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Dep. of Agronomy, Purdue Univ., West Lafayette, IN 47907
* Corresponding author (yjiang{at}purdue.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: AP, ascorbate peroxidase • DR, dehydroascorbate reductase • Eh, soil redox potential • GR, glutathione reductase • MDA, malondialdehyde • MR, monodehydroascorbate reductase • NADPH, nicotinamide adenine dinucleotide phosphate • PAGE, polyacrylamide gel electrophoresis • POD, peroxidase • PVC, polyvinylchloride • ROS, reactive oxygen species • SOD, superoxide dismutase • WL, waterlogging
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Waterlogging changes plant metabolic activity. One of the root metabolic features affected by WL or flooding conditions is antioxidant systems. Waterlogging or flooding generates oxidative stress and promotes the production of reactive oxygen species (ROS) (Yan et al., 1996; Biemelt et al., 2000; Garnczarska and Bednarski, 2004) including superoxide (O2–·), singlet oxygen (1O2), hydroxyl (–OH), and H2O2, which can be detrimental to proteins, lipids, and nucleic acids (Smirnoff, 1993). In plants, enzymatic and nonenzymatic defense systems are involved in ROS scavenging and detoxifying. In enzymatic systems, SOD constitutes the first line of defense against ROS by dismutating O2– to H2O2 (Bowler et al., 1992). Then H2O2 is decomposed by POD and catalase. Superoxide dismutase plays a central role in the enzymatic defense system and is a major enzymatic component of the cellular defense system (Bowler et al., 1992). Superoxide is produced where an electron transport chain is present (Elstner, 1991), so SODs are found in almost all subcellular locations. On the basis of the metal co-factor used by enzymes, three classes of SODs have been identified: Mn-SOD, Fe-SOD, and Cu/Zn-SOD (Alscher et al., 2002).
The ascorbate-glutathione cycle is an important and efficient enzymatic defense system for decomposing H2O2 and maintaining the balance of antioxidants. This cycle involves several enzymes, including ascorbate peroxide (AP), monodehydroascorbate reductase (MR), dehydroascorbate reductase (DR), and glutathione reductase (GR) (Asada, 1992). Ascorbate peroxidase is the first enzyme in this pathway, and its major function is catalyzing the H2O2 to H2O. Glutathione reductase is the last step in the pathway, playing a crucial role in protection against oxidative stress by maintaining a reduced glutathione level (Blokhina et al., 2002).
The activities of antioxidant enzymes in response to WL or hypoxia conditions have been investigated in a number of plant species; however, the results are not consistent. When plant roots are subjected to WL or flooding conditions, SOD activity increased in barley (Hoedrum vulgare L.) roots (Kalashnikov et al., 1994), decreased in mungbean (Vigna radiata L.) leaves (Ahmed et al., 2002), and remained unaffected in tomato (Lycopersicon pimpinellifolium Mill) (Lin et al., 2004) and wheat (Triticum sativum L.) roots (Biemelt et al., 2000). Biemelt et al. (2000) reported that hypoxia did not cause significant changes in the activity and patterns of individual SOD isoforms in wheat roots. Their results also indicated that no significant alterations in transcript and protein levels of SOD isoforms were observed under hypoxia compared with under aerated control. In barley roots, hypoxia causes no changes in the activity of Mn-SOD or the synthesis of Mn-SOD isoforms in mitochondria (Szal et al., 2004). Hypoxia increased AP activity but reduced the activities of GR, DR, and MR in wheat roots (Biemelt et al., 1998). Yan et al. (1996) reported that short-term flooding enhanced SOD, AP, and GR activity in maize (Zea mays L.) leaves, and the extended periods of flooding decreased the activities of these antioxidant enzymes with increasing lipid peroxidation. These studies demonstrated different patterns of antioxidant enzymes in response to WL or flooding conditions. However, the response of antioxidant enzymes to different depths of WL have not been investigated in plant roots, including turfgrass species.
Creeping bentgrass is a widely used cool-season turfgrass on golf greens and fairways in northern regions and transitional zones. Full and partial soil WL occurs in turfgrass sites due to high and frequent precipitation, poor soil quality, or overirrigation followed by slow drainage (Beard, 1973; Carrow, 1996). A reduction in root growth was observed in creeping bentgrass under WL conditions (Huang et al., 1998), but no information is available on how different depths of WL influence the antioxidants of creeping bentgrass. Knowledge of root antioxidant metabolism would provide valuable information for maximizing management programs and enhancing the selection of tolerant cultivars. Our objective was to identify the antioxidant response of creeping bentgrass cultivars to different depths of WL.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Waterlogging Treatments
Waterlogging was imposed by plugging the drainage holes of the PVC tubes. A 1-cm-diam. hole was drilled in the side, 2.5 cm above the bottom, and connected to open-end transparent tubing with the height of the other end adjusted to 15 cm and 1 cm below the soil surface. This transparent tubing was not installed to control the PVC tubes. Water was added daily from the canopy to maintain different WL levels for 21 d because the changes and differences in turf quality were dramatic in two cultivars after 21 d of WL. The WL treatments included: (i) control, drained to field capacity; (ii) water level at 1 cm below the soil surface (WL-1); and (iii) water level at 15 cm below the soil surface (WL-15).
Sampling and Enzyme Assay
The turf quality was visually rated as an integral of color, uniformity, and density on a scale of 1 (brown leaves) to 9 (turgid, green leaves). At the end of the experiment (21d), the roots were washed free from the soil. Samples were then stored at –80°C for further analyses. An additional 1.0 cm-diam. hole was drilled in the side of each PVC tube 15 cm below the soil surface to allow a probe inserted to measure the soil redox potential (Eh). This hole was plugged at all times except during sampling. The soil Eh was measured using Combination Redox Probe (TPS Pty. Ltd., Brisbane, Australia) and the reading was taken after the probe was equilibrated in the soil.
To extract the soluble protein, a frozen sample (0.5 g) from the entire root tissue was ground into fine power using liquid nitrogen and 4-mL of extraction buffer (50 mM potassium phosphate, 1 mM ethylenediaminetetraacetic acid [EDTA], 0.1% triton X-100 (v/v), 1% polyvinylpyrrolidone [PVP], 1 mM dithiothreitol [DTT], and 1 mM phenylmethylsulfonyl [PMSF], pH 7.8) was then added. Samples then were centrifuged at 15 000 x g for 2 x15 min at 4°C, and supernatant was collected for enzyme assay. The protein content was determined using the method of Bradford (1976). All enzyme assays were performed the same day as the Bradford assay using Spectronic Genesys 10 Bio Spectrophotometer (Thermo Electron Corporation, Waltham, MA).
Total SOD activity was measured according to the method of Giannopolities and Rise (1977). The assay medium contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µMp-nitro blue tetrazolium chloride (NBT), 2 µM riboflavin, 0.1 mM EDTA, and 30 to 40 µL of enzyme extract. A reaction mixture was illuminated under 80 to 90 µmol m–2 s–1 for 10 min. The reaction mixture without illumination served as the control, and the mixture lacking of enzyme developed maximum color as maximum reduction of NBT. One unit of SOD activity was defined as the amount of enzymes that can cause 50% inhibition in the rate of NBT reduction.
The activity of AP was assayed by recording the decrease in absorbance at 290 nm for 1 min. The 1.5-mL reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mM EDTA, 0.1 mM H2O2, and 0.15 mL of enzyme. The reaction was started with the addition of 0.1 mM H2O2 (Nakano and Asada, 1981).
The activity of GR was assayed by measuring the decrease in absorbance at 340 nm for 1 min (Cakmak et al., 1993). The reaction mixture contained 0.1 M phosphate buffer (pH 7.8), 1 mM EDTA, 1 mM oxidized glutathione (GSSG), 0.2 mM nicotinamide adenine dinucleotide phosphate (NADPH), and 0.15 mL enzyme extract, with a total volume of 1.5 mL. The reaction was started by adding GSSG.
The activity of POD was determined by an increase in absorbance at 470 nm for 1 min. The assay contained 50 µL of 20 mM guaiacol, 2.83 mL of 10 mM phosphate buffer (pH 7.0), and 0.1 mL of enzyme extract. The reaction was initiated by adding H2O2 (Kochhar et al., 1979).
The H2O2 content was determined using the methods of Bernt and Bergmeyer (1974). Root tissue (1 g) was homogenized in 3 mL of 100 mM sodium phosphate buffer (pH 6.8), and extractions were then centrifuged at 18 000 g for 5 min at 4°C. The 0.17 mL of supernatant was added to 0.83 mL peroxidase reagent containing 83-mM sodium phosphate (pH 7.0), 0.005% (w/v) o-dianisiden, and 40 µg peroxidase/mL. The mixture was incubated at 30°C for 10 min, and 0.17 mL of 1 M perchloric acid was added to stop the reaction. The absorbance at 436 nm was read against blank.
The lipid peroxidation of the root tissue was measured in terms of MDA content (Dhindsa et al., 1981). A 1-mL aliquot of supernatant was mixed with 4 mL of 20% trichloroacetic acid containing 0.5% thiobarbituric acid. The mixture was heated at 100°C for 30 min, quickly cooled, and then centrifuged at 10 000 g for 10 min. The absorbance was read at 532 and 600 nm. The concentration of MDA was calculated using an extinction coefficient of 155 mm–1cm–1 (Health and Packer, 1968).
Native Polyacrylamide Gel Electrophoresis
The procedure of protein extraction was the same as for soluble protein except for using 1.0 g tissue with 2 mL of extraction buffer. The protein fractions were concentrated using Centricon Centrifugal Filter Units (Millipore Corporation, Billerica, MA, USA) with 10 kDa cutoff before loading into the gels. Native polyacrylamide gel electrophoresis (PAGE) was performed by Bio-rad mini-gel system at 4°C, 120 V for 90 min (Laemmli, 1970), except that SDS was omitted. For SOD, AP, and POD, the enzyme extracts were subjected to native PAGE with 10% resolving gel and 4% stacking gel. For GR, isoforms were separated by native PAGE with a 7.5% resolving gel and 3% stacking gel.
The total activity of SOD was stained using the method of Beauchamp and Fridovich (1971), with some modifications. The gels were incubated in 50 mM potassium phosphate buffer (pH 7.5) containing 2.5 mM NBT in dark 25 min. After briefly being washed twice with the same buffer, the gels were soaked in 50 mM potassium phosphate buffer (pH 7.5) containing 30 µM riboflavin and 0.4% N, N, N', N'-tetramethylethylenediamine (TEMED) in the dark for 40 min. The gels were then illuminated for 10 to 15 min with gentle agitation until an appearance of enzyme bands and were transferred to 1% (v/v) acetic acid to stop the reaction. The isoenzymes were identified and characterized by selective inhibition with KCN or H2O2. To inhibit Cu/Zn-SOD and Fe-SOD, 5 mM H2O2 was added during the incubation in 50 mM potassium phosphate buffer (pH 7.5) containing 2.5 mM NBT. For inhibition of Cu/Zn-SOD, the gels were stained using the same procedure, except for the substitution of 5 mM H2O2 with 2 mM KCN (Pitcher et al., 1992).
The activity of POD was detected using the method of Fielding and Hall (1978). The gels were soaked in a sodium phosphate solution (10-mM sodium phosphate and 150-mM sodium chloride, pH 6.0) for 45 min to lower the pH. After briefly being washed with 100 mM potassium phosphate buffer (pH 6.4), the gels were stained in 100 mM potassium phosphate buffer (pH 6.4) containing 20 mM guaiacol and 5.55 mM H2O2 for 5 to 10 min until the bands were clearly visible. The gels were then washed with distilled water to stop the reaction.
The activity of AP was detected using the method of Lopez-Huertas et al. (1999). The gels were preincubated in 50 mM sodium phosphate buffer (pH 7.0) containing 4 mM ascorbate, 2 mM H2O2 for 20 min. The gels were then washed briefly and submerged in 50 mM sodium phosphate buffer (pH 7.8) containing 28 mM TEMED and 1.25 mM NBT for 10 min.
The activity of GR was stained by incubating the gels in a solution of 250 mM Tris (pH 7.8) containing 0.24 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT), 0.4 mM NADPH, 0.34 mM 2,6-dichlorophenolindophenol, and 3.6 mM GSSG for 1 h according to Anderson et al. (1995).
Experimental Design and Statistical Analyses
The experiment was randomized complete block design. Four growth chambers representing four replicates were used in this study. Waterlogged and well-drained tubes were arranged randomly within each chamber. Data from 21 d of the treatment was used to analyze the significance of the WL treatment by comparing the differences with their respective controls at a given cultivar for a given measurement. Statistical Analysis System (SAS 9.1) (SAS Institute Inc. Cary, NC) was used for this significant analysis. The means of the WL treatments were separated using the Least Significant Difference (LSD) at a 0.05 significance level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hydrogen Peroxide and Malondialdehyde Content
Waterlogging did not significantly change the H2O2 and MDA content in cultivars (Table 1). G-6 had an inherently lower H2O2 content. At 21 d of treatment, the H2O2 content increased 11 and 8% for Penncross and 12 and 16% for G-6 under WL-1 and WL-15, respectively. The root MDA content remained unchanged for all treatments in both cultivars.
Antioxidant Enzymes and Isoforms
The activity of SOD increased with the increasing WL level for both cultivars (Fig. 1A
). For Penncross, SOD activities under WL-1 and WL-15 were 32 and 26% higher than those of the control, while SOD activities increased 83 and 44% in WL-1 and WL-15 for G-6, respectively. Native PAGE gels revealed that one isoform of Mn-SOD and four isoforms of Cu/Zn-SOD presented under all treatments (Fig. 2
). The intensity of Mn-SOD and Cu/Zn-SOD increased under WL conditions in Penncross, compared with the control, but similar patterns were not observed in G-6.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Water level at 1 cm below the soil surface had more negative effects on turf quality than WL-15, and SOD and AP activities were generally more affected by WL-1 than WL-15. Malik et al. (2001) reported that the growth rate of wheat was less affected by WL at a 20-cm depth compared with WL at 10 cm or at soil surface. They also found that photosynthesis was reduced by 70 to 80% for the most severe WL treatment, but photosynthesis was little affected when WL occurred at 20 cm below the soil surface. These results indicated that different depths of WL affected plant growth and metabolic activities to different degrees. However, WL at 15 cm deep had similar effects on the HPC and MDA content and activities of GR and POD compared with WL-1 and the control, suggesting that WL up to 21 d did not influence certain antioxidant activities in creeping bentgrass. The patterns of antioxidant enzymes under WL may depend on particular species or cultivars and the duration of stress.
The activity of SOD significantly increased under WL-1 for both cultivars, particularly for the tolerant G-6, which showed 83% higher SOD activity than the control. Yan et al. (1996) found that declines in SOD activity paralleled the enhanced production of O2–· in corn under flooding stress. The high level of SOD may contribute to WL tolerance by improving the detoxification of O2–· induced under WL conditions in this study. In Penncross, the increased SOD activity may be due to the high expression of one Mn-SOD and two Cu/Zn-SODs, although similar patterns were not observed in G-6. Biemelt et al. (2000) reported that hypoxia did not cause significant changes in the activity and patterns of individual SOD isoforms in wheat roots, and that mitochondrial Mn-SOD activity and synthesis of Mn-SOD isoforms were not affected by hypoxia (Szal et al., 2004). The role of SOD on WL tolerance may also depend on the duration of stress; therefore, further research is needed to identify the responses of root Mn-SOD and Cu/Zn-SOD to duration of WL stress in cool-season grasses.
The ascorbate-glutathione cycle is an effective system in the detoxification of H2O2 in leaf chloroplast and in cytosol (Cakmak et al., 1993). The activity of AP and GR is important in determining the efficiency of this pathway. Penncross showed a greater reduction in AP activity than did G-6 under WL-1. Under WL-15, G-6 had 60% higher AP activities than Penncross. The higher AP activity in G-6 indicated a higher potential to eliminate H2O2, which would contribute to WL tolerance. These results suggested that AP activity was associated with the WL tolerance of creeping bentgrass. Lin et al. (2004) indicated that AP activity could serve as criteria for evaluating the flood tolerance of tomato and eggplant (Solanum nielongena L.) roots. Our results agree with Ahmed et al. (2002), who found that WL decreased AP activity in mungbean. The activity of AP also increased with increasing flooding from 3 h to 72 h in eggplant roots (Lin et al., 2004), and a higher AP activity was observed in wheat roots in response to hypoxia (Biemelt et al., 1998). Duration of stress, plant species, and plant organs are the factors influencing AP activity associated with WL tolerance. Native PAGE gel showed that one isoform of AP strongly presented after 21 d of WL in creeping bentgrass (Fig. 3), and its intensity tended to increase under WL conditions. In lupine (Lupinus Luteus L.) roots, five isoforms of AP were shown and one of them disappeared after short-term WL (Garnczarska, 2005). Whether the changing patterns of AP isoforms in response to WL depend on duration of stress is not known in cool-season grasses.
Unlike in AP, the activity of GR remained unchanged under WL conditions. Similar results have been found in other plant species whose activity of GR was unaffected (Lin et al., 2004) or slightly influenced by WL or hypoxia (Biemelt et al., 1998). However, inhibition of GR activities has also been shown under flooding stress (Albrecht and Wiedenroth, 1994; Yan et al., 1996). Although six isoforms of GR generally expressed in roots of two cultivars, one with 120 kDa strongly accumulated. Thus, further study is needed to investigate whether this isoform is associated with antioxidant protection.
The H2O2 content changed slightly for both cultivars. The increase in H2O2 content may be due to a decrease in AP activity in creeping bentgrass. An increased H2O2 content was also found in corn leaves subjected to 5 to 7 d of flooding when a reduction in AP activity was observed, but not under 3 d of treatment (Yan et al., 1996). The content of MDA is often used as an indicator of lipid peroxidation in plant tissue resulting from oxidative stress (Smirnoff, 1995). Hypoxia increased the MDA content in the intolerant species of B. napus L. seedlings (Leul and Zhou, 1999), and the MDA content increased two- to threefold in the roots of peppergrass (Lepidium latifolium L.) when grown in an anaerobic environment (Chen and Qualls, 2003). However, our data showed that there was no significant difference in MDA content among WL treatments, and the results indicated that both WL-1 and WL-15 did not induce membrane lipid peroxidation (Table 1). Hunter et al. (1983) reported that anoxia did not induce lipid peroxidation in German iris (Iris x germanica L.). Lipid peroxidation is also a natural metabolic process under normal aerobic conditions (Blokhina et al., 2002). The unchanged lipid peroxidation level seems to be a characteristic of tolerant plants that cope with environmental stress, such as salinity (Shalata et al., 2001) and anoxia stress (Larson, 1988). Creeping bentgrass has better flooding tolerance than some of the other perennial cool-season grasses (Beard, 1973), and the stable level of lipid peroxidation and H2O2 content under WL conditions suggested that declines in turf quality were associated with changes in antioxidant enzymes.
In summary, WL decreased turf quality and affected root AP and SOD activities, particularly when the water level was at 1 cm below the soil surface. Water level at 15 cm below the soil surface could also significantly reduce turf quality and AP activities, especially in the WL-sensitive cultivar. Waterlogging increased root SOD activity in creeping brentgrass. Also, one Mn-SOD and four Cu/Zn-SODs were identified in creeping bentgrass roots in response to soil WL.
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ACKNOWLEDGMENTS |
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Received for publication July 31, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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