene members of cotton were assessed for their gene structure, location, evolutionary relationships, and transcription expression patterns, and their response to different abiotic stresses was also evaluated. The results obtained should lay the foundation for an in-depth study of the molecular functions of CIPK genes during the later stages of cotton growth.

In the present study, bioinformatic analyses of the D and A genomes of diploid cotton were conducted using the protein sequences of the CIPK gene family of Arabidopsis, rice, and corn as the query sequences. Forty-one gene members were identified in the D genome and 39 members were identified in the A genome of cotton, which are relatively more evolutionarily conserved and have more members in the gene family than in Arabidopsis, rice, and poplar. Gene structure analysis showed that the CIPK gene family of cotton could be divided into two types: one that is intron-rich and the other that is intron-less or has few introns. The intron-rich type contains more than 10 exons, and the no-intron type (intron-less) contains 1-2 exons. Moreover, the CIPK gene family members of cotton also had complex subcellular localization patterns, which might be due to the directional evolution of function and structure of the CIPK genes over long-term. Conserved domain analysis revealed that CIPK members retained relatively conserved domains, and all the sequences of CIPK in the D and A genomes contained a protein kinase domain (PS50011). Most CIPKs contained an NAF domain (PS50816), the active site (PS00108) of serine/threonine protein kinases, the ATP-binding region of protein kinases (PROTEIN_KINASE_ATP, PS00107), and a proton acceptance locus (ACT_SITE). These domains might be an important prerequisite for the functions of the CIPK and might also be a factor in changing the structures and functions of CIPK genes. The participation of various structural domains in metabolic regulation and their specific functions need further experimental verification. The functions of two genes, GrCIPK24 and GaCIPK36, in response to abiotic stress might be due to their special domains, namely EF-type calcium-binding domain (EF_HAND_1, PS00018) of GrCIPK24 and zinc ring-type domain (ZF_RING_2, PS50089) of GaCIPK36. However, further research is needed to confirm this observation.

Although the evolution of one gene family can be regulated by several mechanisms, evolutionary and structural analyses can help in determining the origin and relationships of different species. In this study, the phylogenetic tree analysis showed that the CIPK gene family members of the D and A genomes of cotton were evolutionarily close. The CIPK family members of the dicotyledonous Arabidopsis and poplar were also closely related, unlike the members of the monocotyledonous maize and rice. AtCIPK3 and GrCIPK2/GaCIPK3, AtCIPK6 and GrCIPK26/GaCIPK22, AtCIPK21 and GrCIPK8, and AtCIPK23 and GrCIPK30/GaCIPK16 were clustered together and formed homologous gene pairs. The clustered genes that formed homologous gene pairs between cotton and rice included OsCIPK2 and GrCIPK10/GaCIPK8, and OsCIPK5 and GrCIPK13/GaCIPK38; the homologous gene pairs in the case of cotton and maize were ZmCIPK5 and GrCIPK13/GaCIPK38, ZmCIPK15 and GrCIPK32/GaCIPK13, and ZmCIPK23 and GrCIPK14/GaCIPK36. The results indicate that these genes might have similar functions. Previous studies have revealed that the AtCIPK3 might regulate the expression of the cold resistance gene, RD29A, by regulating the gene for the transcription factor, CBF/DREB1 (Huang et al., 2011). In addition, GrCIPK2 was only upregulated in the shoots after low temperature and was downregulated in response to other stresses (drought and salt stress). GaCIPK3 was upregulated in all the tissues under salt, drought, and low-temperature treatments except in the leaves after low-temperature treatment, where it was downregulated. Although GrCIPK2 and GaCIPK3 could form a gene pair as suggested by their structures, the function of these two genes started to differ, possibly due to stronger resistance and adaptability of Asian cotton (one of the four cultivated species) in the process of long-term artificial selection. AtCIPK6 is involved in the growth and development of plants (Tripathi et al., 2009) and is regulated under different adverse conditions (Chen et al., 2013). GrCIPK26 was up-regulated (by 2.09-times) in the root after salt stress treatment; GaCIPK22 was upregulated in the roots, stems, and leaves after low temperature treatment, and was upregulated in the leaves and stems after drought treatments. The GrCIPK26/GaCIPK22 pair showed similar function as the homologous AtCIPK6 and could be induced under a variety of stress conditions. Gene pair GrCIPK14/GaCIPK36 showed a similar function in response to salt stress and had a significantly different response to drought stress, indicating that the anti-adversity function of these two genes had already begun to differentiate. Furthermore, the expression of OsCIPK2 and OsCIPK5 could be induced by drought stress and ABA (Xiang et al., 2007). The expression of ZmCIPK5, ZmCIPK15, and ZmCIPK23 was upregulated under high- and low-temperature stress (Chen et al., 2011).

These results indicate that many members of the CIPK gene family in cotton play important roles in the growth and development of plants and in their reaction to stress, but the specific mechanism by which they perform these roles needs further elucidation. Cotton fibers originate from the epidermal cells of ovules, and their growth and development are divided into four stages, namely initiation, elongation, secondary cell wall synthesis, and a mature period. The fiber cell differentiation directly affects the length of the mature fibers on the ovules, and the initial stage of fiber development is from 2.0 to 1.0 DPA. The elongation stage is from 2.0 to 8.0 DPA (Wang et al., 2010) and 0-3.0 DPA is the critical period for the formation of fiber (lint) from the differentiated epidermal cells on the ovules. We, therefore, selected 0 DPA as the initial stage and 3.0 DPA as the elongation stage of fiber development for our analyses. The expression analysis of CIPK based on transcriptome sequencing data demonstrated that there were differences in the CIPK transcript levels in the leaves during the initial flowering period and in the ovules at different times of flowering. The differences in the expression were observed for the different CIPK gene family members in the D or A genome, suggesting that these genes had temporal and spatial specificity of expression during the growth and development processes in cotton. However, there were more CIPK members in the A genome whose transcript expression in the ovule was altered, indicating that the role of CIPK genes in the A genome is more important in the development of fiber than those of the genes in the D genome. This could be due to the fact that G. arboreum L. has more well-developed fibrous tissue than G. raimondii and has more genes involved during the developmental process of fiber. This study showed that GrCIPK8 and GrCIPK30 play a role in cotton fiber elongation, and that GrCIPK8/AtCIPK21, and GrCIPK30 and AtCIPK23 are homologous genes. It is reported that AtCIPK23 could regulate potassium uptake and stomatal movement (Xu et al., 2006). Thus, we speculate that the functions of GrCIPK8 are diverse. GrCIPK26 and AtCIPK6 are homologous genes and GrCIPK26 plays a role in the initiation of cotton fiber. AtCIPK6 responds to abiotic stress and is regulated by ABA (Chen et al., 2013), suggesting that GrCIPK26 also has a variety of functions in the growth and development of cotton. The current research demonstrates that the CBL-CIPK system plays an important role in the mechanism of plant resistance to stress; however, research on the CBL-CIPK signal network has mostly been focused on the model plants, like Arabidopsis and rice. Whether the CBL-CIPK signaling system of Arabidopsis and rice exists in other monocotyledonous and dicotyledonous plants, and whether there are differences in the expression and functions of CBL and CIPK of cotton, Arabidopsis, and rice has not yet been determined. If AtCIPK23 could interact with AtCBL1 and AtCBL9 simultaneously and respond to low potassium stress, and AtCIPK23 and GrCIPK30/GaCIPK16 are homologous gene pairs, then whether GrCIPK30 or GaCIPK16 would also simultaneously interact with CBL remains to be studied along with the mechanism of their interaction.

Supplementary material

Table S1. The primers of fluorescence quantitative real-time PCR of the CIPK gene family of diploid cotton under different abiotic stress conditions.

Table S2. Gene pairs in D genome and A genome of the CIPK gene family.

Table S3. The CIPK homologous gene pairs between cotton and other specices.

Table S4. The specific expression of the CIPK gene family in leaves, stems and roots of Gossypium raimondii and Gossypium arboreum L. under different stresses.