Ascorbate-Glutathione Oxidant Scavengers, Metabolome Analysis and Adaptation Mechanisms of Ion Exclusion in Sorghum under Salt Stress

Abstract

Salt stress is one of the major significant restrictions that hamper plant development and agriculture ecosystems worldwide. Novel climate-adapted cultivars and stress tolerance-enhancing molecules are increasingly appreciated to mitigate the detrimental impacts of adverse stressful conditions. Sorghum is a valuable source of food and a potential model for exploring and understanding salt stress dynamics in cereals and for gaining a better understanding of their physiological pathways. Herein, we evaluate the antioxidant scavengers, photosynthetic regulation, and molecular mechanism of ion exclusion transporters in sorghum genotypes under saline conditions. A pot experiment was conducted in two sorghum genotypes viz. SSG 59-3 and PC-5 in a climate-controlled greenhouse under different salt concentrations (60, 80, 100, and 120 mM NaCl). Salinity drastically affected the photosynthetic machinery by reducing the accumulation of chlorophyll pigments and carotenoids. SSG 59-3 alleviated the adverse effects of salinity by suppressing oxidative stress (H₂O₂) and stimulating enzymatic and non-enzymatic antioxidant activities (SOD, APX, CAT, POD, GR, GST, DHAR, MDHAR, GSH, ASC, proline, GB), as well as protecting cell membrane integrity (MDA, electrolyte leakage). Salinity also influenced Na⁺ ion efflux and maintained a lower cytosolic Na⁺/K⁺ ratio via the concomitant upregulation of SbSOS1, SbSOS2, and SbNHX-2 and SbV-Ppase-II ion transporter genes in sorghum genotypes. Overall, these results suggest that Na⁺ ions were retained and detoxified, and less stress impact was observed in mature and younger leaves. Based on the above, we deciphered that SSG 59-3 performed better by retaining higher plant water status, photosynthetic assimilates and antioxidant potential, and the upregulation of ion transporter genes and may be utilized in the development of resistant sorghum lines in saline regions.

Keywords: antioxidants; ion transporters; oxidative stress; proline; reactive oxygen species (ROS); salinity; sorghum.

Figure 1

Outline of the experiment/Two S. bicolor genotypes (SSG 59-3 and PC-5) with varied salt tolerance were phenotypically evaluated based on germination studies and then planted in screen house under different salt concentrations. Leaves and roots from five plants were pooled and considered a biological replicate, and two such replicates were used in the analysis. HPLC was performed to evaluate the accumulation of polyamines under different salt concentrations. Total RNA was isolated from sorghum leaves and the expression pattern of ion transporters was studied.

Figure 2

Effect of salt stress on (a) fresh weight, (b) dry weight, (c) root length, and (d) shoot length of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 3

Effect of salt stress on relative water content, (a) osmotic potential, (b) chlorophyll fluorescence, and (c) chlorophyll stability index (d) of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 4

Effect of salt stress on (a) chlorophyll a, (b) chlorophyll b, and (c) total chlorophyll of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 5

Effect of salt stress on Na⁺:K⁺ in leaves (a), and Na⁺:K⁺ in roots (b) of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 6

Effect of salt stress on superoxide dismutase (SOD, (a,b)), catalase (CAT, (c,d)), peroxidase (POX, (e,f)), and ascorbate peroxidase (APX, (g,h)) of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 6

Effect of salt stress on superoxide dismutase (SOD, (a,b)), catalase (CAT, (c,d)), peroxidase (POX, (e,f)), and ascorbate peroxidase (APX, (g,h)) of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 7

Effect of salt stress on glutathione peroxidase (GPX, (a,b)), glutathione reductase (GR, (c,d)), monodehydroascorbate reductase (MDHAR, (e,f)), and dehydroascorbate reductase (DHAR, (g,h)) of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 7

Effect of salt stress on glutathione peroxidase (GPX, (a,b)), glutathione reductase (GR, (c,d)), monodehydroascorbate reductase (MDHAR, (e,f)), and dehydroascorbate reductase (DHAR, (g,h)) of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 8

Effect of salt stress on total glutathione (a,b), reduced glutathione (GSH, (c,d), and oxidized glutathione (GSSG, (e,f)) of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 9

Effect of salt stress on (a,b) ascorbic acid and (c) carotenoids of sorghum genotypes at 35 DAS and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 10

Effect of salt stress on proline (Pro, (a,b)), glycine betaine (GB, (c,d)), and total soluble carbohydrates (TSC, (e,f)) of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 11

Effect of salt stress on hydrogen peroxide (H₂O₂, (a,b)), membrane injury index (MII, (c,d)), and malondialdehyde (MDA, (e,f)) of sorghum genotypes at 35 and 95 DAS. Post hoc comparisons of the means were performed using Tukey’s HSD test at p < 0.05; different letters (^a–e) indicate significant differences among treatments within each genotype.

Figure 12

Agarose gel: (A) mRNA of salt stress-responsive genes (1: SOS1; 2: SOS2; 3. NHX2; 4: V-PPase-II (leaves); 5: V-PPase-II (roots); 6: CIPK24; 7: Act1; 8: PP2A; L: ladder)), (B) PCR products of the expected sizes (≤ 200 bp; 1: SOS1; 2: SOS2; 3. NHX2; 4: V-PPase-II (leaves); 5: V-PPase-II (roots); 6: CIPK24; 7: Act1; 8: PP2A; L: ladder)), (C–G) melt/dissociation curve of salt stress-responsive genes under saline conditions, (H–M) the expression levels of stress-related ion transporter genes based on qPCR. The PP2A gene was used as the reference gene/internal control. Data are shown as mean ± S.D. (n = 3).

Figure 13

Regulation of Na⁺ and K⁺ ion homeostasis by the SOS pathway. Excess Na⁺ ions elicit a calcium signaling pathway that activates the SOS1–SOS3 protein kinase complex, which stimulates the SOS1 and sodium proton antiporter (NHX-2) exchange activity and, thus, regulates the expression of genes encoding them.