GhCIPK6a increases salt tolerance in transgenic upland cotton by involving in ROS scavenging and MAPK signaling pathways

Abstract

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 GhCIPK6a gene response to salt stress in G. hirsutum. The homologs of GhCIPK6a were analyzed in A₂ (G. arboreum), D₅ (G. raimondii), and AD₁ (G. hirsutum) genomes. GhCIPK6a localized to the vacuole and cell membrane. The GhCBL1-GhCIPK6a and GhCBL8-GhCIPK6a complexes localized to the nucleus and cytomembrane. Overexpression of GhCIPK6a 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 GhCIPK6a overexpressed lines was higher than that of wild-type seeds during imbibed germination stage. The seed germination rates and seedling field emergence percentages of GhCIPK6a overexpressed lines were higher than that of control line under salt stress. Moreover, overexpressing of GhCIPK6a in cotton increased lint percentage, and fiber length uniformity under salt stress.

Conclusions: We verified the function of GhCIPK6a by transformation and RNA-seq analysis. GhCIPK6a overexpressed lines exhibited higher tolerance to abiotic stresses, which functioned by involving in ROS scavenging and MAPK pathways. Therefore, GhCIPK6a has the potential for cotton breeding to improve stress-tolerance.

Keywords: CIPK; Co-expression; Salt stress; Signaling pathway; Upland cotton.

Fig. 1

Multiple sequence alignment of CIPK proteins using ClustalW implemented in DNAMAN. Conserved functional domains are underlined. At, Arabidopsis thaliana; Ca, Cicer arietinum; Pt, Populus trichocarpa; Gh, G. hirsutum; Cotton_A, G. arboreum; Gorai, G. raimondii. Numbers shown in the right indicate amino acid residue positions

Fig. 2

Subcellular localization of the GhCIPK6a:GFP fusion protein in onion epidermal cells. a. Fluorescence microscopy images of cells expressing GFP protein. b. Fluorescence microscopy images of cells expressing the GhCIPK6a:GFP fusion. c. Fluorescence microscopy images of plasmolyzed cells in 30% sucrose solution. The cell membrane is marked with a red arrow, and the vacuole membrane is marked with a yellow arrow. Confocal images of epidermal cells were captured 22–24 h after transformation. Bar = 50 μm

Fig. 3

BiFC assay demonstrated the interaction between GhCIPK6a and GhCBLs in onion epidermal cells. GhCIPK6a interacted with GhCBL1 (a) and GhCBL8 (b) in vivo. Confocal images of epidermal cells were captured 22–24 h after transformation. Bar = 50 μm

Fig. 4

Relative expression of GhCIPK6a in the root tissues of transgenic lines (OE1 and OE2) and wild-type (WT) plants at the three-leaf stage under normal treatment. *, p-value < 0.05; **, p-value < 0.01

Fig. 5

qRT-PCR analysis of GhCIPK6a expression and genes that were co-expressed with GhCIPK6a in different tissues under salt stress. a. Relative expression values of genes co-expressed with GhCIPK6a. b. Heatmap of GhCIPK6a expression and genes co-expressed with GhCIPK6a in different tissues under salt stress. WT, the wild type line, 11-0516; OE2, the transgenic cotton. R, root tissue; S, stem tissue; L, leaf tissue

Fig. 6

Analysis of seed germination rate and electrical conductivity of transgenic lines (OE1 and OE2) and wild-type (WT) line under abiotic stresses. a. Seed germination rate of transgenic lines (OE1 and OE2) and wild-type (WT) line under salt, PEG, and cold treatments. b. Electrical conductivity analysis of transgenic lines (OE1 and OE2) and wild-type (WT) line after soaking for 24 h in 0, 100, and 150 mmol·L^− 1 NaCl solution. *, p-value < 0.05; **, p-value < 0.01

Fig. 7

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

Fig. 8

Statistical analysis of physiological indexes in leaves of transgenic (OE2) line and wild-type (WT) line seedlings under 150 mmol·L^− 1 NaCl treatment. a. The relative content of MDA in the leaves of transgenic line (OE2) and wild-type (WT) line after 2, 5, and 10 days of 150 mmol·L^− 1 NaCl treatment. b. The relative concentration of proline in the leaves of transgenic line (OE2) and wild-type (WT) line after 2, 5, and 10 days of 150 mmol·L^− 1 NaCl treatment. c and d. The relative activity of SOD (c) and POD (d) in the leaves of transgenic line (OE2) and wild-type (WT) line after 2, 5, and 10 days of 150 mmol·L^− 1 NaCl treatment. *, p-value < 0.05; **, p-value < 0.01

Fig. 9

The seedling field emergence percentage of transgenic lines (OE1 and OE2) and wild-type (WT) line under normal and saline conditions in 2016 and 2017. *, p-value < 0.05; **, p-value < 0.01

Fig. 10

The specifically expressed DEGs in OE2 plants analysis. a and b. The DEGs enriched KEGG pathways and expression profile analysis. c. 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 co-expressed genes from down-regulated DEGs. The genes with gray rectangular background were the ones predicted in the same PPI network with GhCIPK6a from up-regulated DEGs. d. Verification of expression profile of the up-regulated DEGs co-expressed with GhCIPK6a. e. Verification of expression profile of the down-regulated DEGs co-expressed with GhCIPK6a