Isolation and basic analysis of GhCIPK6
In present study, cDNA and genomic DNA sequences of GhCIPK6 were isolated from Upland cotton ‘Zhong G5’ . A comparison of the genomic DNA and cDNA sequences using GSDS (http://gsds.cbi.pku.edu.cn/)  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).
To determine genomic location of GhCIPK6, we compared GhCIPK6 to the genome sequences of D5 (G. raimondii) , A2 (G. arboreum) , and AD1 (G. hirsutum L. acc. TM-1) , and searched for homologs (Table 1). We identified four homologs in the D5 genome, including Gorai.001G032000.1, Gorai.009G055400.1, Gorai.010G112400.1, and Gorai.013G216700.1, in which, Gorai.009G055400.1 was the most identified to GhCIPK6 (Additional file 6 Fig. S1). In the A2 genome sequence, three homologs of GhCIPK6 were identified, including Cotton_A_01247, Cotton_A_09063, and Cotton_A_07248, and GhCIPK6 exhibited the highest similarity to Cotton_A_01247. The AD1 genome sequence harbored three homologs of GhCIPK6, namely, Gh_A05G0418, Gh_D05G0536, and Gh_D13G1983. Gh_A05G0418 and Gh_D05G0536 shared more than 99% amino acid sequence identity with GhCIPK6. Phylogenetic analysis showed that all the CIPKs were classified as four groups (Group I to IV, Additional file 6 Fig. S1). GhCIPK6 was classified into Group III. The nucleotide sequence of GhCIPK6 shared 66.22% identity with AtCIPK6 (AT4G30960, http://www.arabidopsis.org/), and 70.72% with CaCIPK6 (EU492906.1, Cicer arietinum, http://www.ncbi.nlm.nih.gov/). Meanwhile, multiple sequence alignment of GhCIPK6 with related proteins were carried out (Fig. 1). These CIPK protein kinases included highly conserved functional domains, such as catalytic domain, activation loop, transmembrane helix domain, and NAF/FISL motif.
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 . So we detected and investigated which GhCBL proteins interact with GhCIPK6 using the BiFC method.
Four GhCBL gene sequences obtained from NCBI (http://www.ncbi.nlm.nih.gov/): GhCBL1 (EU085038.1), GhCBL2 (EU085042.1), GhCBL3 (EU085040.1), and GhCBL8 (EU085041.1). When GhCIPK6-YFPN and GhCBLs-YFPC fusion genes were co-expressed in onion epidermal cells using particle bombardment, yellow fluorescence signals were observed in the nucleus and cell membrane when GhCIPK6 co-expressed with GhCBL1 and GhCBL8 (Fig. 3). By contrast, no signal was observed when GhCIPK6 co-expressed with GhCBL2 or GhCBL3 in onion epidermal cells (data not shown).
GhCIPK6 and salt-response genes significantly up regulated under 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 (T1 generation) were obtained (Additional file 8 Fig. S3a). Through kanamycin resistance assay, PCR analysis and Southern blotting assay, we obtained two T2 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). However, the expression level in OE1 line no significantly 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 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, 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 profiles 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 filter paper placed upright under salt treatment (150 mmol L-1 NaCl) and control (distilled water, CK) (Fig. 6a, Additional file 10 Fig. S5). The seed germination rate of the transgenic cotton lines (48.89% and 53.33%) was significantly higher than that of wild type (17.12%) under salt stress (Fig. 6a, Additional file 10 Figure S5). But the differences between transgenic lines and wild type line in germination potential under salt stress significantly (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 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. 8). Since MDA and proline promote membrane stability, and POD and SOD limit membrane lipid peroxidation by reducing the accumulation of H2O2, So the result suggests that over-expression of GhCIPK6 increases both the POD and SOD activities, and thereby reduces H2O2 accumulation and protects plant seedlings from membrane damage under salt stress.
Improved salt tolerance of GhCIPK6 transgenic cotton in field experiment
To evaluate the salt tolerance of the GhCIPK6-overexpressed cotton plants, we planted the T6 and T7 generation transgenic lines and the control under two different conditions in the field experiment in Handan City, Hebei Province, China. The generation processes of transgenic lines were shown in Additional file 8 Figure S3c and S3d. Table 2 showed the yield and fiber quality traits of transgenic lines and wild type control in 2016 and 2017.
During seedling stage, the seedling field emergence percentage of GhCIPK6 overexpression lines and WT line were similar under normal condition. Under salt stress, the seedling field emergence percentage of OE lines was significantly higher than WT lines over two years (Fig. 9). In the flowering 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 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 2). Therefore, GhCIPK6 overexpressed in cotton increased the seedling field emergence percentage, seed cotton yield under salt 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 11 Fig. S6). 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 . However, the transgenic plants and Z9806, a salt resistant cotton variety, could grow very well, and show no necrosis in leaves (Additional file 11 Fig. S6).
GhCIPK6 involved in MAPK signaling pathway and plant hormone signal transduction pathway to response to salt stress
In order to understand the mechanism of GhCIPK6 overexpressing to improve salt tolerance in transgenic cotton, we analyzed the transcriptome of GhCIPK6-overespression (OE2) line and wild-type plants. A total of 252 genes were up- and 79 genes were down-regulated, respectively, in GhCIPK6-overexpression compare to wild-type plants (Additional file 12 Fig. S7, Additional file 1 Table S1). In 252 up-regulated DEGs, GO-term analysis indicated 78 genes enriched in response to signaling, stress, ROS and stimulus progresses (Additional file 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 file 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 file 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).
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) (Fig. 10c). 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 (Fig. 10d, e, Additional file 14 Fig. S9) and were consistent with the RNA-seq data.
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 significantly higher than wild type under drought (15% PEG 6000) and low-temperature stresses (Fig. 6a).