Knockdown of Cytochrome P450 Genes Gh_D07G1197 and Gh_A13G2057 on Chromosomes D07 and A13 Reveals Their Putative Role in Enhancing Drought and Salt Stress Tolerance in Gossypium hirsutum

Abstract

We identified 672, 374, and 379 CYPs proteins encoded by the CYPs genes in Gossypiumhirsutum, Gossypiumraimondii, and Gossypiumarboreum, respectively. The genes were found to be distributed in all 26 chromosomes of the tetraploid cotton, with chrA05, chrA12, and their homeolog chromosomes harboring the highest number of genes. The physiochemical properties of the proteins encoded by the CYP450 genes varied in terms of their protein lengths, molecular weight, isoelectric points (pI), and even grand hydropathy values (GRAVY). However, over 99% of the cotton proteins had GRAVY values below 0, which indicated that the majority of the proteins encoded by the CYP450 genes were hydrophilic in nature, a common property of proteins encoded by stress-responsive genes. Moreover, through the RNA interference (RNAi) technique, the expression levels of Gh_D07G1197 and Gh_A13G2057 were suppressed, and the silenced plants showed a higher concentration of hydrogen peroxide (H₂O₂) with a significant reduction in the concentration levels of glutathione (GSH), ascorbate peroxidase (APX), and proline compared to the wild types under drought and salt stress conditions. Furthermore, the stress-responsive genes 1-Pyrroline⁻5-Carboxylate Synthetase (GhP5CS), superoxide dismutase (GhSOD), and myeloblastosis (GhMYB) were downregulated in VIGS plants, but showed upregulation in the leaf tissues of the wild types under drought and salt stress conditions. In addition, CYP450-silenced cotton plants exhibited a high level of oxidative injury due to high levels of oxidant enzymes, in addition to negative effects on CMS, ELWL, RLWC, and chlorophyll content The results provide the basic foundation for future exploration of the proteins encoded by the CYP450 genes in order to understand the physiological and biochemical mechanisms in enhancing drought and salt stress tolerance in plants.

Keywords: cytochrome P450 genes; drought and salt stress; oxidant and antioxidant enzymes; stress-responsive genes; virus-induced-gene-silencing.

Figure 1

RNA sequencing analysis for the top 100 upland cotton Cytochrome P450 genes profiled under drought and salt stress conditions. The heat map was visualized using the Mev.exe program (shown by log 10 values) in the control, and in treated samples after 0 h, 1 h, 3 h, 6 h, and 12 h of drought and salt treatment. Yellow—upregulated, blue—downregulated, black—no expression. PEG: Polyethylene glycol.

Figure 2

RT-qPCR analysis of the 30 highly upregulated cotton cytochrome P450 genes as obtained from the RNA sequence data. The heat map was visualized using the Mev.exe program (shown by log 2 values) in control, and in treated samples after 0 h, 1 h, 3 h, 6 h, and 12 h of drought and salt treatment. (A) Osmotic stress treatment by 17% PEG-6000; (B) salt stress treatment by applying 250 mM of NaCl solution. Yellow—upregulated, blue—downregulated, black—no expression.

Figure 3

Phenotype observed in the silenced plants with the TRV2:00 empty vector, wild-type plants and CYP450-silenced plants at 12 days post-inoculation. (A) No obvious symptoms in the leaves of the TRV:00-infected plant (positive control); in each experiment, six plants were used and there were three replicates. (B) RT-qPCR analysis of the changes in the expression level of the two CYP450 genes in the leaf, stem, and root tissues of upland cotton, G. hirsutum, plants treated with VIGS before stress exposure. (C) RT-qPCR analysis of the changes in the expression level of the two CYP450 genes in the leaf tissues of G. hirsutum cotton plants, wild type, TRV:00 and the VIGS plants under drought and salt stress conditions. (D) Gel electrophoresis in determining the effectiveness of gene silencing. TRV2:00 represents the positive control infused with the TRV2 empty vector; TRV2:Gh_Gh_A13G2057 and TRV2:Gh_Gh_D07G1197 represent the CYP450-silenced plants, PDS: phytoene desaturase; WT: wild type (non-VIG plants). Error bars represent the standard deviation of three biological replicates. Letters a/b indicate statistically significant differences (two-tailed, p < 0.01). CK: untreated, Salt: 250 mM NaCl treatment, Drought: 17% PEG-6000 treatment.

Figure 4

Physiological trait measurements in Gh_A13G2057 (CYP450) and Gh_D07G1197 (CYP450)-VIGS cotton plants under salt and drought stress conditions. (A) Representative images of VIGS (TRV: CYP450s, silenced; TRV: 00, control) and the non-VIGS (WT) plants, after eight days of drought and salt stress treatment. Quantitative determination of (B) chlorophyll content, (C) relative water content (RLWC), (D) excised leaf water loss (ELWL), (E) cell membrane stability (CMS) as a measure of ion leakage concentration in leaves of the WT, control and CYP450s-silenced VIGS. All measurements were done after eight days of stress exposure. In (B–E), each experiment was repeated three times. Error bars of the physiological trait measurements represent the standard deviation of three biological replicates. Different letters indicate significant differences between wild-type and CYP450s-VIGS plants (two-tailed; p < 0.01). CK: untreated, Salt: 250 mM NaCl treatment and Drought: 17% PEG-6000 treatment.

Figure 5

Stress-responsive genes (GhSOD, GhP5CS, and GhMYB) expression profiling in CYP450s-VIGS plants. GhActin was used as the reference gene. The letters a/b indicate statistically significant differences (two-tailed, p < 0.01). Error bars of the gene expression levels represent the standard deviation of three biological replicates. CK: untreated, Salt: 250 mM NaCl treatment, Drought: 17% PEG-6000 treatment.