Mutation of SlSBPASE Aggravates Chilling-Induced Oxidative Stress by Impairing Glutathione Biosynthesis and Suppressing Ascorbate-Glutathione Recycling in Tomato Plants

Abstract

Sedoheptulose-1,7-bisphosphatase (SBPase) is a crucial enzyme for photosynthetic carbon assimilation in the Calvin-Benson cycle. Previous studies have shown that overexpression of SBPase is advantageous to chilling tolerance in plants; however, the mechanisms of SBPase acting in the improvement of chilling tolerance remain largely unknown. In the present study, we aimed to uncover the essential role of SBPase in the response of tomato plants to oxidative stress induced by low temperature. To fulfill that, we performed an array of comparative studies between slsbpase mutant plants that we previously generated using CRISPR/Cas9 genome editing system and their wild-type counterparts under chilling stress. It was observed that following a 24 h chilling treatment, slsbpase mutant plants accumulated higher levels of reactive oxygen species (ROS) than wild-type plants and consequently, more severe lipid peroxidation occurred in slsbpase plants. Activity assay of antioxidant enzymes showed that mutation in SlSBPASE significantly decreased activities of peroxidase (POD) and ascorbate peroxidase (APX), but surprisingly did not significantly alter activities of superoxide dismutase (SOD) and catalase (CAT) under the chilling condition. Notably, mutation in SlSBPASE reduced the contents of total ascorbate (AsA) and total glutathione (GSH) and suppressed the recycling of AsA and GSH in chilling-stressed tomato plants. In addition, activities of two GSH biosynthetic enzymes (gamma-glutamylcysteine synthetase and glutathione synthetase) and transcript abundance of their coding genes (GSH1 and GSH2) were markedly reduced in slsbpase mutant plants in comparison with those in wild-type plants under chilling stress. Furthermore, exogenous GSH remarkably mitigated chilling damage in slsbpase plants. Collectively, these results support that mutation in SlSBPASE aggravates chilling-induced oxidative stress by suppressing GSH biosynthesis and AsA-GSH recycling and suggest that SBPase is required for optimal response to chilling stress in tomato plants. The findings also shed light on the idea to mitigate chilling-induced damages by genetically manipulating a photosynthetic enzyme in plants.

Keywords: SBPase; ascorbate; chilling stress; glutathione; oxidative stress; reactive oxygen species; tomato.

Figure 1

Decreased CO₂ assimilation rate (A) and sucrose accumulation (B) as a consequence of SlSBPASE mutation in tomato T2 plants. The results are presented as mean values ± SDs from three independent experiments, and different letters on top of each column represent significantly different values at p < 0.05.

Figure 2

Reactive oxygen species (ROS) accumulation and lipid peroxidation as affected by SlSBPASE mutation in chilling-stressed tomato plants. Lipid peroxidation was assessed by measuring malondialdehyde (MDA) content and electrolyte leakage. (A) H₂O₂; (B) O₂·⁻; (C) MDA content; and (D) electrolyte leakage. At the fourth-leaf stage, slsbpase mutants and wild-type plants were subjected to chilling stress for a period of 24 h. Following that, leaves from different groups were sampled for the quantification of ROS and measurement of lipid peroxidation. The results are presented as mean values ± SDs from three independent experiments, and different letters on top of each column represent significantly different values at p < 0.05 among treatments.

Figure 3

Activities of antioxidant enzymes were differentially affected by SlSBPASE mutation in chilling-stressed tomato plants. (A) Superoxide dismutase (SOD); (B) catalase (CAT); and (C) peroxidase (POD). At the fourth-leaf stage, slsbpase mutants and wild-type plants were subjected to a 24 h chilling stress. Following that, leaves from different groups were sampled for the determination of enzyme activities. The results are mean values ± SDs from three independent experiments, and different letters on top of each column represent significantly different values at p < 0.05 among treatments.

Figure 4

Levels of ascorbate (AsA) and glutathione (GSH) in the AsA-GSH pathway as affected by SlSBPASE mutation in chilling-stressed tomato plants. (A) Total AsA; (B) total GSH; (C) AsA; (D) GSH; (E) AsA/DHA ratio; and (F) GSH/GSSH. At the fourth-leaf stage, slsbpase mutants and wild-type plants were subjected to a 24 h chilling stress. Following that, leaves from different groups were sampled for the determination of AsA and GSH contents. The results are mean values ± SDs from three independent experiments, and different letters on top of each column represent significantly different values at p < 0.05 among treatments.

Figure 5

Activities of enzymes involved in the AsA-GSH pathway as affected by SlSBPASE mutation in chilling-stressed tomato plants. (A) Ascorbate peroxidase (APX); (B) dehydroascorbate reductases (DHAR); (C) monodehydroascorbate reductase (MDHAR); and (D) glutathione reductases (GR). At the fourth-leaf stage, slsbpase mutants and wild-type plants were subjected to a 24 h chilling stress. Following that, leaves from different groups were sampled for the determination of enzyme activities. The results are mean values ± SDs from three independent experiments, and different letters on top of each column represent significantly different values at p < 0.05 among treatments.

Figure 6

Activities of gamma-glutamylcysteine synthetase (γ-ECS; A) and glutathione synthetase (GS; B) in the pathway of GSH biosynthesis and relative transcript abundance of GSH1 (C) and GSH2 (D). At the fourth-leaf stage, slsbpase mutants and wild-type plants were subjected to a 24 h chilling stress. Following that, leaves from different groups were sampled for the determination of enzyme activities and transcript abundance. The expression level in wild-type leaves under control conditions was set to 1, and the relative expression level in the rest of samples was calculated accordingly. The results are mean values ± SDs from three independent experiments, and different letters on top of each column represent significantly different values at p < 0.05 among treatments.

Figure 7

Effects of exogenous GSH on chilling damage as represented by relative electrolyte leakage. At the fourth-leaf stage, slsbpase mutants and wild-type plants were sprayed with or without GSH on 3 consecutive days and then subjected to a 24 h chilling stress. Following that, leaves from different groups were sampled for the determination of electrolyte leakage. The results are mean values ± SDs from three independent experiments, and different letters on top of each column represent significantly different values at p < 0.05 among treatments.

Figure 8

Simplified schematic representation of the importance of sedoheptulose-1,7-bisphosphatase (SBPase) in the mitigation of oxidative stress triggered by low temperature.