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. 2024 Jul 30;25(15):8308.
doi: 10.3390/ijms25158308.

Calcium-Dependent Protein Kinase GhCDPK16 Exerts a Positive Regulatory Role in Enhancing Drought Tolerance in Cotton

Mengyuan Yan  1 , Meijie Chai  1 , Libei Li  1 , Zhiwei Dong  1 , Hongmiao Jin  1 , Ming Tan  1 , Ziwei Ye  1 , Shuxun Yu  1 , Zhen Feng  1
Affiliations

Calcium-Dependent Protein Kinase GhCDPK16 Exerts a Positive Regulatory Role in Enhancing Drought Tolerance in Cotton

Mengyuan Yan et al. Int J Mol Sci. .

Abstract

Cotton is essential for the textile industry as a primary source of natural fibers. However, environmental factors like drought present significant challenges to its cultivation, adversely affecting both production levels and fiber quality. Enhancing cotton's drought resilience has the potential to reduce yield losses and support the growth of cotton farming. In this study, the cotton calcium-dependent protein kinase GhCDPK16 was characterized, and the transcription level of GhCDPK16 was significantly upregulated under drought and various stress-related hormone treatments. Physiological analyses revealed that the overexpression of GhCDPK16 improved drought stress resistance in Arabidopsis by enhancing osmotic adjustment capacity and boosting antioxidant enzyme activities. In contrast, silencing GhCDPK16 in cotton resulted in increased dehydration compared with the control. Furthermore, reduced antioxidant enzyme activities and downregulation of ABA-related genes were observed in GhCDPK16-silenced plants. These findings not only enhanced our understanding of the biological functions of GhCDPK16 and the mechanisms underlying drought stress resistance but also underscored the considerable potential of GhCDPK16 in improving drought resilience in cotton.

Keywords: GhCDPK16; Gossypium hirsutum L.; drought stress; osmotic adjustment; reactive oxygen species (ROS).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
GhCDPK16 possesses typical characteristics of CDPK proteins. (A) Conservative domains of GhCDPK16 protein. (B) Model of the GhCDPK16 protein predicted by AlphaFold3. (C,D) Cartoon of S_TKc (C) and EFh (D) structures. (E) Docking of Ca2+ in the EFh domain of GhCDPK16.
Figure 2
Figure 2
Expression analysis of GhCDPK16 during cotton development and hormone treatment. (A) The expression profile of GhCDPK16 in different tissues. (B–F) Transcription level of GhCDPK16 in leaves of cotton after ABA (B), MeJA (C), SA (D), GA (E), and IAA (F) treatments. (G) Expression patterns of GhCDPK16 under PEG-induced drought stress. Values represented the mean ± SD from three biological replicates. ** p < 0.01 and *** p < 0.001 by Student’s t test.
Figure 3
Figure 3
Subcellular localization of GhCDPK16. GFP-GhCDPK16 and GhCDPK16-GFP fusion proteins were transiently expressed in N. benthamiana leaf cells. The empty GFP protein served as the positive control. Bars = 10 μm.
Figure 4
Figure 4
Overexpression of GhCDPK16 increased drought resistance in Arabidopsis. (A) Seed germination of wild-type (WT) and GhCDPK16-overexpressing Arabidopsis on 1/2 MS agar plates with 0 mM, 100 mM, 200 mM, and 300 mM mannitol. Photographs were taken one week after mannitol treatments. OE1–OE3 represented independent homozygous Arabidopsis lines overexpressing GhCDPK16. Bars = 1 cm. (B–D) Phenotypes of WT and overexpressing lines after 10 days of treatment with 0 mM (B), 100 mM (C), and 200 mM (D) mannitol, and the statistics of primary root length under different concentration of mannitol treatments. Bars = 1 cm. Values represented the mean ± SD from three biological replicates. ** p < 0.01 and *** p < 0.001 by Student’s t test.
Figure 5
Figure 5
Overexpression of GhCDPK16 enhanced antioxidant capacity and water retention ability. (A–C) The analysis of the relative water content (A), proline content (B), and MDA content (C) of WT and transgenic lines with or without drought treatment for 10 days. (D–F) Measurements of SOD (D), POD (E), and CAT (F) activities in leaves with WT and GhCDPK16-overexpressing lines after drought treatment. OE1–OE3 represented independent homozygous Arabidopsis lines overexpressing GhCDPK16. Values represented the mean ± SD from three biological replicates. Columns with different letters indicated significant differences (p < 0.05, Student’s t test).
Figure 6
Figure 6
Silencing of GhCDPK16 reduced drought tolerance in cotton. (A) Phenotypes of control and GhCDPK16-silenced plants via VIGS technology. The GhPDS gene was used as a marker, revealing an albino leaf phenotype following VIGS in cotton. Numbers 1 to 3 represented three distinct plants. (B) Relative expression levels of GhCDPK16 in control and TRV:GhCDPK16 plants. (C) Phenotypes of control and GhCDPK16-silenced plants before and after drought treatment. (D–J) The analysis of chlorophyll content (D), relative water content (E), proline content (F), MDA content (G), and activities of SOD (H), POD (I), and CAT (J) in control and GhCDPK16-silenced plants leaves after drought treatment. Values represent the mean ± SD from three biological replicates. ** p < 0.01 by Student’s t test. Columns with different letters indicate significant differences (p < 0.05, Student’s t test).
Figure 7
Figure 7
Expression levels of abiotic stress-related genes in control plants and GhCDPK16-silenced plants. (A–E) Relative expression levels of several ABA-related genes, including GhABA1 (A), GhABA2 (B), GhAAO3 (C), GhCDPK1 (D), and GhDi19 (E) in control and GhCDPK16-silenced plants after drought treatment. Numbers 1 to 3 represented three distinct GhCDPK16-silenced plants. Values represent the mean ± SD from three biological replicates. ** p < 0.01 by Student’s t test.
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