A CBL-interacting Protein Kinase Gene, GhCIPK6, Increases Salt Tolerance in Transgenic Upland Cotton by Involving in Multiple Stress Signaling Pathways


 Background: Salt stress is one of the most damaging abiotic stresses in production of Upland cotton (Gossypium hirsutum). Upland cotton is defined as a medium salt-tolerant crop. Salinity hinders root development, shoots growth, and reduces the fiber quality.Results: Our previous study verified a GhCIPK6 gene response to salt stress in G. hirsutum. The homologs of GhCIPK6 were analyzed in A2 (G. arboreum), D5 (G. raimondii), and AD1 (G. hirsutum) genomes. GhCIPK6 localized to the vacuole and cell membrane. The GhCBL1-GhCIPK6 and GhCBL8-GhCIPK6 complexes localized to the nucleus and cytomembrane. Overexpression of GhCIPK6 enhanced expression levels of co-expressed genes induced by salt stress, which scavenged ROS and involved in multiple signaling pathways verified by RNA-seq analysis. Water absorption capacity and cell membrane stability of seeds from GhCIPK6 overexpressed lines was higher than that of wild-type seeds during imbibed germination stage. The germination rates and field emergence percentage of GhCIPK6 overexpressed lines were higher than that of control line under salt stress. Moreover, overexpressing of GhCIPK6 in cotton increased lint percentage, and fiber length uniformity under salt stress. Conclusions: We verified the function of GhCIPK6 by transformation and RNA-seq analysis. GhCIPK6 overexpressed lines exhibited higher tolerance to abiotic stresses, which functioned by involving in multiple stress responsive pathways. Therefore, GhCIPK6 has the potential for cotton breeding to improve stress-tolerance.

In Arabidopsis, overexpression of AtCIPK6 gene increased the tolerance to salt stress [52,53].
AtCBL4/SOS3 interacts with AtCIPK24/SOS2 in the roots to mediate Na + extrusion via the action of the H + /Na + antiporter SOS1 at the plasma membrane in SOS signaling pathway in Arabidopsis under salt stress [54,55]. There were a novel SOS pathway, CBL10-CIPK8-SOS1, functions to transport accumulated Na + out of cells to regulate salt tolerance [51]. The AtCBL1/9-AtCIPK23 complex activates the AKT1 channel at the plasma membrane to enhance K + uptake under low K + condition [24,56,57]. Meanwhile, AtCIPK23 and AtKC1 act synergistically to modulate the activity of AKT1 [58].
In our previous study, we characterized a salt-responsive gene from cDNA libraries and Microarray results, GhCIPK6 (Accession number in GenBank: HM002633), homologous to AtCIPK6 [42]. GhCIPK6 also differently expressed in salt-tolerant and salt-sensitive Upland cotton varieties under salt stress by transcriptome analysis [73]. We isolated the full length of the GhCIPK6 gene and characterized its function in Upland cotton. Changes in physiological indexes showed that over-expression of GhCIPK6 in Upland cotton significantly strengthened the salt tolerance, which were verified that overexpressed GhCIPK6 enhanced co-expressed genes expression levels in stress signaling pathways by RNA-seq results. Meanwhile, the transgenic cotton grew better than wild type in saline field.,. Here we elucidate the function of GhCIPK6 and the potential use in transgenic cotton.

Isolation and basic analysis of GhCIPK6
In present study, cDNA and genomic DNA sequences of GhCIPK6 were isolated from Upland cotton 'Zhong G5' [42]. A comparison of the genomic DNA and cDNA sequences using GSDS (http://gsds.cbi.pku.edu.cn/) [74] revealed that there was no intron in the genomic sequence of

GhCIPK6.
GhCIPK6 contained an open reading frame (ORF) of 1,296 bp and encoded a protein of 431 amino acids and molecular weight 48.637 kD. Using ScanProsite online software (http://au.expasy.org/tools/scanprosite/), motif scan analysis showed that GhCIPK6 included a Serine/Threonine protein kinase catalytic domain at the N-terminal, which contained an ATP-binding region and an active site, and a CBL-interacting NAF/FISL module at the C-terminal. GhCIPK6 contains a transmembrane helix domain between amino acid residues 198 and 217 (Fig. 1).

Subcellular localization of GhCIPK6
Subcellular localization analysis using SubLoc v1.0 (http://www.bioinfo.tsinghua.edu.cn/SubLoc/) indicated that GhCIPK6 was localized to the cytoplasm, which was confirmed by generating a construct by fusing GFP to C-terminal end of GhCIPK6 under the control of the CaMV 35S promoter, and transiently expressing the construct in onion epidermal cells. Indeed, fluorescence was specifically localized to the cytoplasm (Fig. 2b). To establish whether the GhCIPK6: GFP fusion was present at the cell membrane; the onion epidermal cells were plasmolyzed in sucrose solution. The analysis demonstrated that the fusion protein was restricted to the vacuole and cell membrane (Fig.   2c).

Verification of the Interaction between GhCIPK6 and GhCBLs in Vivo
The activation of CIPKs was regulated by binding to one or more CBL proteins. It was previously reported that GhCIPK1 interacted with GhCBL2 and GhCBL3 [72]. So we detected and investigated which GhCBL proteins interact with GhCIPK6 using the BiFC method.

Expression levels of GhCIPK6 and salt-response genes significantly increased by salt treatment
In the cultivar 'Zhong G5', GhCIPK6 transcript accumulated to higher levels in the root, and expression level significantly increased by salt treatment for 1, 6, 12, and 24 hours. In stem tissue, the GhCIPK6 expression was induced after a longer period of salt treatment than that in the root, and the fold-change was less than in the root (Additional file 7 Fig. S2a).
To investigate whether overexpression of GhCIPK6 in cotton enhanced salt tolerance, we transformed GhCIPK6 into Upland cotton cultivar '11-0516'. Eleven transgenic cotton individuals (T 1 generation) were obtained (Additional file 8 Fig. S3a). Through kanamycin resistance assay, PCR analysis and Southern blotting assay, we obtained two T 2 generation transgenic progeny plants that harbored two copies of insert fragment, which named by OE1 and OE2 (Additional file 8 Fig. S3b). We examined the expression level of GhCIPK6 in roots of transgenic and control plants at the three-leaf stage by qRT-PCR analysis. GhCIPK6 expression was significantly higher in OE2 than that in wild type line without treatment (Fig. 4). Meanwhile, the expression level in OE1 line no significantly increased than wildtype cotton. Therefore, we further analyze the functions of GhCIPK6 gene using OE2 transgenic line. We also examined the expression level of GhCIPK6 in different tissues of transgenic cotton under salt stress (Additional file 7 Fig. S2b). Overexpressed GhCIPK6 strongly induced by salt stress in roots of transgenic cotton, especially after 1, 3, 6, and 12 h salt treatments. In addition, the expression profiles of GhCIPK6 gene in other tissues were similar to that of wild type. Moreover, GhCIPK6 expression increased with increasing duration of exposure to salt stress (Fig. 5). To determine the function of GhCIPK6 under multiple abiotic stresses, we analyzed the cotton expression profiles of GhCIPK6 using public datasets from PLEXdb and GEO. GhCIPK6 gene was analyzed under multiple abiotic stresses, such as ABA, cold, drought, salinity, and alkalinity (pH) in G. hirsutum (Additional file 9 Fig. S4).
CIPKs interact with CBLs and PP2Cs to form a complex that regulates the activity of K + transporters [24,75]. Therefore, we examined the expression level of GhAKT1 in transgenic cotton and wild type under salt stress and control (in hydroponic growth, Fig. 5) conditions. GhAKT1 expression only increased strongly in the root of the OE2 line at three-leaf stage after 12 h salt stress treatment, which exhibited similar tendency in stem. After salt treatment at three-leaf stage, GhAKT1 expression was strongly induced in all tissues of the wild-type line, especially the leaf. The increase in expression level might be associated with maintaining K + homeostasis in root upon exposure to salt stress, which would enhance the salt tolerance of transgenic line in turn.
GhCIPK6 interacts with GhCBL1/GhCBL8, SnRK2.6 and PP2C proteins, respectively, to regulate the expression of downstream genes [24,75]. Then, we analyzed the expression profiles of GhCBL1, GhCBL8, GhPP2C (DQ303437.1), and GhSnRK2.6 (JN872373) [12,76] (Fig. 5). The transcript levels of GhCBL1 and GhCBL8 rose sharply soon after exposure to salt stress, and then decreased at 12 h after salt stress in leaves. During the same time, GhCBL1 expression was induced by salt stress in the stem of the transgenic line, OE2. GhPP2C was upregulated in all tissues of OE2 after salt treatment, especially the leaves, except in the roots. GhSnRK2.6 was strongly induced in all tissues of OE2 at one and three hours after treatment, but transcripts only accumulated in the leaves after 6 and 12 h stresses (Fig. 5).
GhCIPK6 enhanced salt tolerance of transgenic cotton Under salt stress, germination and emergence of cotton were the key stages. We determined the germination potential and germination rate of cotton seed using rolls of filter paper placed upright under salt treatment (150 mmol L -1 NaCl) and control (distilled water, CK) ( Fig. 6a, b). Germination potential and germination rate of the transgenic cotton were significantly higher than those of wild type under salt stress. In addition, we also obtained the homozygotes of transgenic Arabidopsis,  Table 1). The cell membrane of the transgenic lines (OE1 and OE2) was more stable than that of wild type, and water absorption capacity was higher than that of wild type during imbibed germination stage. Thus, transgenic cottonseeds may maintain a higher germination rate under salt stress due to increased stability of cell membrane, which ensured that water was absorbed at normal rates under salt stress. Therefore, overexpression of GhCIPK6 in Upland cotton improved salt tolerance in seed germination stage through increasing the stability of cell membrane.
To confirm that overexpression of GhCIPK6 enhanced salt tolerance during seedling stage, we treated transgenic and wild-type seedlings at three-leaf stage by soaking the roots in hydroponic solution with 150 mmol L -1 NaCl. We determined the MDA and proline contents, and SOD and POD activities of seedlings exposed to salt stress. After 2, 5, and 10 d of salt treatment, the transgenic line maintained lower relative content of MDA than control, but relative content of proline and relative activities of POD and SOD were higher than those of wild-type (Fig. 7). Since MDA and proline promote membrane stability, and POD and SOD limit membrane lipid peroxidation by reducing the accumulation of H 2  During seedling stage, the field emergence percentage of GhCIPK6 overexpression lines and WT line were similar under normal condition. Under salt stress, the field emergence percentage of OEs lines was significantly higher than WT lines over two years (Fig. 8). In the mature period, there was a hail disaster on June 28, 2016, which caused the boll number of OEs and WT lines less than that in 2017 (Table 3). Meanwhile, the OE lines recovered better than the WT line after hail disaster, especially under salt stress. The stronger resilience of OE lines was shown in more boll number and higher lint percentage than that in WT line, under salt condition in 2016 (Table 3). Otherwise, the fiber uniformity rate of GhCIPK6 overexpressed lines was significantly higher than that in WT line, which indicated that GhCIPK6 overexpressed lines showed stronger adaptability and resilience in extreme environments.
In 2017, the boll number of transgenic lines was significantly higher than that in WT line under normal condition, which was no significant difference under salt stress. In addition, the boll weight and lint percentage were no significant differences between OE and WT lines. It can be speculate that GhCIPK6 overexpressed in Upland cotton could increase yield under normal condition. There was no influence in fiber quality trait. Under salt stress, there were also no differences in yield and fiber quality traits between overexpression and wild type lines (Table 3). Therefore, GhCIPK6 overexpressed in cotton increased the field emergence percentage, seed cotton yield under normal condition, and maintained the stability of yield and fiber quality traits under extreme treatments.
We also evaluated the salt tolerance of transgenic and control cottons during flowering and boll setting stage in the field in Akesu City, Xinjiang Province, China in 2013 (Additional file 10 Fig. S5).
The salt content of the soil under the surface 5 to 10 cm was approximately 0.92%, which was significantly higher than the tolerance of cotton. Under severe salt stress, there were extensive necrosis in the leaves occurred in the wild type plants and ZG5, a salt sensitive cotton variety [73]. Protein interactions among DEGs was detected using the online STRING program, there were 23 genes co-expressed with GhCIPK6 within 78 up-regulated genes, and two genes co-expressed with GhCIPK6 in 79 down-regulated DEGs (Additional file 5 Table S5). Combined the information of edges and nodes of up-and down-regulated genes, the PPI network was conducted using Cytoscape software (Fig. 10).
In PPI network, there were eight genes predicted to co-express with GhCIPK6 directly, six up-and two down-regulated DEGs. Six up-regulated DEGs were Gh_A03G0309 (18.5 kDa class I heat shock protein, HSP18.5-C), Gh_A05G3030 (Protein phosphatase 2C 37, PP2CA), Gh_A12G2380 (Probable protein phosphatase 2C 75, AHG1), Gh_A13G1741 (Protein phosphatase 2C 56, ABI1), Gh_D04G0015 (Probable protein phosphatase 2C 8, PP2C8), and Gh_D09G1525 (18.2 kDa class I heat shock protein, HSP18.2), respectively. All PP2C genes were involved in MAPK signaling pathway (ko04016) and plant hormone signal transduction pathways (ko04075). Two HSP genes were involved in Protein processing in endoplasmic reticulum (ko04141). Two down-regulated DEGs were Gh_D05G1740 (Serine/threonine-protein kinase STY46) and Gh_D07G1409 (Probable serine/threonine-protein kinase), which were no KEGG annotation, both enriched in the MAPKKK activity process (GO:0004709). All DEGs in the PPI network were verified using qRT-PCR ( Overexpressed GhCIPK6 also improve the tolerance to osmotic and low-temperature stresses To investigate the tolerance to abiotic stresses of transgenic cotton lines, we also determined seed germination potential and seed germination rate using rolls of filter paper placed upright under drought (15% PEG 6000) and low-temperature stresses (distilled water, at 15°C), respectively (Fig. 6a,   b). Germination potential and germination rate of transgenic cotton were significantly higher than wild type (the control) under osmotic and low-temperature stresses.

Discussion
To develop the gene sources for molecular breeding is a popular strategy used in genetic improvement to improve abiotic stresses. Here, GhCIPK6 (HM002633) has the potential to improve the salt tolerance in cotton. GhCIPK6 shared 66.22% nucleotide sequence identity with AtCIPK6.
GhCIPK6 expression was significantly increased by salt treatment (Additional file 8 Fig. S3).
Overexpression of GhCIPK6 enhanced the germination rate under various stress treatments.

GhCIPK6 (HM002633) is a novel gene differed from KC465063_GhCIPK6
A GhCIPK6 gene (GenBank accession number is KC465063) was previously reported in Upland cotton [43,45]. The GhCIPK6 (HM002633) identified here shared 78.56% nucleotide sequence identity with KC465063_GhCIPK6, and 90.49% amino acid sequence identity. We further analyzed the GhCIPK6 (HM002633) and KC465063_GhCIPK6 sequences in the A 2 , D 5 , and AD 1 genomes (Additional file 3 Table S3). The homolog IDs of GhCIPK6 (HM002633) in the A 2 , D 5 and AD 1 genomes are Cotton_A_01247, Gorai.009G055400.1, Gh_A05G0418 and Gh_D05G0536, respectively. There are three homologs of KC465063_GhCIPK6, Gorai.010G112400.1 in the D 5 genome, Gh_A06G0873 and Gh_D06G1020 in AD 1 genome, but there was no homolog in the A 2 genome. Each predicted chromosome locations of KC465063_GhCIPK6 and GhCIPK6 (HM002633) in A 2 , D 5 , and AD 1 genomes were shown in Table 2. Multiple sequence alignment and phylogenic analysis confirmed that the two GhCIPK6s were two different genes in sequence. To confirm that the differences between GhCIPK6 (HM002633) and KC465063_GhCIPK6 not only involved their nucleotide sequences, we analyzed the phosphorylated sites in amino acid sequences (Additional file 14 Fig. S9, Additional file 5 Table S5).
Differences between amino acid sequences caused differences in the phosphorylated sites that might activate downstream genes.
Using public data, we analyzed the relative expression levels of KC465063_GhCIPK6 and GhCIPK6 (HM002633) in different cotton tissues and at different stages of fiber development subjected to various stresses (Additional file 9 Fig. S4). We found that the expression level of GhCIPK6 (HM002633) was strongly higher than KC465063_GhCIPK6 in different tissues under different treatments. GhCIPK6 (HM002633) was more sensitive to salt stress than KC465063_GhCIPK6. The transcripts of GhCIPK6 (HM002633) was up-regulated to a greater extent in roots of salt-tolerant and salt-sensitive cotton cultivars after different salt treatment times. Heterologous expression of KC465063_GhCIPK6 significantly enhanced tolerance to salt, drought, and ABA treatments in transgenic Arabidopsis, but there was no evidence that this gene could increase abiotic tolerance when overexpressed in cotton. Therefore, abiotic stress had a greater effect on GhCIPK6 (HM002633) expression than that on KC465063_GhCIPK6, and these two genes may contribute to salt stress tolerance via different regulatory pathways.
GhCIPK6 can be used in cotton breeding to improve the stress-tolerance Germination stage is the most sensitive stage to salt stress for cotton development [77], and the germination rate of over-expressing GhCIPK6 lines was higher than that of wild type under abiotic stress. The effect of salt stress on cell membrane permeability was detected by determining the electrical conductivity after soaking the seeds for 24h in different concentrations of NaCl solution since cotton is particularly sensitive to abiotic stresses at the germination stage. Compared with control, the transgenic line showed higher germination rate under varied stresses. In imbibed germination stage, the water absorbency rate of transgenic cotton also maintained a normal and stable level under salt stress. After 24 h of soaking in NaCl solution, the cell membrane of transgenic line (OE2) was more stable than control (Fig. 6c, Table 1).
Seed germination indexes include root length and hypocotyl length, which were measured at 9 d after germination under salt and control treatments. There was no significant difference between the transgenic and wild-type cotton in seedlings growth under salt treatment and normal conditions (data not shown). These results suggest that overexpression of GhCIPK6 does not relieve the inhibitory effect to seedlings growth of salt stress.
Under consumingly salt stress, these overexpressed GhCIPK6 cotton plant could grow better than wild type cotton (Additional file 10 Fig. S5). The transgenic lines, and receptors, with salt sensitive and resistive varieties were planted in the natural salinity field in Akesu, Xinjiang Province, China in 2013, which salt content of the soil below the surface 5 to 10 cm was approximately 0.92%. The OE2 line could grow better than other varieties in the salinity field. The damage degree of salt stress of OE2 cotton was lighter than controls (Additional file 10 Fig. S5). Meanwhile, germination, growth, yield and fiber quality traits of transgenic cotton lines were not decreased compare to wild type cotton, especially overexpressed GhCIPK6 plants showed higher adaptive capacity than receptor plants under extreme environments (Fig. 8, Table 3).

GhCIPK6 involved in multiple salt responsive pathways to response salt stress in Upland cotton
In present study, we determined relative content of MDA in the shoots of transgenic and wild-type cotton under salt stress and normal conditions (Fig. 7). Under salt stress, relative MDA content in the OE2 line was maintained at around 1.00. However, in wild-type cotton, relative content of MDA increased with treatment time lasted. After 10 d exposure to salt stress, the MDA content of wild-type cotton was approximately 1.40-fold higher than control. The proline content in the OE2 and wild-type lines were both increased, but the increase was greater in OE2. Soil salt causes changes in physiological indexes while plants come across stress. Physiological indexes, including MDA, proline content, and the activity of antioxidant enzymes (POD and SOD), are typical parameters for evaluating abiotic stress tolerance. Plants with lower relative levels of MDA and higher levels of proline tend to maintain cell membrane stability under stresses [78]. Under salt treatment, the salttolerance cotton genotype displayed higher plant dry weight, photosynthesis and antioxidant enzymes activities [79]. Similarly, the activities of POD and SOD were higher in OE2 than wild type under salt stress, which implied that the transgenic cotton had a greater ability to scavenge the ROS in response to salt stress. Furthermore, the higher the activity of POD and SOD under stresses, the better the plant is able to scavenge over-produced ROS and protect its cells from ROS damage [16].
Among RNA-seq analysis results, there were some DEGs involved in the response to reactive oxygen species pathway (GO:0000302) in OE2 plants (Additional file 12 Fig. S7), which demonstrated GhCIPK6 overexpression could improve the express levels of peroxidase related genes to enhance ROS scavenging ability.
CIPKs are involved in a variety of stress responsive processes, function in multiple regulatory pathways, such as the SOS pathway, the low-K + response pathway [24,75], and the ABA signaling pathway [80]. GhPP2C and GhSnRK2.6 were both strongly induced in OE2 plants under salt stress.
Also in RNA-seq analysis, there were four PP2C genes predicted to co-express with GhCIPK6 in transgenic plants under salt treatment ( Fig. 10; Additional file 3 Table S3). It was speculated that GhCIPK6 is involved in the MAPK signaling pathway and plant hormone signal transduction pathway, by co-expressed with GhPP2C proteins.
The CIPK family of protein kinases regulates ion homeostasis by forming a complex with CBL proteins.
The CBL4/CIPK6 complex in Arabidopsis modulates the AKT2 potassium channel in the plasma membrane [81]. In vitro CBL1, CIPK23, and AKT1 can act together to mediate the CBL-dependent enhancement of phosphorylation of target proteins by CIPKs [82]. CIPKs modulate the activity of the genes encoding K + channels to mediate root K + uptake, such as AtAKT1 and AtAKT2 in Arabidopsis [24,56,81,83]. A signaling network consisting of CBL-CIPK-PP2C has not been shown to activate the AKT1 channel [24]. Overexpression of GhCIPK6 in cotton might enhance tolerance to abiotic stress by interacting with GhCBLs, GhPP2C, and GhSnRK2.6, also regulates peroxidase activity, which were also verified using RNA-seq analysis between OE2 and WT cottons ( Fig. 9; Additional file 12 Fig. S7). Thus, GhCIPK6 probably functions in various signal pathways that mediate response to abiotic stresses in cotton. In conclusion, GhCIPK6 could function in multiple stress responsive pathways, such as K + uptake pathway (Fig. 5), ROS scavenging pathway ( Fig. 7; Additional file 12 Fig. S7a), MAPK signaling pathway and plant hormone signal transduction pathway (Fig. 9), to enhance salt tolerance in transgenic cotton. In one word, this work shows that GhCIPK6 overexpression can improve the salt tolerance in cotton.

Conclusions
We isolated the GhCIPK6 from Upland cotton under salt stress, and verified the function of GhCIPK6 by transformation and RNA-seq analysis. GhCIPK6 overexpressed lines exhibited higher germination rate than wild-type cotton under abiotic stresses, which functioned by involving in multiple stress responsive pathways, such as the K + uptake pathway, the ROS scavenging pathway, MAPK signaling pathway and plant hormone signal transduction pathway. Moreover, GhCIPK6 overexpressed lines increased lint percentage, and fiber length uniformity under salt stress in the field. Therefore, GhCIPK6 has the potential for cotton breeding to improve stress-tolerance.

Plant materials and plant growth conditions
The Upland cotton cultivar '11-0516' (a line bred from CCRI 12) was used for genetic transformation.
To amplify candidate gene sequences, cDNA and genomic DNA were isolated from the Upland cotton cultivar 'Zhong G5'.
Upland cotton seedlings were grown in modified 1/2 Hoagland solution. Plants were grown at 28/20°C, a light intensity of 600 mol·cm 2 •s -1 , and a photoperiod of 14h light/10h dark [42]. At the three-leaf stage, half of the seedlings were transferred to plates containing 150 mmol L -1 NaCl solution in the growth medium; the remaining seedlings was grown in normal nutrient solution as control. Roots, stems, and leaves of the treated seedlings and control ones were sampled separately at 1, 2, 3, 6, and 12 h after exposure to salt stress, immediately frozen with liquid nitrogen and stored at −80°C for RNA extraction and qRT-PCR analysis.
Total RNA was extracted, qualified, and quantified, contaminating genomic DNA was digested with DNase I, and the RNA was reverse transcribed into cDNA following a previously described method [42].

Isolation and bioinformatics analysis of full-length GhCIPK6
The primers which were designed using Primer Premier 5 software (PREMIER Biosoft International) and shown in Additional file 1 Table S1, were used to amplify the full-length cDNA of a previous reported CBL-interacting protein kinase (GW691274) from Upland cotton roots [42]. The fragment was obtained using a TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio Inc.). The amplification conditions were as follows: 94°C for 5 min, then 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min, followed by 72°C for 10 min. Then the fragment was purified and sequenced.
Multiple sequence alignment of GhCIPK6 with CIPK proteins of other species was performed using the ClustalW program implemented in DNAMAN software, and the phylogenetic tree was constructed using MEGA5.2 software with the neighbor-joining method and the 1,000 bootstrap test replicates.

Subcellular localization of GhCIPK6
To make GFP-tagged GhCIPK6, the coding region of GhCIPK6 was amplified with primers containing KpnI and BamHI restriction sites, and the PCR product was first cloned into pMD 18-T vector (TaKaRa), and then sequenced. After KpnI and BamHI digestion, the PCR fragment was cloned into the p3301-GFP plasmid, resulting in p3301-GhCIPK6: GFP.
For subcellular localization in onion (Allium cepa) epidermal cells, the fusion construct p3301-GhCIPK6: GFP was introduced into onion epidermal cells by particle bombardment using the PDS-1000 System (Bio-Rad) at 1,100 psi helium pressure. The p3301-GFP vector was also introduced as control.
GFP expression level was observed using a fluorescence microscope (Nikon, EZ C1) after incubation for 22-24 h at 28°C. The onion epidermal cells were plasmolyzed in 30% sucrose solution.

BiFC analysis of interaction between GhCIPK6 and GhCBLs
The fusion vectors pUC-GhCIPK6-YFP N and pUC-GhCBLs-YFP C were constructed using gene-specific primers with restriction sites (Additional file 1 Table S1). The constructs were transiently co-expressed in onion epidermal cells by particle bombardment using the PDS-1000 System (Bio-Rad) at 1,100 psi helium pressure. After incubation for 22-24 h at 28°C, YFP fluorescence in the transformed cells was observed by confocal laser scanning microscopy (Nikon, EZ C1).

Overexpression vector construction and cotton transformation
The coding sequence of GhCIPK6 was amplified by PCR using LA-Taq DNA polymerase and genespecific primers containing restriction enzyme sites (Additional file 1 Table S1). GhCIPK6 was cloned and inserted into the Xba I-Sac I site of the pBI121 vector, under the control of the CaMV 35S promoter. The recombinant plasmid was named pBI-GhCIPK6. The construct was transferred into cotton cultivar (11-0516) using the pollen tube-pathway method [84]. Seedlings of independent lines of GhCIPK6 transgenic plants were identified using 5g L -1 kanamycin sulfate solution, and were further confirmed by amplification of NPTII, which primers were shown in Additional file 1 Table S1. The copy number of the inserted fragment was determined by Southern Blotting analysis.

Physiological index determination
To investigate the tolerance of transgenic cotton lines to abiotic stress, germination assays were performed using filter paper roll upright method. Twenty seeds of each transgenic or control cotton line were placed in a roll of filter paper, supplemented with distilled water (Control), NaCl solution (150 mmol L -1 ) and PEG6000 solution (15%, w/w), respectively, and maintained at 28°C for 9 days, three replicates. Root length and hypocotyl length of seedlings grown under salt stress were measured on the ninth day. In addition, to evaluate the chilling tolerance of the transgenic lines, seeds were placed at 15°C for 15 d and the germination rate was obtained, three replicates. To determine the effect of salt stress on germination stage, water absorptivity rates were measured during the imbibition stage. Twenty seeds of each transgenic or control line exposed to solution containing different NaCl concentrations (0, 100, 150, and 200 mmol L -1 ), and maintained at 28°C, four replicates. The weight of seeds were determined each two hours.
For seedling growth assay, transgenic seedlings and control were grown in modified 1/2 Hoagland solution. At the three-leaf stage, transgenic and control cotton seedlings were transferred to 1/2 Hoagland solution or solution supplemented with 150 mmol L -1 NaCl. Roots of salt-stressed and control seedlings were sampled at 1, 2, 3, 6, and 12 h after treatment, immediately frozen in liquid nitrogen, and then stored at -80°C until used for RNA extraction and qRT-PCR analysis. After growing for 2, 5, and 10 days under salt stress, leaves were sampled for determination of physiological indexes, including the MDA and proline contents, and antioxidant enzyme (SOD and POD) activity, followed previous methods [85][86][87]. All assays include three biological replications with six plants each replication and error bars indicate the standard deviation (SD). The field design followed a randomized complete block design with four replications, respectively.
Field management followed conventional standard field practices except irrigation. The saline field was irrigated with 0.4% saline water before sowing, and the control field was irrigated with fresh water. The receptor cultivar '11-0516' was used as control in normal and salinity conditions.  [73], and expression profiles of candidate genes were also analyzed. All microarray data was normalized using robust multichip analysis (RMA). The expression profiles of several candidate genes were examined using quantitative RT-PCR in transgenic cotton plants exposed to salt stress. The heatmap was constructed by Mev software (http://mev.tm4.org/).

RNA sequencing and analysis
Total RNA was extracted from the roots samples of OE2 transgenic line and the receptor cultivar '11-0516' seedlings, which were treated for 0, 1, 6, 12, 24 and 48 hours, respectively, and three biological replicates. RNA-seq libraries were prepared and sequenced on an Illumina Hiseq2500 platform (Biomarker Technology Corporation, Beijing, China). Significant differential expression analysis was using the DEGSeq method [88], defined as the fold change (salt treated cv 0h) > 2 and FDR < 0.01, that analysis was performed using BMKCloud (www.biocloud.net). Gene Ontology (GO) analysis was conducted by WEGO online software. The DEGs enriched pathways analysis was used Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Protein-protein interaction (PPI) networks was established using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) v11 (http://string-db.org/) and Cytoscape app (http://apps.cytoscape.org/apps/stringapp) [89,90]. A combined score ≥ 0.4 was chosen for PPI network construction.

Quantitative RT-PCR analysis
Quantitative RT-PCR analyzed the expression profiles of candidate genes in transgenic cotton plants and wild type subjected to salt stress. Quantitative RT-PCR was performed in triplicate with an ABI PRISM ® 7500 Real-Time PCR System using the SYBR ® Premix Ex Taq ™ Kit (TaKaRa, DRR041A), according to the manufacturer's instruction. The gene-specific primers were shown in Additional file 1 Table S1. The GhUBQ7 transcript was used as a normalization control to quantify relative levels .
Relative expression levels were calculated using the 2 -∆∆t method [91,92]. Standard deviation was calculated from three biological replicates.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are available from the corresponding author on reasonable request.         The specifically expressed DEGs in OE2 plants enriched KEGG pathways and expression profile analysis.

Figure 10
The PPI network predicted using STRING program among the DEGs and GhCIPK6. A. The PPI network predicted using STRING program. The genes with red rhombus background were co-expressed genes in up-regulated DEGs. The genes with green oval background were coexpressed genes from down-regulated DEGs. The genes with gray rectangular background were the ones predicted in the same PPI network with GhCIPK6 from up-regulated DEGs. B.
Verification of expression profile of the up-regulated DEGs co-expressed with GhCIPK6. C.
Verification of expression profile of the down-regulated DEGs co-expressed with GhCIPK6.

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