A Multifactorial Regulation of Glutathione Metabolism behind Salt Tolerance in Rice

Abstract

Knowledge of the stress-induced metabolic alterations in tolerant and sensitive plants is pivotal for identifying interesting traits that improve plant resilience toward unfavorable environmental conditions. This represents a hot topic area of plant science, particularly for crops, due to its implication in food security. Two rice varieties showing dissimilar resistance to salt, Baldo and Vialone Nano, have been studied to investigate the mechanisms underpinning tolerance toward salinity, and these studies have focused on the root system. A detailed analysis of the salt stress-dependent modulation of the redox network is here presented. The different phenotype observed after salt exposure in the two rice varieties is coherent with a differential regulation of cell-cycle progression and cell-death patterns observed at root level. Baldo, the tolerant variety, already showed a highly responsive antioxidative capacity in control conditions. Consistently, stressed Baldo plants showed a different pattern of H₂O₂ accumulation compared to Vialone Nano. Moreover, glutathione metabolism was finely modulated at transcriptional, post-transcriptional, and post-translational levels in Baldo. These results contribute to highlight the role of ROS and antioxidative pathways as a part of a complex redox network activated in rice toward salt stress.

Keywords: Oryza sativa; abiotic stress; cell cycle; glutathione metabolism; hydrogen peroxide; miRNA395; rice; salt stress.

Figure 1

Effect of high salinity on rice seedlings. (A–C). Baldo and Vialone Nano plants in control and stress conditions. Seedlings were grown to the 2nd leaf stage (V2 stage) and then treated with 100 mM NaCl for 7 days. (D) Color of the 2nd leaf of Baldo and Vialone Nano in control and treated conditions recorded in the CIELab color space and converted to the red–blue–green scale. CIELAB expresses leaf color as three components: L component represents the perceptual lightness; A negative value was for green light component; A positive value was for red light component; B negative value was for blue light component; B positive value was for yellow light component. CIELab color values of the 2nd leaf were reported. The reported values are the means of four independent experiments ± standard deviation. Statistical significance was determined by one-way ANOVA followed by a Tukey test (p < 0.05). Different letters indicate significant difference. The statistical analysis was performed by comparing the values obtained for each light component (L/A/B) in leaves derived from NaCl plants over treatment time.

Figure 2

Morpho-Phenotypical Characterization of rice seedlings subjected to salt treatment. Baldo and Vialone Nano were grown under 100 mM NaCl for 7 days after salt treatment. (A) Leaf chlorophyll content; (B) Maximum shoot length; (C) Shoot dry weight; (D) Maximum root length; (E) Root dry weight. The reported values are the means of four independent experiments ± standard deviation. Statistical significance was determined by two-way ANOVA followed by a Tukey test (p < 0.01). Different letters indicate significant difference.

Figure 3

Cell cycle regulation in rice roots subjected to salt stress. Flow cytometric analysis of cell cycle of Baldo and Vialone Nano roots in control and salt stress conditions at 1 day and 7 days after salt treatment. All samples were stained with PI. Cell cycle progression in apical root meristem in response to salinity treatment. The analysis reflects the relative number of cells according to DNA content in each cell. The first peak (2n) represents the G1-phase and the second peak (4n) represents the S/G2 phase. Corresponding diagrams report the proportion of cells in G1 and S/G2 phases in Vialone Nano and Baldo under control and stress conditions. Data are presented as an average estimated from each treatment in five biological replicates with 30,000 events each. Student’s t-test was used to evaluate the significant variations between control and treated conditions for each variety. The reported values are the means of four independent experiments ± standard deviation. Statistically significant variations are shown (*) with p-value ≤ 0.05.

Figure 4

Cell death evaluation on salt-treated Baldo and Vialone Nano root apparatus. (A) Relative Root Mortality Ratio (NaCl/C plants) obtained from quantification of Evan’s Blue-stained root extract. (B) Expression of programmed cell death-related gene OsPDCD5 in control and treated plants. Data are presented as the mean standard deviation of at least three biological replicates. In (A) statistical significance determined by two-way ANOVA followed by a Tukey test (p < 0.01). Different letters indicate significant difference. In (B) Student’s t-test was used to evaluate the significant variations between control and treated conditions for each variety. Asterisks indicate significant differences between treatment and control by t-test with p < 0.01. ns, non-significant. The reported values are the means of four independent experiments ± standard deviation.

Figure 5

Redox markers in rice roots under salt stress. (A) Changes in H₂O₂ content; (B) levels of malondialdehyde (MDA) and (C) total antioxidant activity in Baldo and Vialone Nano roots subjected to salt stress. The reported values are the means of four independent experiments ± standard deviation. Statistical significance was determined by two-way ANOVA followed by a Tukey test (p < 0.01). Different letters indicate significant difference.

Figure 6

ASC and GSH levels in rice roots under control and salt conditions. (A) Ascorbate content in control and treated roots (100 mM NaCl). Changes in total levels of ascorbate (reduced plus oxidized forms; ASC + DHA) pools induced by 100 mM NaCl and determined at 24 h after treatment. (B) Glutathione content in control and treated roots (100 mM NaCl). Changes in total levels of glutathione (reduced plus oxidized forms; GSH + GSSG) pools induced by 100 mM NaCl and determined at 24 h after treatment. The reported values are the means of four independent experiments ± standard deviation. Statistical significance was determined by one-way ANOVA followed by a Tukey test (p < 0.01). Different letters indicate significant difference.

Figure 7

Transcriptional and post-transcriptional regulation of GSH-related genes in rice cultivars dealing with salt stress. Expression profile of genes involved in glutathione biosynthesis, (A) γ-glutamylcysteine (γ-EC), and (B) glutathione synthetase 1 (GS1) 24 h after salt treatment. The results were normalized for the expression of the housekeeping gene OsACT1. (C) Expression profile of miRNA395f gene. Student’s t-test was used to evaluate the significant variations between control and treated condition for each variety. Statistically significant variations are shown (*) using p-value ≤  0.01. ns, non-significant. The reported values are the means of four independent experiments ± standard deviation.

Figure 8

S-Glutathionylation of root proteins in rice seedlings subjected to salt treatment. Densitometric analysis of S-glutathionylation profile of rice proteins extracted from roots at 24 h from salt exposure. The densitometric analysis was performed and quantified by using ImageLab. The density of the blotting signals was normalized to Ponceau S. The reported values are the means of four independent experiments ± standard deviation. Statistical significance was determined by one-way ANOVA followed by a Tukey test (p < 0.01). Different letters indicate significant difference.

Figure 9

Redox strategy underpinning salt tolerance in rice. A differential modulation of the redox balance is pivotal for the protection against salt stress in the tolerant rice variety. In Baldo, a rapid and effective modulation of the antioxidative systems allows a delay of the oxidative damage to the cellular components and consequent impairment of cell-cycle progression and cell viability due to a shift toward an ROS signaling mode instead of a damaging one. In this context, glutathione metabolism was identified as a possible pathway, which contributes to conferring salt tolerance on rice. A variety-specific modulation of the expression of the enzyme γ ECS and regulation, at a post-transcriptional level, of sulfur assimilation, mediated by osa-miR395, can contribute to the delayed decrease in plant growth and viability observed in tolerant Baldo rice variety.