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Environmental Sciences
Procedia Environmental Sciences 11 (2011) 1346 - 1351
Conference Title
Preconditioning Alters Antioxidative Enzyme Responses in Rice Seedlings to Water Stress
Xuemei Li 1,a, Lihong Zhang2,b, Yueying Li1,c
1College of Chemical and Life Science, Shenyang Normal University, Shenyang, China 2Department of Environmental Science, , Liaoning University, Shenyang, China aLxmls132@163.com, bLihongzhang132@163.com, cyueyinglicn@yahoo.com.cn
Abstract
Rice seedlings (Oryza sativa L.) were grown in a controlled environment and divided into control seedlings (CK1: 80% field capacity was always held), preconditioned seedlings (PT, 6 days mild drought for preconditioning—3 days re-watering—intermediate drought stress) and non-preconditioned seedlings (CK2, 9 days 80% field capacity and immediately followed by intermediate drought). Antioxidative enzyme (i.e., peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT)) activities, as well as malondialdehyde (MDA) content were measured in CK1 and PT after preconditioning and re-watering as well as CK1, PT and CK2 after intermediate drought. Mild drought preconditioning increased POD activity and MDA content, but no effects were observed on SOD and CAT activities. After re-watering, POD activity and MDA content of PT were similar to CK1. Intermediate drought stress significantly decreased SOD activity but increased POD and CAT activities, as well as MDA content in CK2. Compared to CK2, POD and SOD activities significantly increased, and no effect was observed on MDA content in PT. CK2 suffered more serious injuries than PT as indicated by higher MDA content, suggesting that mild drought preconditioning made rice seedlings modulate their defense response such that they could acclimatize more successfully to intermediate drought stress environment.
© 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of the Intelligent Information Technology Application Research Association.
Keywords: antioxidative enzymes; malondialdehyde; drought stress; re-watering; oryza sativa
1. Introduction
Plant growth is greatly affected by drought stress, and plants must adapt to this stress to survive. Rice is one of the most important food crops in the world, and staple for more than half of the global population. Rice demands tremendous amounts of water during growth. Improvements in the tolerance of rice cultivar to drought stress are important and can help increase crop yield under stressing environments.
Drought induced stomatal closure and therefore reduces photosynthesis [1]. It also can cause lipid peroxidation, protein degradation, chlorophyll bleaching and changes in antioxidants by forming reactive oxygen species (ROS) [, 3]. According to Horling et al. [4], plant response to water deficit also includes accumulation of osmoprotectants and up-regulation of oxidative stress protectors. Drought stress results in stomatal closure, which limits CO2 fixation and decreases NADP+ regeneration. These adverse
1878-0296 © 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of the Intelligent Information Technology Application
Research Association.
doi:10.1016/j.proenv.2011.12.202
conditions increase the rate of reactive oxygen species production (ROS) such as hydrogen peroxide (H2O2), superoxide (O-2) and hydroxyl (-OH) radicals, by enhanced leakage of electrons to molecular oxygen. ROS can destroy normal metabolism through oxidative damage to lipids, proteins and nucleicacids [5]. To be able to endure oxidative damage under such an adverse condition, plants possess antioxidative enzymes such as peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT). The balance between ROS production and activities of antioxidative enzyme determines whether oxidative signaling and/or damage will occur [6]. These antioxidant enzymes are reported to increase under various environmental stresses [7]. There are many reports in the literature that underline the intimate relationship between enhanced or constitutive antioxidant enzyme activities and increased resistance to environmental stress [8, 9].
Higher plants have intricate mechanisms enabling them to respond to environmental changes. Many reports showed that pre-treatment with low, nonfreezing temperature can enhance frost resistance in many species [10' 11]. When wheat subjected to a second period of moderate water stress, there was a general rearrangement in the substrates and scavenging enzymes of the H2O2 detoxification cycle that might be related to the decreased oxidative threat [1 ]. Martínez-Domínguez and others [13] reveal that Spartina densiflora undergoes oxidative stress in its polluted location but is able to rapidly modulate its redox status when cultured under the presence or absence of polluting agents. If a more efficient regulation of these preexisting mechanisms could be induced, crop tolerance could be increased to a reasonable extent by inducing individual plant adaptation. The immersion of whole seedlings in stressful solutions containing PEG, NaCl or a mixture of both has been proven to be efficient for increasing salt tolerance in short-term experiments carried out with tomato and lettuce [14' 15].
If rice has the capacity for some form of ''stress imprint'' from previous water stress, we can expect that rice would rapidly modulate their defense system when water stress recurs. Therefore, the present study was aimed to examine the comparative potential of preconditioned and non-preconditioned of mild drought to overcome the negative effects of intermediate drought in rice seedlings. The experimental results showed that preconditioning treatment modulates antioxidant system, and hence, it can be adopted as an effective method for alleviating the intermediate drought stress.
2. Materials and methods
2.1. Plant material and treatments
Rice seeds were surface sterilized in 2.65 % sodium hypochlorite for 10 min and then washed thrice thoroughly in distilled water. Seeds were then soaked in sterile deionized water at 28 °C for 6 h and then transferred to two sheets of sterile filter paper moistened with deionized water. The seeds were germinated at 28 C for 48 h in the dark. On the next day, the germinated seeds were grown in pots filled with vermiculite under well-watered conditions in a growth chamber (27 C day/20C night, 16 h/8 h light/dark period, 800 ^mol m-2 s-1 PPFD and 80% relative air humidity). Seedlings were irrigated with nutrient solution. Seedlings were grown for 8 days and then divided into three groups. (1) CK1: control seedlings, maintained at about 80% field capacity throughout the 15 days experiment by daily watering. (2) CK2: non-preconditioned seedlings, well watered 9 days and then stressed by 40% field capacity (denoted as Intermediate drought). (3) PT: preconditioned seedlings, preconditioned by exposure to mild drought (50% field capacity) for 6 days, re-watered to 80% field capacity for 3 days and then exposed to 40% field capacity for 6 days as for CK2. The experiment design is illustrated in Table 1.
Table 1. Scheme of the experimental design
Phase 1, 6 days Phase 2, 3 days Phase 3, 6 days
CK1 Well watered
PT Mild Re-watering Intermediate drought
CK2 Well watered Intermediate
2.2 Enzyme extraction and assay
Fresh sample was homogenized in extraction buffer (0.1 M phosphate buffer pH 6.8) with mortar and pestle on ice. The homogenate was then centrifuged at 12,000g for 15 min at 4°C and the supernatant was used as the crude extract for SOD, CAT, POD and lipid peroxidation assay.
Total SOD activity was assayed by measuring the ability of the crude enzyme in inhibiting the photochemical reduction of nitroblue tetrazolium (NBT) by superoxide radicals generated photochemically. The reaction mixture (3 ml ) contained 130 mM methionine, 750 ^M NBT, 100 ^M EDTA, and 0.05 ml of enzyme extract in 0.1M PBS (pH 6.8). The reaction was started with adding 20 ^M riboflavin to the reaction mixture in the cuvette which was exposed to a 15-W circular "white light" tube for 15 min and then absorbance was measured at 560 m. One unit of SOD was defined as the amount of enzyme required to inhibit the reduction rate of NBT by 50% at 25 C.
CAT activity was determined using the method described by Aebi [16]. The reaction was started with the addition of the supernatant, and the decomposition rate of H2O2 was followed at 240 nm.
POD activity was determined according to the change in absorption at 470 nm due to guaiacol oxidation. The reaction mixture (3 ml) contained 0.1 M PBS (pH 6.0), 30 % H2O2, and guaiacol. The reaction was started by adding 20 ^l of enzyme extract. The total POD activity was determined by spectrophotometry following the rate of formation of tetraguaiacol at 470 nm.
2.3Mansuoadamts cf poclima ocmtamt
Lipid peroxidation was determined by estimating the content of malondialdehyde (MDA) produced by the thiobaTbituric acid reaction. MDA was determined by a colour reaction with thiobarbituric acid [17]. To 1.0 ml aliquot of the supernatant, 4.0 ml of 0.5 % thiobarbituric acid (TBA) in 20% TCA was added. The mixture was heated at 95 °C for 30 min. MDA content was then determined spectrophotometrically at 532 nm and corrected for nonspecific turbidity at 600 nm.
2.4 Statistical analysis
The results were subjected to analysis of independent samples T Test between CK1 and CK2 or PT in each measuring date. The data analysis was carried out using statistical package SPSS 7.5. Comparisons with P < 0.05 were considered significantly different.
3. Results
3. C Chnmgas cf antioxidative enzyme activities
POD activity in PT increased significantly following mild drought, subsequently it declined to the control values after re-watering (Fig 1). After intermediate drought, POD activity significantly increased in PT and CK2.
Figure 1. Changes in peroxidase (POD) activity of rice leaves subjected to mild drought for 6 days, re-watering for 3 days and intermediate drought for 6 days. Data are expressed as the mean ± standard deviation (SD) of three replicates. An asterisk symbol denotes significance at p<0.05 and two asterisk symbols (^ JK) denote significance at p<0.01 between CK2 or PT vs CK1.
The activities of CAT and SOD was not affected by mild drought and re-watering (Fig 2, 3). Following intermediate drought, preconditioned seedling significantly increased in SOD activity but had no change in CAT activity, while CK2 had significantly lower SOD activity and higher CAT activity than CK1.
Figure 2. Changes in superoxide dismutase (SOD) activity of rice leaves subjected to mild drought for 6 days, re-watering for 3 days and intermediate drought for 6 days.
Figure 3. Changes in catalase (CAT) activity of rice leaves subjected to mild drought for 6 days, re-watering for 3 days and intermediate drought for 6 days.
3.2 Change of malondialdehyde (MDA) content
MDA content was higher in seedlings subjected to mild drought than CK1. But subsequently the difference diminished after re-watering and had no increase after intermediate drought (Fig 4). In contrast to PT, CK2 had significantly higher MDA content than CK1 after exposure to intermediate drought. This indicates that preconditioning improved the resistance of seedlings to recurred drought.
Mild drought Re-watering Intermediate drought
Figure 4. Change in MDA content of rice leaves subjected to mild drought for 6 days, re-watering for 3 days and intermediate drought for 6 days.
4. Discussion
Water stress is one of the major stress factors on plant metabolism affecting plant growth,
photosynthesis and lipid peroxidation. Plant drought tolerance has very complex mechanisms. Plants had to develop various morphological, biochemical and physiological mechanisms to respond and adapt to drought stress and thus acquire drought tolerance. Adaptation to stress has been suggested to be mediated by both preexisting (constitutive) and induced (acclimated) defenses [18-20].
Drought stress causes increase in reactive oxygen species production [21]. There is increasing evidences suggesting that the oxidative stress is a major damaging factor in plants under different environmental stresses [22]. Plants respond to elevated levels of oxidative stress by activating their antioxidative defense systems [23]. Within a cell, SOD and CAT constitute the first line of defense against ROS. On the other hand, unspecific peroxidases such as guaiacol peroxidase are considered to be stress related enzymes and can be used as stress marker [24]. Our results show that mild drought and re-watering did not change SOD and CAT activity in PT. Bian and Jiang [25] also reported that the SOD, CAT and POD activities remained unchanged in the leaves of Kentucky bluegrass under drought stress as well as re-watering. Intermediate drought induced a significant increase in POD and SOD activities in PT and a significant increase in POD and CAT activities in CK2 suggesting that drought lead to defensive responses.
To estimate oxidative stress, MDA content had been widely used as an indicator of lipid peroxidation and, thereby, of oxidative damage in drought-exposed plants [26]. Our results in MDA content (Fig. 4) showed that mild drought induced a significant increase in the amount of lipid peroxidation product. Three days re-watering allowed the recovery of MDA content to CK1 level. It suggests that change in lipid peroxidation caused by mild drought was fully reversible. However, in spite of no changes were observed in CAT activity, membrane lipid peroxidation in PT was apparently lower than CK2. These results seem to indicate that preconditioned seedling has developed an efficient antioxidative machinery (POD and SOD) able to counteract the harmful effect of intermediate drought, thus preventing major physiological damages. CK2 suffered more serious injuries than PT as indicated by higher MDA content. Although POD and CAT activities increased but SOD activity decreased in CK2, the equilibrium between ROS generation and scavenging may be perturbed by intermediate drought stress.
We propose that the phase of mild drought of seedlings might cause the epigenetic changes to occur and activate set of genes to combat the stress. This sort of drought stress might be optimum in preconditioned plants that allowed the epigenetic changes to occur with maximum efficiency. In conclusion, our study shows that the main advantage of this method of seedling preconditioning is that agronomic drought tolerance can be directly improved by inducing adaptation in whole plants and large populations.
Acknowledgment
This study was supported by the National Natural Science Foundation of China (30870205, 31070285) and the Liaoning province Natural Science Foundation (20092070, 20102205). The authors wish to thank Prof. Tao for his help in revision of the manuscript.
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