GhCIPK6 Increases Salt Tolerance in Transgenic Upland Cotton by Involving in ROS Scavenging and MAPK 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 A 2 ( G. arboreum ), D 5 ( G . raimondii ), and AD 1 ( 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 MAPK 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 seed germination rates and seedling field emergence percentages 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 ROS scavenging and MAPK pathways. Therefore, GhCIPK6 has the potential for cotton breeding to improve stress-tolerance.

(KC465063) in Arabidopsis enhanced tolerance to salt treatment, and in transgenic cotton, this gene mediates the uptake of K + under Ca 2+ -de cient condition [42,44].
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 [41]. GhCIPK6 also differently expressed in salttolerant and salt-sensitive Upland cotton varieties under salt stress by transcriptome analysis [72]. 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 signi cantly strengthened the salt tolerance, which were veri ed 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 eld. Here we elucidate the function of GhCIPK6 and the potential use in transgenic cotton breeding.

Isolation and basic analysis of GhCIPK6
In present study, cDNA and genomic DNA sequences of GhCIPK6 were isolated from Upland cotton 'Zhong G5' [41]. A comparison of the genomic DNA and cDNA sequences using GSDS (http://gsds.cbi.pku.edu.cn/) [73] 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 con rmed 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, uorescence was speci cally 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).
Veri cation 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 [71]. So we detected and investigated which GhCBL proteins interact with GhCIPK6 using the BiFC method.
GhCIPK6 and salt-response genes signi cantly up regulated under salt treatment In the cultivar 'Zhong G5', GhCIPK6 transcript accumulated to higher levels in the root, and expression level signi cantly 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 le 7 Fig. S2a).
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 le 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 signi cantly higher in OE2 than that in wild type line without treatment (Fig. 4). However, the expression level in OE1 line no signi cantly increased than wild-type 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 (OE2) under salt stress (Additional le 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 pro les 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 pro les 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 le 9 Fig. S4).
CIPKs interact with CBLs and PP2Cs to form a complex that regulates the activity of K + transporters [24,74]. 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 [24,74]. Then, we analyzed the expression pro les of GhCBL1, GhCBL8, GhPP2C (DQ303437.1), and GhSnRK2.6 (JN872373) [12,75] (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 during germination and seedling stages Under salt stress, germination and emergence of cotton were the key stages. We determined the germination potential and seed germination rate of cotton seed using rolls of lter paper placed upright under salt treatment (150 mmol L -1 NaCl) and control (distilled water, CK) (Fig. 6a, Additional le 10 Fig. S5). The seed germination rate of the transgenic cotton lines (48.89% and 53.33%) was signi cantly higher than that of wild type (17.12%) under salt stress (Fig. 6a, Additional le 10 Figure S5). But the differences between transgenic lines and wild type line in germination potential under salt stress signi cantly (data not shown). In addition, we also obtained the homozygotes of transgenic Arabidopsis, analyzed seed germination rate and growth under salt treatment. The transgenic Arabidopsis maintained higher seed germination rate and grew normal under NaCl concentration up to 200 mmol L -1 as well (data not shown).
Meanwhile, we analyzed the changes in water absorbency rate of transgenic and wild type cotton seeds in imbibed germination stage (Fig. 7), and the effect of salt stress on cell membrane permeability by determining the electrical conductivity after soaking for 24 h in different concentrations of NaCl solution (Fig. 6b). 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 maintain a higher seed 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 con rm that overexpression of GhCIPK6 enhanced salt tolerance during seedling stage, we treated transgenic and wildtype 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. 8). Since MDA and proline promote membrane stability, and POD and SOD limit membrane lipid peroxidation by reducing the accumulation of H 2 O 2, So the result suggests that over-expression of GhCIPK6 increases both the POD and SOD activities, and thereby reduces H 2 O 2 accumulation and protects plant seedlings from membrane damage under salt stress. During seedling stage, the seedling eld emergence percentage of GhCIPK6 overexpression lines and WT line were similar under normal condition. Under salt stress, the seedling eld emergence percentage of OE lines was signi cantly higher than WT lines over two years (Fig. 9). In the owering and boll periods , 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 2). Meanwhile, the transgenic 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 2). Otherwise, the ber uniformity rate of GhCIPK6 overexpressed lines was signi cantly 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 signi cantly higher than that in WT line under normal condition, which was no signi cant difference under salt stress. In addition, the boll weight and lint percentage were no signi cant 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 in uence in ber quality trait. Under salt stress, there were also no differences in yield and ber quality traits between overexpression and wild type lines (Table 2). Therefore, GhCIPK6 overexpressed in cotton increased the seedling eld emergence percentage, seed cotton yield under salt condition, and maintained the stability of yield and ber quality traits under extreme treatments.
We also evaluated the salt tolerance of transgenic and control cottons during owering and boll setting stage in the eld in Akesu City, Xinjiang Province, China in 2013 (Additional le 11 Fig. S6). The salt content of the soil under the surface 5 to 10 cm was approximately 0.92%, which was signi cantly 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 [72]. However,  Table S1). In 252 up-regulated DEGs, GO-term analysis indicated 78 genes enriched in response to signaling, stress, ROS and stimulus progresses (Additional le 13 Fig. S8a, b). Among the 78 candidate genes, there were 33 genes response to salt, osmic, and drought stresses (Fig. 10a, b), eight genes involved in MAPK cascade (GO:0000165). The gene Gh_A11G1875 predicted in activation of MAPKK activity process (GO:0000186) and regulation of stomatal closure (GO:0090333). There was one gene, Gh_D02G0057, response to ABA stimulus (GO:0071215), and seven genes were response to gibberellin acid (GO:0009739). KEGG pathways analysis showed the 78 genes mainly enriched in to signal transduction pathways, especially in MAPK signaling pathway (ko04016) and plant hormone signal transduction pathway (ko04075) (Fig. 10a, b, Additional le 13 Fig. S8c).
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 le 2 Table S2). Combined the information of edges and nodes of up-and down-regulated genes, the PPI network was conducted using Cytoscape software (Fig. 10c).
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 under drought (15% PEG 6000) and low-temperature stresses (distilled water, at 15°C), respectively. Seed germination rate of transgenic cottons were signi cantly higher than wild type under drought (15% PEG 6000) and low-temperature stresses (Fig. 6a).

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 signi cantly increased by salt treatment (Additional le 7 Fig. S2). Overexpression of GhCIPK6 enhanced the seed germination rate under various stress treatments.
To con rm that the differences between GhCIPK6 (HM002633) and GhCIPK6 (KC465063) not only involved their nucleotide sequences, we analyzed the phosphorylated sites in amino acid sequences (Additional le 3 Table S3, Additional le 15 Fig. S10). 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 GhCIPK6 (HM002633) and GhCIPK6 (KC465063) in different cotton tissues, at different stages of ber development and subjected to various stresses (Additional le 9 Fig.  S4). We found that the expression level of GhCIPK6 (HM002633) was strongly higher than GhCIPK6 (KC465063) in different tissues under different treatments. GhCIPK6 (HM002633) was more sensitive to salt stress than GhCIPK6 (KC465063). The transcripts of GhCIPK6 (HM002633) was up-regulated to a greater extent in roots of salt-tolerant and saltsensitive cotton cultivars after different salt treatment times. Heterologous expression of GhCIPK6 (KC465063) signi cantly 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 GhCIPK6 (KC465063).
GhCIPK6 can be used in cotton stress-tolerance breeding Germination stage is the most sensitive stage to salt stress for cotton development [76], and the seed 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 24 h 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 seed 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.   6b, Fig. 7).
Seed germination indexes include root length and hypocotyl length, which were measured at nine days after germination under salt and control treatments. There was no signi cant difference between the transgenic and wild-type cotton in seedlings growth under salt treatment and normal conditions (data not shown). In the normal and saline elds, the growth performance during the seedling and owering stages were investigated, between GhCIPK6 overexpression lines and wild type line. The growth of transgenic and wild-type cottons were inhibited by salt stress without signi cant difference between them (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 le 11 Fig. S6). The transgenic lines, and receptors, with salt sensitive and resistive varieties were planted in the natural salinity eld in Akesu, Xinjiang Province, China in 2013, which soil salt content below the surface 5 to 10 cm was approximately 0.92%. The OE2 line could grow better than other varieties in the salinity eld. The damage degree of salt stress of OE2 cotton was lighter than controls (Additional le 11 Fig. S6). Meanwhile, germination, growth, yield and ber 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. 9, Table 2).

GhCIPK6 involved in multiple salt responsive pathways to response salt stress
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. 8). 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 [77]. Under salt treatment, the salt-tolerance cotton genotype displayed higher plant dry weight, photosynthesis and antioxidant enzymes activities [78]. 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 le 13 Fig. S8), 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 [24,74], and the ABA signaling pathway [79]. 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 coexpress with GhCIPK6 in transgenic plants under salt treatment (Fig. 10, Additional le 2 Table S2). 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 [80]. In vitro CBL1, CIPK23, and AKT1 can act together to mediate the CBL-dependent enhancement of phosphorylation of target proteins by CIPKs [81].
CIPKs modulate the activity of the genes encoding K + channels to mediate root K + uptake, such as AtAKT1 and AtAKT2 in Arabidopsis [24,55,80,82]. In wild halophyte Hordeum brevisubulatum, HbCIPK2, combined with HbCBL1, could activate HbVGKC1 to absorb K + , combined with HbCBL4/10 could modulate HbSOS1L to exclude Na + [83]. 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 veri ed using RNA-seq analysis between OE2 and WT cottons (Fig. 10, Additional le 13 Fig. S8). 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. 8, Additional le 13 Fig. S8a), MAPK signaling pathway and plant hormone signal transduction pathway (Fig. 10a, b), 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 veri ed the function of GhCIPK6 by transformation and RNA-seq analysis. GhCIPK6 overexpressed lines exhibited higher seed 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 ber length uniformity under salt stress in the eld. 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 modi ed 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 [41]. 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, quali ed, and quanti ed, contaminating genomic DNA was digested with DNase I, and the RNA was reverse transcribed into cDNA following a previously described method [41].

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 le 4 Table S4, were used to amplify the full-length cDNA of a previous reported CBL-interacting protein kinase (GW691274) from Upland cotton roots [41]. The fragment was obtained using a TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio Inc.). The ampli cation 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 puri ed 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 neighborjoining method and the 1,000 bootstrap test replicates.

Subcellular localization of GhCIPK6
To make GFP-tagged GhCIPK6, the coding region of GhCIPK6 was ampli ed with primers containing KpnI and BamHI restriction sites, and the PCR product was rst 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 uorescence 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-speci c primers with restriction sites (Additional le 4 Table S4). 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 uorescence 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 ampli ed by PCR using LA-Taq DNA polymerase and gene-speci c primers containing restriction enzyme sites (Additional le 4 Table S4). 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 identi ed using 5g L -1 kanamycin sulfate solution, and were further con rmed by ampli cation of NPTII, which primers were shown in Additional le 4 Table S4. 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 lter paper roll upright method. Thirty seeds of each transgenic or control cotton line were placed in a roll of lter 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 nine days, three replicates. The seed germination rate was investigated on the ninth day. The seed germination rate = the number of germinated seeds at the ninth day / the total number of seeds × 100%. Root length and hypocotyl length of seedlings 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. The water absorbency rate = (the weight of seeds after water absorption -the initial weight of seeds before soaking) / the initial weight of seeds before soaking × 100%.
For seedling growth assay, transgenic seedlings and control were grown in modi ed 1/2 Hoagland solution. At the threeleaf 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).

Field phenotype assay
To evaluate the salt tolerance of transgenic cotton lines during the whole growth and development period, eld trials were carried out in Quzhou Experimental Station of China Agricultural University at Handan City (36°78′N, 114°92′E), Hebei Province, China, in summer season of 2016 to 2017. Cotton plants were planted in four row plots with 13 holes each row. Plots were 4 m in length with 80 cm row spacing for the experiment. Plants were spaced 33 cm in rows. Each hole planted eight seeds. The eld design followed a randomized complete block design with four replications, respectively. Field management followed conventional standard eld practices except irrigation. The saline eld was irrigated with 0.4% saline water before sowing, and the control eld was irrigated with fresh water. The receptor cultivar '11-0516' was used as control in normal and salinity conditions. The seedling eld emergence percentage was investigated after sowing 30 days, which were used to re ect the status of the seedling emergency and survival. The seedling emergency percentage = the number of survival seedling / the number of total sowing seeds (eight seeds per hole×13 holes per row × four rows) × 100%. Boll number was surveyed from 44 individuals in each plot on Sept 15 th . Yield related traits consisted of boll weight (g) and lint percent (%). Fiber length (mm), ber length uniformity (%), ber strength (cN·tex -1 ), ber elongation (%), and Micronaire were determined with HVI 1000 (Uster ® HVISPECTRUM, Spinlab, USA) by the Cotton Quality Supervision, Inspection and Testing Center, Ministry of Agriculture, Anyang, Henan Province, China.
Expression analysis based on public expression data of cotton Public cotton expression data (Additional le 5 Table S5) were obtained from PLEXdb (http://www.plexdb.org/index.php) and Gene expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). Transcriptome differences between the root tissues of two cotton cultivars (salt-tolerant 'Zhong 07' and salt-sensitive 'Zhong G5') were evaluated after 3, 12, and 48 h of exposure to salt stress [72], and expression pro les of candidate genes were also analyzed. All microarray data was normalized using robust multichip analysis (RMA). The expression pro les 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). Signi cant differential expression analysis was using the DEGSeq method [88], de ned 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 pro les 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-speci c primers were shown in Additional le 4 Table S4. 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.

Statistical analysis
Statistical analyses were performed using SPSS software (13.0 for Windows Evaluation Version, SPSS Inc., LEAD Technologies, USA). All signi cant differences were identi ed using a t-test and marked as *, p-value <0.05; **, p-value <0.01.

Supplementary Information
Supplementary information accompanies this paper at Additional le 1 Table S1. Up-and down-regulated DEGs in OE2 cotton treated by salt stress screening from RNA-seq analysis.
Additional le 2 Table S2. The edges and nodes information of Co-expression genes with GhCIPK6 in PPI network predicted using STRING online program.
Additional le 3 Table S3 Full-length amino acid sequences were aligned using the integrated ClustalW and phylogenetic tree was constructed using the neighbor-joining method implemented in MEGA5.2 (1,000 bootstrap test replicates). Black triangles indicated GhCIPK6 (HM002633) and homologs from the A 2 , D 5 , and AD 1 genomes, respectively. Black circles indicated GhCIPK6 (KC465063), and homologs from the D 5 and AD 1 genomes, respectively. The percentage of replicate trees was shown at the branches.

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Availability of data and materials
All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.      The water absorbency rate in imbibed germination stage of transgenic lines (OE1 and OE2) and wild-type (WT) line exposed to solution containing different NaCl concentrations (mmol L-1). A, control, represented the water absorbency rates of transgenic lines and wild type line exposed to distilled water (0 mmol L-1 NaCl concentration). B, C, and D represented the water absorbency rates of transgenic lines and wild type line exposed to solution with different NaCl concentrations, (B) 100 mmol L-1, (C) 150 mmol L-1, and (D) 200 mmol L-1, respectively. *, p-value <0.05; **, p-value <0.01.

Figure 10
The speci cally expressed DEGs in OE2 plants analysis. A and B. The DEGs enriched KEGG pathways and expression pro le analysis. C. The PPI network predicted using STRING program. The genes with red rhombus background were coexpressed genes in up-regulated DEGs. The genes with green oval background were co-expressed genes from downregulated DEGs. The genes with gray rectangular background were the ones predicted in the same PPI network with GhCIPK6 from up-regulated DEGs. D. Veri cation of expression pro le of the up-regulated DEGs co-expressed with GhCIPK6. E. Veri cation of expression pro le of the down-regulated DEGs co-expressed with GhCIPK6.

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