CBL-Interacting Protein Kinases 18 (CIPK18) Gene Positively Regulates Drought Resistance in Potato

Abstract

Sensor-responder complexes comprising calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs) are plant-specific Ca²⁺ receptors, and the CBL-CIPK module is widely involved in plant growth and development and a large number of abiotic stress response signaling pathways. In this study, the potato cv. "Atlantic" was subjected to a water deficiency treatment and the expression of StCIPK18 gene was detected by qRT-PCR. The subcellular localization of StCIPK18 protein was observed by a confocal laser scanning microscope. The StCIPK18 interacting protein was identified and verified by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). StCIPK18 overexpression and StCIPK18 knockout plants were constructed. The phenotypic changes under drought stress were indicated by water loss rate, relative water content, MDA and proline contents, and CAT, SOD and POD activities. The results showed that StCIPK18 expression was upregulated under drought stress. StCIPK18 is localized in the cell membrane and cytoplasm. Y2H shows the interaction between StCIPK18 and StCBL1, StCBL4, StCBL6 and StCBL8. BiFC further verifies the reliability of the interaction between StCIPK18 and StCBL4. Under drought stress, StCIPK18 overexpression decreased the water loss rate and MDA, and increased RWC, proline contents and CAT, SOD and POD activities; however, StCIPK18 knockout showed opposite results, compared with the wild type, in response to drought stress. The results can provide information for the molecular mechanism of the StCIPK18 regulating potato response to drought stress.

Keywords: StCIPK18; drought stress; potato; protein interaction.

Figure 1

Analysis of potato StCIPK18 gene expression. (A) The relative expression level of StCIPK18 in different organs of potatoes. (B) Relative expression of the StCIPK18 gene under drought stress. CK: control; WS1: water stress group 1; WS2: water stress group 2; WS3: water stress group 3. Relative expression levels, determined by qRT-PCR, relative to the expression of the StEFla gene, are expressed as 2^−ΔΔCt. Each column represents the mean values ± SE (n = 3; * p < 0.05; ** p < 0.01).

Figure 2

Subcellular localization of the StCIPK18 protein in tobacco leaf cells. The EGFP and StCIPK18-EGFP fusion protein transiently expressed in tobacco. GFP: EGFP fluorescence signal in the dark field; Auto: Autofluorescence of chlorophyll; Bright: Cell morphology under bright field; Merged: Combination field. The scale bale represents 10 μm.

Figure 3

Interaction between StCIPK18 and StCBLs, demonstrated by yeast two-hybrid assay and BiFC. (A) Self-activation experiment of StCIPK18 and StCBLs. (B) Y2H assay analyzing the interaction between StCIPK18 (bait) and StCBLs (prey). (C) BiFC assay of the interaction between StCIPK18 and StCBL4. StCIPK18 was introduced into the pSPYCE vector and fused with C-terminal YFP; StCBL4 was introduced into the pSPYNE vector and fused with N-terminal YFP. pSPYCE-StCIPK18 + pSPYNE was used as a negative control. The scale bale represents 20 μm.

Figure 4

Acquisition and identification of transgenic plants. (A,B) Callus and differentiated buds. (C,D) Rooting and screening transgenic plants. WT: Wild-type plant “Atlantic”; OE: Transgenic plant “Atlantic” carrying recombinant plasmids pCAMBIA1300-35S-StCIPK18; RNAi: Transgenic plant “Atlantic” carrying recombinant plasmids pCPB121-StCIPK18. (E,F) PCR detection of transgenic plants. M: DL 2000 marker; P: Positive control plasmid; WT: Negative control; 1–6: Transgenic lines. (G,H) The relative expression level StCIPK18 in the transgenic plants and WT plants. WT: Wild-type plants of “Atlantic”; OE-1~OE-3: Transgenic plants of “Atlantic” carrying recombinant plasmids pCAMBIA1300-35S-StCIPK18; RNAi-1~RNAi-3: Transgenic tubers of “Atlantic” carrying recombinant plasmids pCPB121-StCIPK18. Each column represents the mean values ± SE (n = 3; ** p < 0.01).

Figure 5

Overexpression of StCIPK18 improved drought tolerance after 14 days of water deficiency. (A) Phenotypic differences between WT, OE-StCIPK18 and RNAi-StCIPK18 lines after 14 days of water deficiency. The scale bale represents 10 cm. (B) Water loss from detached leaves. (C) Leaf relative water content under non-stress and drought conditions. (D) CAT activity. (E) SOD activity. (F) POD activity. (G) Proline content. (H) MDA content. Each column represents the mean values SE (n = 3; * p < 0.05; ** p < 0.01).

Figure 6

Prediction pathway model of potato StCIPK18 response to drought stress. StCIPK18 regulates the ability of plants to remove ROS to improve drought tolerance. CBLs can receive Ca²⁺ signal and then combine with StCIPK18 to form a complex, which can regulate the drought resistance of plants by regulating the expression of downstream stress-responsive genes and improving the activity of antioxidant enzymes. The solid line indicates the pathways that have been tested to have a direct effect and the dashed line indicates the pathways that have an indirect effect and require further verification.

Figure 7

RNAi-mediated gene silence in transgenic potato plants. (A) Schematic illustration of the engineered pCPB121-StCIPK18 vector. (B) Schematic illustration of the target region of the StCIPK18 gene.