Glutathione Status and Protein Synthesis during Drought and Subsequent Rehydration in Tortula ruralis

Abstract

Glutathione status and its relationship to protein synthesis during water deficit and subsequent rehydration have been examined in the drought-tolerant moss, Tortula ruralis. During slow drying there is a small decrease in total glutathione but the percentage of oxidized glutathione (GSSG) increases. During rapid drying there is little change in total glutathione but a small increase in GSSG. On rehydration of slowly dried moss, GSSG rapidly declines to normal level. But when rapidly dried moss is rehydrated, there is an immediate, sharp increase in GSSG as a percentage of total glutathione. After 2 hours of rehydration GSSG starts declining and reaches a normal level in about 6 hours. When an increasing degree of steady state water deficit is imposed on the moss tissue with polyethylene glycol 6000, there is a progressive decrease in protein synthesis but an increase in oxidized glutathione. When 5 milimolar GSSG is supplied exogenously during rehydration of rapidly dried or slowly dried moss, protein synthesis is strongly inhibited. In vitro protein synthesis supported by moss mRNA is also inhibited by more than 85% by 150 micromolar GSSG. The role of glutathione status in water deficit-induced inhibition of protein synthesis is discussed.Drought-tolerant moss, Tortula ruralis, can be dried rapidly or slowly to less than 20% of its original fresh weight without loss of its ability to recover on subsequent rehydration (2). However, the rates of metabolic responses on rehydration vary with the speed of the previous drying. For example, the consumption of 02 ( 17) Administration of Steady State Stress. Polythylene glycol 6000 was used as osmoticum to impose steady state water stress as described previously (7). Five hundred mg fresh moss was placed in 5 ml of PEG solution with varying water potential. After 1.5 h, 20 ,uCi of [4,5-3H]leucine (60 Ci/mmol) was added in experiments when rate of protein synthesis was studied. In a parallel experiment after 1.5 h ofpreincubation ofmoss in PEG solution, the level of GSSG as percent oftotal glutathione was determined.Determination of Total Glutathione and Oxidized Glutathione. Enzymic methods described recently (1) were used to determine total glutathione (GSH + GSSG) in terms of GSH equivalents, and oxidized glutathione (GSSG). Tissue samples consisted of 0.5 g fresh moss or equivalent weight of dried moss. Tissue was homogenized in 2.5 ml of 5% 5-sulfosalicylic acid and a 10,000g supematant was obtained and used to determine total glutathione in GSH equivalents by the DTNB-GSSG reductase method. A standard curve was obtained by using authentic GSH at concentrations of 0 to 5 gM. The assay was monitored by following the rate of formation of TNB2 at 412 nm.For the determination of GSSG, 2-vinylpyridine was used to trap the GSH present in the 5-sulfosalicylic acid supematant 2 Abbreviations: TNB, 5-thio-2-nitrobenzoate; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid). Figure IA. It can be seen that during slow drying there is a steady increase in GSSG in the first 4 ...

Drought Stress, Enzymes of Glutathione Metabolism, Oxidation Injury, and Protein Synthesis in Tortula ruralis

“…(Gaertn, Meyer, and Scherb) were collected, stored, and prepared for experiments as described by Bewley (2) (5). GSSG content was calculated as percentage of the total glutathione.…”

“…The homogenate was centrifuged at 15,000g for 10 min and the supernatant obtained was used as enzyme source. The protein content of the enzyme extract was determined as above (5).…”

“…Fresh moss (250 mg) or its equivalent in dried moss was placed in S mL distilled water containing 20 ,uCi of [4,5-3H] leucine (60 Ci/mmol) for 2 h. When a longer treatment with another chemical or period of rehydration was required, radiolabeled leucine was added for the last 2 h. At the end ofincorporation, tissue was washed with cold (4°C) solution of 100 ,ug/mL carrier leucine. Procedures for extraction of proteins and for determination of protein content and radioactivity were as described (5).…”

“…Polyribosomes are conserved during rapid drying but not during slow drying (7). But the rate of protein synthesis on rehydration is considerably lower in RD2 than in SD moss (5). It has been suggested that GSSG, which inhibits in vivo and in vitro protein synthesis in this moss and accumulates during rehydration following rapid drying (5), mediates the drought stress-induced inhibition of protein synthesis.…”

Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide

“…The H 2 O 2 (0.5 mM/1h treatment) used to induce oxidative stress was probably detoxified in part through the action of the enzyme glutathione-peroxidase (GPx) and the concomitant conversion of GSH into GSSG 29 . Increasing GSSG levels can potentially inhibit protein synthesis in animal and plant cells 40,41 , and because of this, Candida cells are likely to export GSSG under conditions of oxidative stress, resulting in a decrease in total intracellular glutathione levels.In yeasts, peroxide resistance has been associated with intracellular GSH levels [42][43][44] . Further, it has been previously proposed that the rate of removal of H 2 O 2 is a direct function of GPx activity × GSH 29 .…”