Effect of short-term water deficit stress on antioxidative systems in cucumber seedling roots
© Fan et al.; licensee Springer 2014
Received: 18 March 2014
Accepted: 2 May 2014
Published: 23 May 2014
Cucumber is one of the most popular vegetables, and have little tolerance for water stress. The antioxidant defense system is one of major drought defense and adaptive mechanisms in plants, however, relatively few data are available regarding antioxidant systems in responses of cucumber to water deficit. The effect of short-term drought stress on the antioxidant system, lipid peroxidation and water content in cucumber seedlings roots was investigated.
The results showed that polyethylene glycol (PEG) induced water stress markedly decreased water content of cucumber seedling roots after treatment of 36 h, and caused excessive generation of reactive oxygen species (ROS) including superoxide (O2.−), hydrogen peroxide (H2O2). Meanwhile, malondialdehyde (MDA) content increased. Antioxidant enzymes including superoxide dismutases (SOD), peroxidases (POD), catalase (CAT) and ascorbate peroxidase (APX) activities increased in different time and different extent under water stress, while ascorbate (AsA) and glutathione (GSH) content, glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) activities all decreased when compared to control.
Therefore, it can be concluded that water stress strongly disrupted the normal metabolism of roots and restrained water absorption, and seemingly enzymatic system played more important roles in protecting cucumber seedling roots against oxidative damage than non-enzymatic system in short-term water deficit stress.
KeywordsCucumis sativus Antioxidative enzymes Antioxidants Reactive oxygen species Drought stress
Water deficit is a major environmental factor restricting plant growth, development and productivity, particularly in arid regions more than any other single environmental factor. It seems the worldwide losses in crop yields from water deficit probably exceed the cumulative loss of all other stresses (Kramer ). Seasonal drought is becoming a severe challenge in agriculture production in the Yangtze River Basin in China. It usually happens from June to August when is critical time for most crops, even worse, it tends to come along with high temperature (Huang et al. ). It is well known that drought stress impairs numerous metabolic and physiological processes in plants (Mahajan and Tuteja ). Water stress results in significant declines in water potential and relative water content (RWC) and directly affects many aspects of plant physiology (Correia et al. ). Drought could induce excessive generation of reactive oxygen species (ROS) including superoxide anion (O2.−), hydrogen peroxide (H2O2) and hydroxyl radical (HO−), which could cause deterioration of membrane lipids, proteins and nucleic acids, leading to increased membrane leakage of solutes (Huang et al. ; Shehab et al. ). Thus, oxidative stress is one of the major causes of cellular damage in plants during stress (Miller et al. ). However, to remove ROS and maintain redox homeostasis, plants have evolved a complex array of antioxidant defense systems to prevent oxidative injury resulting from high levels of ROS, which includes antioxidative enzymes superoxide dismutases (SOD), peroxidases (POD), catalase (CAT), glutathione reductase (GR) and ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) (Asada ), low molecular mass antioxidants ascorbate (AsA), glutathione (GSH), and some compatible solutes such as betaines and proline (Foyer and Noctor ; Veljovic-Jovanovic et al. ). Previous studies have shown that drought tolerant cultivars generally have enhanced constitutive antioxidant enzyme activity under drought stress in comparison with sensitive cultivars (Sun et al. ). Maintaining a higher level of antioxidative enzyme activities may contribute to drought induction by increasing the capacity against oxidative damage (Sharma and Dubey ).
Cucumber is one of the most popular vegetables, and it is considered a shallow-rooted crop and has been reported to have little tolerance for water stress. The antioxidant defense system is one of major drought defense and adaptive mechanisms in plants (An and Liang ). To the best of our knowledge, however, relatively few data are available regarding antioxidant systems in responses of cucumber to water deficit. Therefore, in the present study, we investigated the effect of water deficit on antioxidant systems of cucumber seedlings.
Plant material and treatments
The experiments were carried out in the environment-controlled greenhouse. Cucumber (Cucumis sativus L., cv. Jinyou No. 1) seeds were placed in sterile Petri plates on filter paper moistened with distilled water. They were allowed to germinate in the dark in a thermostatically-controlled chamber at 29 ± 1°C for approximately 30 h. The germinated seeds were sown in 50 cells plug trays containing peat moss and perlite (1:1, v/v) at 1200 μmol m−2 s−1 photosynthetic photo flux density and 14/10 h (25–30/16–20°C) day/night regime. Relative humidity fluctuated between 60 and 75%. At the two-leaf stage, seedlings were removed from the plastic plates, and the roots were rinsed with distilled water. Uniformly sized healthy seedlings were selected and transferred into troughs (40 × 30 × 13 cm) filled with 10 L of full-strength Hoagland’s nutrient solution, which was aerated for 40 min each hour. After pre-culturing for 3 d, the seedlings were treated with the following methods: (1) control [CK (full-strength Hoagland’s nutrient solution)]; (2) 5% polyethylene glycol 6000 (PEG) treatment [5% PEG (full-strength Hoagland’s nutrient solution containing 5% PEG)]; (3) 10% PEG treatment [10% PEG (full-strength Hoagland’s nutrient solution containing 10% PEG)]; each treatment has nine containers, and each container includes 6 plants, providing a total of 54 plants per treatment. Root samples of CK and 10% PEG treatment were harvested in triplicate at 0, 12, 24 and 36 h after the treatment initiation and immediately frozen in liquid nitrogen, and stored at −80°C for the subsequent analyses. After 36 h of treatment, plant roots per treatment were collected for the determination of water content (WC).
Determination of WC of root
Roots of cucumber seedlings were washed with tap water two to three times, rinsed twice with distilled water, gently blotted dry with a paper towel, and weighed for fresh weight. Oven dried at 70°C to constant dry weight. WC of roots was calculated according to the formula WC = [(Fresh weight-dry weight)/ Fresh weight] × 100%.
Enzyme extraction and assays
For the enzyme assays, 0.3 g roots were ground with 2 mL ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM ascorbate and 2% PVP. The homogenates were centrifuged at 4°C for 20 min at 12,000 × g and the supernatant was used for enzyme activity assay. All steps in the preparation of the enzyme extract were carried out at 4°C. SOD activity assay was based on the method described by Giannopotitis and Ries (). POD activity was measured using modification of the procedure of Egley et al. () , the reaction mixture in a total volume of 2 mL contained 25 mM (pH 7.0) sodium phosphate buffer, 0.1 mM EDTA, 0.05% guaiacol (2-ethoxyphenol), 1.0 mM H2O2 and 100 μL enzymes extract. The increase of absorbance due to oxidation of guaiacol was measured at 470 nm. APX activity was determined according to Nakano and Asada (). CAT activity was done according to Cakmak and Marschner (). GR activity was measured as a decrease in A340 due to oxidation of NADPH according to Foyer and Halliwell (). MDHAR activity was determined following Hossain et al. () with slight modification. The reaction mixture contained 50 mM of Tris–HCl buffer (pH 7.5), 1 mM of NADH, 5 mM of ascorbic acid, 0.15 U of ascorbate oxidase and 30 μL of enzymes extract. The activity was calculated from the change in absorbance at A340 for 2 min when the extinction coefficient was 6.2 mM−1 cm−1. DHAR activity was determined following Nakano and Asada () with some modifications. The reaction mixture contained 50 mM of KH2PO4 buffer (pH 7.0), 50 mM of GSH, 10 mM of DHA and 50 μL of enzyme extract. The activity was calculated from the change in absorbance at A265 for 2 min when the extinction coefficient was 14 mM−1 cm−1.
Determination of free radical production
The O2.− production rate was measured according to Elstner and Heupel () by monitoring the nitrite formation from hydroxylamine in the presence of O2.−, with some modifications. One g root segments was homogenized with 3 mL of 65 mM phosphate buffer (pH 7.8), 0.1 mL of 10 mM hydroxylamine hydrochloride, and 1 mL supernatant. After incubation at 25°C for 20 min, ethyl ether in the same volume was added and centrifuged at 1,500 × g for 5 min. A standard curve with NO2− was used to calculate the production rate of O2.− from the chemical reaction of O2.− and hydroxylamine. H2O2 content was measured by the titanium method (Patterson et al. ). MDA content was measured by the thiobarbituric acid reaction method (Heath and Packer ).
Assay of antioxidants
All data presented are the mean values. All experiments were conducted using three replicates at least. All data were statistically analyzed by the analysis of variance (ANOVA) with SAS software (SAS Institute, Cary, United States) using Duncan’s multiple range test at the 0.05 level of significance.
Results and discussions
Water state and oxidative free radical production in the roots of cucumber seedlings subjected to a short term water stress
The enzymatic antioxidation system in cucumber seedling roots in responses to a short term water stress
Responses of the non-enzymatic antioxidation systems in cucumber seedling roots to a short term water stress
In conclusion, the present results suggested that water deficit interrupted the ROS balance between generation and elimination in cucumber roots, which strongly disrupted the normal metabolism of roots and restrained the water absorption. Finally, plants become wilted and their stems did not stand upright. Interestingly, we also found that the enzymatic system (SOD, POD, CAT and APX), partially, enhanced the ROS-scavenging capacity to some extent in short-term water deficit stress, while non-enzymatic system (ASA and GSH) exhibited a relative decease of ROS-scavenging in this experiment. Seemingly it showed that the enzymatic system played more important roles in protecting cucumber seedling roots against oxidative damage than non-enzymatic system in short-term water deficit stress. However, extended studies are needed to understand a precise mechanism.
LD and XW performed these experiments. HFF and CXD designed the research, analyzed data and wrote the paper. All the authors read and approved the final manuscript.
Reactive oxygen species:
This research was supported by the National Natural Science Foundation of China (No. 31101539; No. 31201658).
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