Knockdown of NtCPS2 promotes plant growth and reduces drought tolerance in Nicotiana tabacum

Abstract

Drought stress is one of the primary environmental stress factors that gravely threaten crop growth, development, and yields. After drought stress, plants can regulate the content and proportion of various hormones to adjust their growth and development, and in some cases to minimize the adverse effects of drought stress. In our previous study, the tobacco cis-abienol synthesis gene (NtCPS2) was found to affect hormone synthesis in tobacco plants. Unfortunately, the role of NtCPS2 genes in the response to abiotic stress has not yet been investigated. Here, we present data supporting the role of NtCPS2 genes in drought stress and the possible underlying molecular mechanisms. NtCPS2 gene expression was induced by polyethylene glycol, high-temperature, and virus treatments. The results of subcellular localization showed that NtCPS2 was localized in the cell membrane. The NtCPS2-knockdown plants exhibited higher levels of gibberellin (GA) content and synthesis pathway genes expression but lower abscisic acid (ABA) content and synthesis pathway genes expression in response to drought stress. In addition, the transgenic tobacco lines showed higher leaf water loss and electrolyte loss, lower soluble protein and reactive oxygen species content (ROS), and lower antioxidant enzyme activity after drought treatment compared to wild type plants (WT). In summary, NtCPS2 positively regulates drought stress tolerance possibly by modulating the ratio of GA to ABA, which was confirmed by evidence of related phenotypic and physiological indicators. This study may provide evidence for the feedback regulation of hormone to abiotic and biotic stresses.

Keywords: NtCPS2-knockdown; abscisic acid; drought stress; genome editing; gibberellin.

Figure 1

Transcript profiles of tobacco copalyl diphosphate synthase 2 (NtCPS2) genes in response to abiotic stress or biological stress. Ten-week-old tobacco seedlings were treated with drought, 45°C heat, PVY or TMV, the photos were taken after 6 hours of drought stress, 48 hours of heat stress, 7 days of PVY inoculation or 7 days of TMV inoculation (A–D). And seedlings were sampled after photography to extract RNA, and then transcript levels of NtCPS2 were checked by quantitative PCR (RT-qPCR) under drought stress (E), heat stress (F), PVY (G) or TMV (H). In each quantitative reverse transcription quantitative PCR (RT-qPCR), the transcript levels of the tobacco reference gene L25 in different samples were also evaluated. Three technical replicates were performed for each experiment. The data shown are the mean ± SD of three independent experiments. Statistical analysis was performed using the ANOVA test (p< 0.05) and significant differences are indicated by different letters.

Figure 2

Subcellular location of NtCPS2 in tobacco epidermal cells. (A) Green fluorescence of 35S::NtabDOG1L-T-GFP; (B) Red fluorescence of 35S::NtabDOG1L-T-GFP; (C) Chlorophyll fluorescence; (D) bright-field images; (E) merged images. Bars = 7.5 μm.

Figure 3

The phenotypic analyzes of NtCPS2 transgenic plants and WT plants. (A) Phenotype of WT and transgenic seeds sown for 7, 21, and 70 days, respectively. (B) Germinative force of WT and transgenic seeds sowed for 7 days. (C) Germinative rate of WT and transgenic seeds sowed for 14 days. (D) Fresh weights of 70-day-old WT and transgenic plants. Bars = 0.1 cm, 0.5 cm or 1.0 cm. Error bars represent means ± SD. Statistical analysis was performed using the ANOVA test (p< 0.05) and significant differences are indicated by different letters. .

Figure 4

NtCPS2 functions in endogenous hormone. (A, B) The content of gibberellin and abscisic acid. (C) Gibberellin to abscisic acid ratio. Error bars represent means ± SD. Statistical analysis was performed using the ANOVA test (p< 0.05) and significant differences are indicated by different letters.

Figure 5

(A) The relative expression of ent- acid synthase gene KS. (B) The relative expression of ent- acid oxidase gene KO. (C) The relative expression of ent-acid oxidase gene KAO. (D) The relative expression of GA2-oxidase gene GA2ox. (E) The relative expression of zeaxanthin epoxidase gene ZEP. (F) The relative expression of 9-cis epoxycarotenoid dioxygenase gene NCED. Error bars represent means ± SD. Statistical analysis was performed using the ANOVA test (p< 0.05) and significant differences are indicated by different letters.

Figure 6

NtCPS2 functions in drought tolerance. (A) Stomata of WT and transgenic plants under desiccation treatment. Scale bar =200 μm. (B) Ratio of stomata of WT and transgenic lines after dehydration. (C) Water loss rates of leaves detached from WT and transgenic lines. (D) Stomatal conductance (gs) of WT and transgenic lines after 6 h of drought treatment. (E) Transpiration rate (E) of WT and transgenic plants after 6 h of drought treatment. Error bars represent means ± SD. Statistical analysis was performed using the ANOVA test (p< 0.05) and significant differences are indicated by different letters.

Figure 7

NtCPS2 functions in antioxidant capacity. After 6 h of drought treatment, WT and transgenic plants were stained with DAB (A) and subjected for H₂O₂ content determination (B). The activity levels of antioxidant enzymes CAT (C), POD (D) in three transgenic plants and WT plants were treated with drought for 6 h. Scale bar =0.2 cm. Error bars represent means ± SD. Statistical analysis was performed using the ANOVA test (p< 0.05) and significant differences are indicated by different letters.

Figure 8

NtCPS2 functions in membrane permeability. (A) Comparisons of relative electrolyte leakage rates in the transgenic lines and WT. (B) The content of soluble protein (sPRO) in the transgenic lines and WT. Error bars represent means ± SD. Statistical analysis was performed using the ANOVA test (p< 0.05) and significant differences are indicated by different letters.

Figure 9

Metabolic network of the effect of NtCPS2 gene on drought resistance in tobacco.