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. 2021 Feb 18;27(2):359–368. doi: 10.1007/s12298-021-00943-1

Genome-wide analysis of CBL and CIPK family genes in cotton: conserved structures with divergent interactions and expression

Weinan Sun 1, Bing Zhang 1, Jinwu Deng 1, Lin Chen 1, Abid Ullah 2,✉, Xiyan Yang 1,✉
PMCID: PMC7907412  PMID: 33707874

Abstract

Calcineurin B-like proteins (CBLs) interact with CBL-interacting protein kinases (CIPKs) to form complex molecular modules in response to diverse abiotic stresses. Although previous studies demonstrated that the CBL-CIPK networks play a crucial role in plants response to abiotic stresses, however, little is known about their functions in cotton. In the present study, a total of 22 GhCBL and 79 GhCIPK gene family members were identified in upland cotton (Gossypium hirsutum Linn). Synteny analysis revealed that most genes of GhCBL and GhCIPK exist in pairs between At sub-genome and Dt sub-genome. Interaction analysis between GhCBL and GhCIPK proteins by yeast two-hybrid (Y2H) suggested that the GhCBL-GhCIPK networks were complex, and exhibited functional redundancy in cotton. Quantitative expression analysis by public transcriptome datasets revealed that some GhCBL and GhCIPK genes are differentially expressed under abiotic stress treatments, and especially under drought stress. Our results not only contribute to understanding the structural features of GhCBL and GhCIPK genes but also provide the basis for in-depth functional studies of GhCBL-GhCIPK networks in stress response for plants.

Supplementary information

The online version contains supplementary material available at (doi:10.1007/s12298-021-00943-1).

Keywords: Abiotic stresses, Cotton, Transcription profile, GhCBL, GhCIPK, Protein interaction

Introduction

Plants suffer from various abiotic stresses, such as drought, salt, cold and heat during their life cycles. However, they developed a series of signal transduction mechanisms for protection against deleterious environmental effects. The calcium ion (Ca2+), as a ubiquitous second messenger, plays an important role in the response to multiple abiotic stresses. Initially, calcium signals are transduced via Ca2+ sensors, which include three types: calcium-binding proteins calmodulin (CaM) and CaM-like proteins (CMLs), calcium-dependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs) (Kudla et al. 2010). Among these, CBLs are unique plant proteins sharing sequence similarity with mammalian calcineurin B, which harbor conserved structural components, and contain four EF-hand motifs with Ca2+-binding capability (Sánchez-Barrena et al. 2005). The PFPF motif, which contains conserved serine residues, is used for phosphorylation by CIPKs, and this phosphorylation mechanism can promote the interaction between CBLs and CIPKs (Du et al. 2011). In addition, some CBLs harbors an N‐terminal MGCXXS/T motif that allows for lipid modification by myristoylation and S‐acylation. It is required for subcellular localization for AtCBL1 (Hashimoto et al. 2012). CBLs relay Ca2+ signals by interacting with and activating the CBL-interacting protein kinases (CIPKs), which are plant specific serine-threonine protein kinases. CIPKs interact with CBLs through an essential C-terminal NAF or FISL motif, which generates a complex signaling network in response to abiotic stresses. The NAF domain has dual functions, including both self-inhibition and specific binding with CBLs to activate CIPKs activity (Guo et al. 2001; Hashimoto et al. 2012). Moreover, other CIPKs may have a PPI domain that can interact with a type of protein phosphatase PP2C in the C-terminal, such as ABI1 and ABI2 (Ohta et al. 2003).

Previous studies have demonstrated that the CBL-CIPK complex plays significant roles in the developmental process, as well as plants responses to multiple abiotic stresses and nutrient signaling cascades (Li et al. 2009; Yu et al. 2014). A well-known SALT OVERLY-SENSITIVE (SOS) signaling pathway CBL4 (SOS3)-CIPK24 (SOS2)-NHX7 (SOS1) were demonstrated in Arabidopsis, in which AtCBL4 recruits AtCIPK24 to the plasma membrane and activates the plasma membrane Na+/H+-antiporter AtNHX7, thus maintaining ion homeostasis for salt tolerance (Qiu et al. 2002). In another way, AtCBL10-AtCIPK24 mainly served to exclude excess Na+ from the cytoplasm into vacuoles to maintain the stability of Na+ in the cytoplasm by targeting vacuole localized K(Na)/H antiporter AtNHX1 (Kim et al. 2007; Quan et al. 2007). While, CBL1/9-CIPK23 activated K+ transporter AKT1 and enhanced K+ uptake, particularly under low-K+ stress, in Arabidopsis and rice (Cheong et al. 2007; Li et al. 2014; Xu et al. 2006). AtCBL9-AtCIPK3 formed a complex that reacted to ABA signals, and functions as a negative regulator in seed germination (Pandey et al. 2008). There are 10 CBLs and 26 CIPKs which build multiple interaction networks to convey Ca2+ signals in Arabidopsis (Manik et al. 2015). The diversity of the expression patterns, subcellular localizations and their targets of CBL-CIPK modules makes the networks flexible and complex, but also redundant (Batistic et al. 2010; Tang et al. 2015; Yasuda et al. 2017). Due to the highly conserved structures, the functions of some CBLs and CIPKs or CBL-CIPKs are supposed to be similar in various plants. For instance, the SOS pathways were conserved in rice, tomato and poplar (Fukuda et al. 2004; Huertas et al. 2012; Tang et al. 2010). Overexpression of OsCIPK23 in rice induced expression of genes related to effective responses against drought (Yang et al. 2008). TaCBL9 also participates in the response to drought stress (Sun et al. 2015). CBL2-CIPK6 involves in plant sugar homeostasis via interacting with tonoplast sugar transporter TST2 (Deng et al. 2020).

Cotton, as a textile source, is an important natural fiber crop globally. Although cotton possesses a higher drought tolerance than other crops, persistent water shortage seriously influences its productivity and quality (Ullah et al. 2017). Importantly, the functions and mechanisms of CBLs-CIPKs in cotton remain poorly understood, especially under drought stress. To fill this gap knowledge, a genome-wide identification of CBL and CIPK genes was performed in upland cotton (Gossypium hirsutum Linn), and the structures, the interactions between the two families and the expression patterns were also analyzed. The results revealed that the structures of CBLs and CIPKs were conserved between cotton and Arabidopsis. The yeast-2-hybrid assay demonstrated that most of the interactions occurred between GhCBL1 and GhCIPK1 sub-groups in cotton. Moreover, GhCBLs and GhCIPKs are differentially expressed under a range of different abiotic stresses, especially drought stress.

Materials and methods

Identification of GhCBL and GhCIPK family genes in G. hirsutum

To obtain candidate gene sequences, 10 AtCBL and 26 AtCIPK gene sequences were downloaded from the TAIR database (https://www.Arabidopsis.org/index.jsp). The sequences of GhCBL and GhCIPK were download from the CottonFGD website (http://www.cottonfgd.org) by querying the Arabidopsis AtCBLs and AtCIPKs proteins in cotton genome database using batch BLAST program (https://www.cottongen.org/tools/batch_blast). The names for GhCBLs and GhCIPKs were assigned based on their homologous genes in Arabidopsis, according to the phylogenetic tree. The molecular weight (MW) and isoelectric point (PI) of each amino acid were calculated by using the ExPASY program (https://web.expasy.org).

For phylogenetic analysis, the protein sequence of all identified CBLs and CIPKs from cotton and Arabidopsis were used for multiple sequence alignment by ClustalX (ver. 1.83). Phylogenetic trees were constructed using the MEGA7.0 software by the Neighbor-Joining method with 1000 bootstrap replicates. The evolutionary distances were completed using the Poisson correction method.

Gene structure and conserved motif analysis of GhCBLs and GhCIPKs

The coding sequence (CDS) and genome sequence of GhCBL and GhCIPK genes were used to analyze gene structure by using the Gene Structure Display Server (GSDS 2.0, http://gsds.cbi.pku.edu.cn/). The amino acid sequences of all GhCBLs and GhCIPKs were used for prediction of motifs with the Multiple Em for Motif Elicitation (MEME) program (http://meme-suite.org/tools/meme). The optimum width of motifs was set to range from 4 to 33, the maximum number of motifs was 18, and default values for other parameters.

Synteny analysis and chromosomal location of GhCBLs and GhCIPKs

GhCBL and GhCIPK genes were obtained from G. hirsutum genomic data and were distributed to the At and Dt subgenomes, according to the phylogenetic analysis. These homologous gene pairs were mapped to syntenic blocks for intra- and inter-genomic comparison. The Circos software was used to draw the syntenic diagram (Gu et al. 2014). The chromosomal locations of GhCBL and GhCIPK genes were verified from the database: Gossypium_hirsutum_v1.1.gene.gff3 (http://mascotton.njau.edu.cn/info/1054/1118.htm) (Zhang et al. 2015). The chromosomal images were drawn using MapChart software. Tandem duplications of GhCIPK genes were determined based on genes located on the same chromosome within a 100 kb genomic window.

Yeast two-hybrid (Y2H) assay

A GAL4-based yeast two-hybrid system was used and the yeast two-hybrid assay was conducted using the Matchmaker™ Gold Yeast Two-Hybrid system (Cat. No. 630489, Clontech). The full length cDNA of GhCBLs were inserted into the pGADT7 vector, and the full length cDNA of GhCIPKs were inserted to the pGBKT7 vector. Gene-specific primers were listed in Online Resource 1. The prey and bait vectors were transformed into Y187 and Y2H gold strains. The interactions between GhCBLs and GhCIPKs after mating were determined by the growth on SD medium with the—Trp/-Leu/-His/ + X-α-Gal (SD-3) and -Trp/-Leu/-His/-Ade/ + X-α-Gal (SD-4) assay as described by the instruction manual (Clontech, PT1172-1, PT4084-1). The positive control was the diploid hybrid yeast containing pGBKT7-53 and pGADT7-T.

Expression analysis of GhCBL and GhCIPK genes

To investigate the expression pattern of GhCBL and GhCIPK genes, the TM-1 transcriptome datasets corresponding to the expression abundances in various tissues and abiotic stresses were acquired from NCBI (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA248163/). The heatmap was generated using HemI software (http://hemi.biocuckoo.org/index.php) (Deng et al. 2014).

To verify the genes regulated by drought stress, three-week-old seedlings of cotton (Gossypium hirsutum L. J668) were cultivated in soil under 16 h light/8 h dark conditions. Three-week-old seedlings were treated by withdrawal water for one week (mild drought condition) and two weeks (severe drought condition), and then rewatered to recover the growth. Leaves and roots were collected from the seedlings at mild drought conditions, severe drought conditions, 2 days after rewatering, and 4 days after rewatering for RT-PCR and qRT-PCR analysis. The seedlings grown under normal conditions were sampled as control. Total RNA was extracted from the samples using a modified guanidine thiocyanate method, as previously described (Tu et al. 2007). For RT-PCR and qRT-RCR analyses, RNA was reverse transcribed to cDNA using SuperScript III Reverse Transcriptase, according to the manufacturer’s instructions. The specific primers for qRT-PCR were designed using Primer Premier 5.0 and synthesized commercially based on the CDS of genes. qRT-PCR analysis of selected GhCBLs and GhCIPKs was performed using an ABI Prism 7500 system (Applied Biosystems). GhUBQ7 (GenBank accession No. DQ116441) was used as the internal control to calculate expression levels. Relative expression values were calculated using the 2−∆∆CT methods. The mean values of three biological replicates were calculated. The primers used for RT-PCR and qRT-PCR were listed in Online Resource1.

Results

Genome-wide identification of CBL and CIPK genes in G. hirsutum

Based on the multiple sequence alignment analysis, a total of 22 GhCBL (Online Resource 2) and 79 GhCIPK genes (Online Resource 3) were identified from the G. hirsutum genome. The cotton CBLs and CIPKs were named according to the nomenclature rules of Arabidopsis, in which A and D represented the A and D subgenome of G. hirsutum. The predicted molecular features (molecular weights and isoelectric points) of GhCBL and GhCIPK proteins were shown in Online Resource 2 and 6. Because cotton is an allotetraploid with At and Dt subgenomes, individual AtCBL and AtCIPK have multiple corresponding cotton homologs. To investigate evolutionary relationships between GhCBLs and GhCIPKs with Arabidopsis CBL and CIPK proteins, Neighbor-Joining phylogenetic trees were constructed based on their protein sequences. GhCBLs were clustered into four groups (I–IV; Fig. 1A). The 10 AtCBLs had their counterparts in cotton, and some AtCBLs presented multiple homologous GhCBLs in cotton, though not for AtCBL3, AtCBL6, and AtCBL7 (Fig. 1A; Online Resource 2). GhCIPKs were divided into three groups (i–iii; Fig. 1B). Similarly, the 26 AtCIPKs proteins had their GhCIPK counterparts in cotton, among them, AtCIPK6 possessed the most homologue in cotton. It was worth noting that there was no GhCIPK homologue that corresponded to AtCIPK19 (Fig. 1B; Online Resource 3). The differences of groups on GhCIPK genes in Arabidopsis and cotton indicated the variant evolution of GhCIPKs in cotton might result in different functions of some GhCIPKs in cotton from Arabidopsis.

Fig. 1.

Fig. 1

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Phylogenetic tree of CBL (A) and CIPK (B) proteins across cotton and Arabidopsis using MEGA7.0 software through the Neighbor-Joining method. A GhCBLs were clustered into four groups (I–IV). B GhCIPKs were divided into three groups (i–iii)

The structures of GhCBL and GhCIPK genes were analyzed. The numbers of introns in all GhCBLs ranged from 6 to 10 (Online Resource 4A). Genes in group II have seven introns. The intron/exon structure of GhCIPKs can be divided into two types, whereby GhCIPKs in group ii and group iii possess few or no introns, while genes in group i have multiple introns (Online Resource 4B).

Conserved motifs of GhCBL and GhCIPK family proteins in G. hirsutum

The conserved motifs were determined using MEME software in order to further investigate the structural features of GhCBL and GhCIPK proteins. Ten motifs were detected in GhCBL proteins (Fig. 2A; Fig. Online Resource 5A, B). All the GhCBL proteins contain motif 1, 2 and 3, which were reported as the four EF-hand motifs (Fig. 2B). Most of the GhCBL proteins in group I–III possess the conserved PFPF motif (Fig. 2A; Online Resource 5A). The myristoylation site motif was also conserved in the GhCBL proteins in group I–II. However, GhCBL10 (A1/D1/A2D2) had a specific motif 10 in at N terminus.

Fig. 2.

Fig. 2

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Conserved motifs of GhCBL and GhCIPK proteins. A Schematic representation of GhCBL proteins. 1–10 with different color represent motifs identified using the MEME program. B Composition of amino acid in some conserved motifs of GhCBL. C Schematic representation of GhCIPK proteins. 1–18 with different color represent motifs identified using the MEME program. D Composition of amino acid in some conserved motifs of GhCIPKs

Eighteen motifs were identified for the GhCIPK proteins, and the detailed information was presented in Online Resource 6A, B. The N-terminal structure of CIPK proteins was conserved, which harbored the activation loop (motif 6). However, the C-terminal structure was divergent among groups (Fig. 2C). Most of the GhCIPK proteins contained NAF domain (motif 9) and PPI domain (motif 8) which were responsible for the interaction with CBLs or protein phosphatases. Surprisingly, the ATP binding site (motif 17) only exists in group iii (Fig. 2C, D). These differences might indicate the functional divergent among different groups.

Chromosomal localization and Synteny analysis of GhCBLs and GhCIPKs

Chromosomal localization revealed that all GhCBL or GhCIPK genes were distributed on most of chromosomes, except Dt04 (Online Resource 7). As expected, most of the homologous genes in GhCBL and GhCIPK families were distributed to homologous chromosomes. It was observed that more GhCIPK genes were distributed on chromosomes A/D05, A/D06, and A/D09 than other chromosomes, while no GhCIPK gene was distributed on chromosomes A/D01 and A/D04 (Online Resource 7). However, some genes, such as GhCBL1A1, GhCBL7A1, GhCIPK9A1, and GhCIPK10D3 had no counterpart in the homologous chromosomes. The asymmetrical distribution between A and D subgenome indicates that these genes might experience different evolution processes in cotton.

Genome synteny of GhCBLs and GhCIPKs was analyzed to explore the relationships among the genes in GhCBL and GhCIPK families. The results revealed that through intergenomic comparison, there was seldom synteny between GhCBL genes due to the fewer numbers of the GhCBL gene family, while strong synteny was found among the GhCIPK genes (Fig. 3).

Fig. 3.

Fig. 3

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Synteny analysis of GhCBLs (red) and GhCIPKs (dark) in G. hirsutum. A01–A13 and A01–D13 represent the 26 chromosomes in G. hirsutum

GhCBLs physically interact with GhCIPKs in vivo

CBL and CIPK proteins were supposed to interact physically via the conserved NAF domain found in CIPKs. Here, yeast two-hybrid system assays were employed to elucidate the interactions between 12 GhCBL and 35 GhCIPK proteins selected from different groups in cotton (Fig. 4). Numerous interactions were identified on SD-3 (-Leu/-Trp/-His/ + X-α-Gal). One GhCBL interacted with multiple GhCIPKs from different groups. GhCBL1A1 interacted with GhCIPK6A3, GhCIPK10D2, GhCIPK14A2, GhCIPK20D1 and GhCIPK24A1. While no interactions were found between GhCIPK5D1, GhCIPK5A2, GhCIPK6D2, GhCIPK6D3 or GhCIPK6A4, GhCIPK10D3 with any of the GhCBLs (Fig. 4A). Not surprisingly, many interactions were reduced when assays were carried out on SD-4 (-Leu/-Trp/-His/-Ade/ + X-α-Gal) (Fig. 4B). It was also demonstrated that the extensive interactions were occurred group I GhCBLs and group I GhCIPK1 sub-groups. However, the specific interaction between CBL1/2 and CIPK23, which was well illustrated in Arabidopsis, was not observed in cotton. Moreover, among the five CIPK6 homologues tested, only GhCIPK6A3 displayed interactions with some GhCBLs (Fig. 4B). These results also suggested either the functional redundancy or functional divergence existed between homologous genes, which contributes to an intricate and complex interaction network between CBLs and CIPKs.

Fig. 4.

Fig. 4

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Divergent interactions between GhCBLs and GhCIPKs by yeast -2-hybrid on SD-3 and SD-4 medium. SD-3 represents the SD medium with -Leu/-Trp/-His + X-α-Gal. SD-4 represents the SD medium with -Leu/-Trp/-His/-Ade + X-α-Gal. Mating with pGADT7 or pGBKT7 empty vector was used as the negative control. “Po” represents positive control, and “Ne” represents negative control

Expression profiles of GhCBLs and GhCIPK

CBLs and CIPKs were reported to participate in abiotic stress response in plants. To investigate the expression patterns of GhCBLs and GhCIPKs in cotton, we detected their transcriptional levels using RT-PCR. Some genes of GhCBLs and GhCIPKs that were significantly altered by drought stress were identified (Fig. 5A, B). Our results showed that GhCBL1A1 and GhCBL2A1 were up-regulated after mild drought conditions in the leaf (L3). However, GhCBL4A1, GhCBL4D1 and GhCBL8D1 were down-regulated after mild and severe drought conditions (R2-R3) in the root, with relatively low expression in leaf (Fig. 5A). RT-PCR analysis demonstrated that some GhCIPK genes showed diverse expression patterns under drought conditions, either in leaf or root (Fig. 5B). It was worth noting that GhCIPK6D1 was significantly up-regulated in leaf under severe drought conditions, it was extremely low expression under normal and after rewatering. GhCIPK5D2, GhCIPK5A2, GhCIPK8D2, GhCIPK14A2, and GhCIPK14D2 were also up-regulated under drought treatments in leaf. While GhCIPK17D1 was down-regulated under drought treatments. GhCIPK5D1 and GhCIPK5A1 were up-regulated in the root, but GhCIPK9A2 and GhCIPK9D2 were down-regulated in root after drought treatments (Fig. 5B).

Fig. 5.

Fig. 5

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Expression profiles of GhCBLs and GhCIPKs. (A–B) Expression analysis of selected GhCBL genes in drought response by RT-PCR. (C–D) Heat-map of selected GhCBLs and GhCIPKs in various tissues and under PEG stress treatments based on transcriptome datasets. (E–F) Expression analysis of selected GhCBL genes in drought response by qRT-PCR. L/R1: mock (seedings were under normal conditions), L/R2-5 represent leaf or root under mild drought condition, severe drought condition, 2 days after rewatering, and 4 days after rewatering

To further clarify the expression patterns of GhCBLs and GhCIPKs, all of GhCBL and GhCIPK genes were analyzed for cotton, using public transcriptome datasets represent the transcription patterns under different tissues and various abiotic stresses (Online Resource 8, 9). Some of the GhCBL genes showed higher expression in the stem than other tissues. And GhCBL1A1 exhibited relatively high expression levels in a range of tissues. Moreover, GhCBL1A1 and GhCBL2A1 were significantly up-regulated after 3 h PEG treatment, and GhCBL4A1 and GhCBL4D1 also were relatively up-regulated after 3 h and 6 h PEG treatments (Fig. 5C), which were concordant with the results from RT-PCR. As shown in Fig. 5D, most GhCIPKs exhibited high expression in various tissues. Moreover, GhCIPK5A2, GhCIPK5A1, GhCIPK9A2, GhCIPK16A1, GhCIPK14A2, GhCIPK14D2 and GhCIPK17D1 were highly expression after 12 h PEG treatment, while GhCIPK6D1 showed higher expressions after 3 h and 6 h PEG treatments than 1 h and 12 h.

qRT-PCR analysis was further conducted to verify the expression profiles of GhCBL and GhCIPK genes (Fig. 5E, F). It was showed that GhCBL1A1 was up-regulated after a severe drought in leaf and root, especially in the root (Fig. 5E). Three genes of GhCIPK were up-regulated under drought conditions both the leaf and root. Moreover, the expression of GhCIPK6D1 could be hardly found in leaf under normal conditions and rewatered (Fig. 5F). Those results suggest that the function of GhCBL and GhCIPK proteins can, in combination, potentially regulate a wide range of responses to abiotic stresses, perhaps in tissues-specific ways.

Discussion

Drought is one of the most important abiotic stress limiting plant growth, development and yield in many crops. In this regard, understanding of regulatory genes underlying drought stress is important for developing transgenic crop to cope with drought stress (Ilyas et al. 2020; Ullah et al. 2020). Calcium and its signaling pathway plays important role in regulating stress responses. Calcineurin B-like proteins (CBLs) are specific Ca2+ sensors of plants, decoding calcium transients by specifically interacting with and regulating a family of protein kinases (CIPKs) (Luan 2009).

In the current study, 22 GhCBL and 79 GhCIPK genes were identified from the upland cotton genome. Every AtCBLs and AtCIPKs had homologous sub-group genes in cotton, except for AtCIPK19 (Fig. 1), with counterpart homologue from either At subgenome or Dt subgenome, since the allotetraploid for cotton genome, which might be the results of the segmental duplication events contributed to the gene families (Cui et al. 2020).

Conserved domains in protein families among different plants might represent their conserved functions in physiological and biochemical processes. Conserved motif analysis revealed that all GhCBLs had four EF-hands that were supposed to capture Ca2+, which was demonstrated in Arabidopsis and other species (Mohanta et al. 2015). GhCBL10 (A1/D1/A2/D2) had a specific motif 10 at the N terminus, which might be a potential transmembrane domain, as indicated in Arabidopsis (Batistic et al. 2010). The conserved N-terminal myristoylation site of GhCBL proteins in groups I and II might be engaged in their subcellular localizations. The conserved NAF or PPI domain of all GhCIPKs also could be used to predict their interactions with GhCBLs and PP2C. In addition, GhCIPK family proteins have an ATP binding site in the N-terminal, which is also consistent with the previous research (Xi et al. 2017). While GhCIPK14A1 lacks the domains, it is highly homologous to GhCIPK14D1 based on phylogenetic analysis, and thus is considered to be a valid GhCIPK (Fig. 1).

The expression patterns of numerous GhCBLs and GhCIPKs were determined under drought stress. The results showed that some GhCBLs and GhCIPKs were differentially expressed under drought stress in leaf or root (Fig. 5). GhCBL2A1, a homolog of AtCBL2, was significantly up-regulated under drought stress in leaf, which was recently verified for its function in sugar homeostasis via interacting with GhCIPK6A3 (Deng et al. 2020). Previous studies suggested the heterologous expression of GhCIPK6A3 significantly enhances the tolerance to drought in Arabidopsis (He et al. 2013). GhCBL4A1 and GhCBL4D1, a pair of homologue genes of AtCBL4, with down-regulated expression under mild and severe drought conditions, displayed similar expression patterns under drought stress (Fig. 5). GhCIPK6D1, a homologue of GhCIPK6A3, was markedly up-regulated both in leaf and root under mild and severe drought conditions, with decreased expression after rewatering. The expression of some GhCIPK genes, whose homologous genes were rarely reported in other studies, was regulated by drought stress. For example, GhCIPK9A2 and GhCIPK9D2, GhCIPK5A1 and GhCIPK5D1 being remarkably up-regulated (Fig. 5). These genes might play a role in drought tolerance mechanisms and constitute a basis for developing transgenic material with drought resistance.

The yeast two-hybrid assay suggested no unique interaction between GhCBLs and GhCIPKs, presumably due to their functional redundancy. The main interactions were found in the GhCBL1 sub-group and the GhCIPK1 sub-group (Fig. 4), which was different from other studies. Previous research showed that AtCBL1/9 could either interact with AtCIPK23 to enhances K + uptake under low-K + stress or interact with AtCIPK26 to activate NADPH oxidase RBOHF (Drerup et al. 2013; Xu et al. 2006). In the current study, GhCBL1A1 was found to either interact with GhCIPK23 or GhCIPK26, which might indicate the conserved functions in different plants. However, GhCBL9A1 could interact with GhCIPK26, but not GhCIPK23 on SD-4 medium, revealing the divergent functions in different plants. SOS2/CIPK24 and SOS3/CBL4 constitute a typical pathway in plants stress response, and the interaction between GhCBL4D1 and GhCIPK24A1 could be detected on SD-3 medium. Nonetheless, we identified some new interactions between GhCBLs and GhCIPKs. For example, GhCBL1/9-GhCIPK6D1, GhCBL9A1-GhCIPK5A2 and GhCBL1D1-GhCIPK7D1. Furthermore, these pairs of genes were differentially expressed under drought stress conditions, which could be used in further study for their functions on the drought stress response.

In conclusion, the phylogenetic relationship and structural features of the GhCBL and GhCIPK gene families and their encoded proteins were analyzed. In addition, their interactions and expression pattern in drought response have also been studied. This study identified candidate genes for further functional analysis of specific GhCBLs and GhCIPKs under drought stress in cotton.

Supplementary information

ESM1 (28488 kb) (27.8MB, docx)

Funding

This work was supported by funding from the National Key Project of Research and the Development Plan (2016YFD0101006) and National Natural Science Foundation of China (31371675).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflicts of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Abid Ullah, Email: abidhzau@webmail.hzau.edu.cn.

Xiyan Yang, Email: yxy@mail.hzau.edu.cn.

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