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USDA-ARS, Plant Stress and Water Conservation Lab., 3810 4th St., Lubbock, TX 79415
* Corresponding author (jmahan{at}lbk.ars.usda.gov).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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5x) seasonally, but again not in response to water deficits. The amount of ascorbate and the activity of ascorbate peroxidase were increased under water stress ( 5x and 1.5x, respectively). The amount of MDA (an indicator of oxidative damage) varied seasonally ( 2x), though not in response to water stress. While glutathione metabolism appears sufficient for oxidative stresses resulting from field-water deficits, altered ascorbate metabolism may be a response to water deficits in the field. Malondialdehyde amount did not increase with water stress, suggesting that antioxidant metabolism was sufficient for the oxidative stresses. It is apparent that antioxidant metabolism in these plants exposed to water deficits under production conditions was sufficient to protect against oxidative damage.
Abbreviations: DOY, day of year • GSH, reduced glutathione • GSSG, oxidized glutathione • PET, potential evapotranspiration • ROS, reactive oxygen species
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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-tocopherol, ascorbate peroxidase, and glutathione reductase than salt-sensitive cultivars when grown under salt stress. Oxidative stress is a term commonly used to describe the adverse effects of ROS on plants. A variety of enzymatic and nonenzymatic mechanisms exist to metabolize ROS into less harmful chemical species. The term antioxidant metabolism describes the detoxification of ROS, and the chemicals involved are generally referred to as antioxidants. Recent reviews by Foyer (2001) and Blokhina et al. (2003) summarize a great deal of the current knowledge of antioxidant metabolism in plants.
Efforts to limit water-stress related oxidative damage in plants generally fall into two broad areas: management of water to moderate the water stresses, and the development of germplasm with enhanced antioxidant metabolism. Success in both approaches will be enhanced by improved knowledge of when and to what extent antioxidant metabolism is a limiting factor in the plant's response to environmental stress.
Glutathione is one of several chemical compounds in plants that are involved in the detoxification of ROS. Glutathione can be oxidized directly by oxidants and also as a component of the Halliwell-Asada cycle that maintains the cellular ascorbate pool in a reduced state (Noctor et al., 1998). Previous studies on the role of glutathione in oxidative stress protection have generally been based on oxidative stresses generated by acute exposures to chemical oxidants (Iturbe-Ormaetxe et al., 1998), gaseous pollutants (Grill et al., 1982; Foster and Hess, 1980), high air temperatures (Nieto-Sotelo and Ho, 1986), and water stresses (Loggini et al., 1999). However, studies involving antioxidants in plants grown under field conditions and subjected to stresses in the field are rare.
The goal of this study was to determine the timing and extent of limitations in glutathione metabolism in cotton grown under water stresses typical for a production environment. The study involved a seasonal analysis of antioxidant metabolism in cotton grown under water stress in Lubbock, TX, during the summers of 1996 and 1997. Glutathione was the only antioxidant monitored in Year 1. The absence of water-stress related variation in glutathione in that year prompted the expansion of the study in 1997 to include the imposition of another level of water stress, and additional measurements of antioxidant metabolism. In both years, cotton plants were grown in the field under conditions representative of cotton production in semiarid regions like the Texas High Plains, and the imposed water treatments resulted in the development of relatively severe water stresses.
It is hypothesized that oxidative stress resulting from water stress will occur during the growing season. Knowledge of the extent and pattern of oxidative stress in field-grown cotton will provide valuable information on the best means of improving antioxidant activity in cotton. It will provide an understanding of when, how much, and for how long antioxidant metabolism should be altered to improve plant performance under stress in the field.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Irrigation 1996
Two irrigation treatments were established in Year 1: dryland and full irrigation. Both treatments received preplant irrigations of 127 mm and rain of 300 mm. The full-irrigation treatment also received subsequent irrigations on a regular basis throughout the season. Irrigation scheduling was automated on a 3-d interval and was initiated on DOY 173. The full-irrigation treatment received 16 irrigations of 21 mm. Estimates of potential evapotranspiration (PET) were provided by the PET Network of the Texas Agricultural Experiment Station, Lubbock, TX.
Irrigation 1997
Three irrigation treatments were established in Year 2; dryland, deficit irrigation, and full irrigation, receiving rain, a fraction of PET, and full PET, respectively. All treatments received preplant irrigations of 127 mm and rainfall of 256 mm. The deficit and full-irrigation treatments also received irrigations on a regular basis throughout the season. Irrigation scheduling was automated on a minimum interval of 3 d, and irrigation was initiated on DOY 181. The full-irrigation treatment received 21 irrigations of 21 mm, and the deficit irrigation treatment received 21 irrigations of 7 mm. Estimates of PET were provided by the PET Network of the Texas Agricultural Experiment Station, Lubbock, TX.
Leaf Water Potential
Leaf water potential was determined in Year 2 on a weekly basis in all treatments between 1400 and 1500 h (when differences among treatments would be largest). Measurements were made on leaves collected from the third node below the apex using a pressure chamber (Model 3005 Plant Water Status Console from Soilmoisture Equipment Corp., Santa Barbara, CA). A minimum of three samples were analyzed for each measurement.
Collection of Leaf Samples
All leaf samples for metabolic analyses were collected between 0800 and 0830 h. Samples were collected early in the day to minimize diurnal differences in antioxidant metabolism that were not related to water status. Leaves were collected from the third node below the apex from plants in all treatments. Ten samples per treatment were placed in plastic bags with a wet paper towel for transport from the field to the laboratory (generally < 15 min), frozen in liquid nitrogen, and stored at –90°C until analysis.
Glutathione Reductase Assay
Glutathione reductase was extracted from cotton leaf material by grinding the tissue in a mortar and pestle to a powder in liquid nitrogen. The powder was then ground to a paste with ice-cold buffer [0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 20% (v/v) glycerol, 1% (w/v) polyvinylpolypyrrolidone, 1% (v/v) Triton X, 5 mM ethylenediamine tetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride] at a ratio of 4 mL g–1 fresh wt. The extract was centrifuged (15000 x g) for 30 min at 4°C. Polyethylene glycol (8000 MW) was added to 20% saturation, and the mixture stirred slowly for 3 h at 4°C. The mixture was centrifuged (15000 x g) for 30 min at 4°C. The pellet was resuspended in 2 mL of buffer (0.05 M Tricine pH 7.5 containing 0.012 mM flavin adenine dinucleotide, 0.065 mM DTT, and 20% glycerol (prepared at time of use). The resuspended pellet was used in the assays.
The glutathione reductase assay was modified from Mahan et al. (1990). The 1-mL assay contained 0.5 mM oxidized glutathione (GSSG) and 0.1 mM NADPH in a buffer (0.125 M HEPES pH 8). The assay was initiated with the addition of 0.5 mL of the leaf extract that contained approximately 0.03 units of glutathione reductase. Enzyme activity was monitored by the decrease in absorbance at 340 nm for 30 s at 25°C and the initial rate determined. A unit of activity is the amount of enzyme that will catalyze the reduction of 1 µmol of GSSG min–1 at 25°C.
Glutathione Measurements
The amount and form of glutathione were determined by a modification of the method of Anderson (1985), which measured total glutathione [reduced glutathione (GSH), and GSSG] by a glutathione reductase catalyzed reaction. Removal of GSH from the sample by chemical derivatization provided for the quantification of GSSG in the sample.
Leaf tissue was ground to a powder in liquid nitrogen in a prechilled mortar and pestle. The powder was then ground to a paste with 10% 5-sulfosalicylic acid (5 mL g–1 fresh wt. of tissue) to reduce the oxidation of GSH. The extract was centrifuged (15000 x g) for 30 min at 4°C and used in subsequent assays.
The 1-mL volume assay for total glutathione contained the following: 0.28 mM NADPH, 0.6 mM 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB), the plant extract or glutathione standard, and 0.2 units per assay glutathione reductase from yeast in 0.143 M potassium phosphate pH 7.5, with 6.3 mM EDTA). The assay mixture (minus glutathione reductase and leaf extract) was allowed to equilibrate at 30°C for 5 min. After the addition of the glutathione-containing extract and glutathione reductase, the decrease in absorbance at 412 nm was monitored for 30 s. A reaction blank was prepared by replacing the plant extract with 10% 5-sulfosalicylic acid. The concentration of glutathione was determined through comparison with a standard curve of reaction rate as a function of the concentration of GSSG (0.07–1.68 µg mL–1 in 10% 5-sulfosalicylic acid). Glutathione values are expressed as GSH equivalents (1 mol GSSG is equivalent to 2 mol GSH). Each reported value represents the mean of a minimum of three determinations.
The amount of oxidized glutathione in the extract was determined by removing GSH by derivatization with 2-vinylpyridine. The derivatizations were performed immediately after leaf extractions to minimize the oxidation of GSH in the sample. Plant extract (300 µL) and 6 µL of 2-vinylpyridine were mixed in a microcentrifuge tube, and 34 µL of 1 M triethanolamine was added to the side of the tube and the solution was mixed vigorously. The final pH of the mixture was measured, and if >7, the derivatization was repeated using less triethanolamine. The derivatizations were performed at room temperature for 1 h, and the resultant solution was then used as plant extract for the glutathione assay previously described.
Ascorbate Assay
The ascorbate extraction and assays are described in Lukaszewski and Blevins (1996). Ascorbate was extracted by homogenizing 0.1 g of previously frozen cotton leaves with a tissue homogenizer in 1.0 mL of 5.0% meta-phosphoric acid on ice. The acid extract was centrifuged (15000 x g) for 30 min at 4°C. The supernatant fraction was used immediately in assays. The assay for total ascorbate (1 mL) contained 0.1 M citrate with leaf extract in 0.2 M potassium phosphate buffer pH 6.2. The assay was initiated with the addition of 1 unit of ascorbic acid oxidase, and the decrease in absorbance at 265 nm was monitored for 30 s at 25°C. The reaction rate is proportional to the amount of ascorbate, as described by a standard curve of ascorbate (0–160 mg in 5.0% meta-phosphoric acid). Ascorbate concentration was expressed as nanomoles per gram fresh weight.
Ascorbate Peroxidase Assay
The extraction and assay procedures for ascorbate peroxidase are described in Allen (1995). Cotton leaf material (0.5 g) was ground to a powder in a mortar and pestle using liquid nitrogen. The powder was homogenized in 2 mL extraction buffer with a tissue homogenizer on ice. The extraction buffer consisted of 50 mM HEPES, pH 7, 1 mM ascorbate, 1 mM EDTA, and 1% (v/v) Triton X, and was prepared immediately before the extraction. The extract was centrifuged (15000 x g) for 15 min at 4°C. The supernatant fraction was used for the assays. All reagents and the extract were prepared fresh before the assay. The assay reaction mixture contained 50 mM HEPES, pH 7.0, 1 mM EDTA, 1 mM hydrogen peroxide, 0.5 mM ascorbate, and 30 µL of the extract in a total volume of 1 mL. The assay was allowed to equilibrate at 25°C for 1 min before the addition of hydrogen peroxide, which initiated the reaction. The reaction was monitored as a decrease in absorbance at 290 nm for 60 s at 25°C, and the initial rate determined. A control reaction was prepared by replacing the ascorbate with the HEPES buffer. A unit of ascorbate peroxidase is defined as the amount necessary to oxidize 1 µmol of ascorbate min–1 at 25°C (290 nm extinction coefficient of 2.8 L mmol–1 cm–1).
Malondialdehyde Quantification
The lipid peroxidation assays were performed according to a modification of the method of Draper and Hadley (1990). Previously frozen cotton leaf tissue (0.1 g) was ground in liquid nitrogen and homogenized in 1.2 mL 5% tetrachloroacetic acid plus 0.12 mL of 0.05% methanolic butylated hydroxytoluene with a tissue homogenizer. The homogenate was heated for 30 min at 100°C to release protein-bound MDA. After cooling, the homogenate was centrifuged (15000 x g) for 15 min and the extract was used for the assays. The extract (0.5 mL) was reacted with 0.5 mL of 0.67% thiobarbituric acid at 100°C for 30 min. A sample blank consisted of 0.5 mL of the extract plus 0.5 mL of water incubated at 100°C for 30 min. The assay mixture was cooled to room temperature and the absorbance of each sample was determined at 532 nm and 600 nm. The absorbance at 600 nm was subtracted from the 532-nm reading, and the concentration of MDA was calculated using an extinction coefficient of 155 L mmol–1 cm–1.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Figure 2 shows the seasonal pattern of leaf water potential in Year 2 treatments. As a result of rain, the water potentials in all three treatments were similar before DOY 205. From DOY 205 to 269, the dryland treatment generally showed the lowest leaf water potential values, with the deficit irrigation treatment intermediate between dryland and full irrigation. At the end of the season, by DOY 269, the differentials were eliminated by a combination of rain events and reduced evaporative demand due to cooler air temperatures at the end of the season.
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Glutathione and Glutathione Reductase
Seasonal variation in the amount and form of glutathione during Year 1 is shown in Fig. 3
. The amount of glutathione varied across the season in both the dryland and full-irrigation treatments with no significant (P > 0.05) differences associated with the amount of water received. In both treatments at all dates, the reduced form was predominant, with no pronounced differences in the reduced and oxidized forms that could be associated with the water treatments.
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Poole and Rennenberg (1992) studied variation in the glutathione content of needles on a seasonal basis from spruce trees grown at three altitudes. Higher levels were found at the highest altitude, and lower levels at the lower altitudes, and it was concluded that increased glutathione content was an adaptive response to increased stresses experienced by the plants at the higher altitudes. It might be expected that the antioxidant metabolism of plants would respond differently to high altitude oxidative stress that is constant and long term, as compared with water stress that is variable and transient.
In general, the majority of glutathione in an unstressed plant cell is found in the reduced form (GSH) typically comprising 70 to 90% of the total (Bielawski and Joy, 1986; Smith, 1985). Under oxidative stress, if the rate of glutathione oxidation exceeds the rate of glutathione reduction, an increase in the fraction of glutathione in the oxidized form (GSSG) can result (Sgherri and Navari-Izzo, 1995; Smith, 1985). Sgherri and Navari-Izzo (1995) reported changes in the glutathione metabolism of sunflowers under water stress in the field. Changes in glutathione content and form resulting from oxidative stresses were identified in experiments involving glutathione metabolism in catalase-deficient barley by Smith et al. (1985), who reported increases in the total glutathione content and oxidized glutathione in H2O2–stressed barley plants. Increased glutathione content has been reported to be related to a feedback inhibition of glutathione synthesis by GSH (Richman and Meister, 1975; Hell and Bergmann, 1990; Schneider and Bergmann, 1995) and the depletion of GSH has been postulated to initiate increased glutathione synthesis under oxidative stress. Since there was no water-stress-related increase in oxidized glutathione in this study, an increase in total glutathione pool would not be anticipated.
Seasonal variation in glutathione content was reported in spruce [Picea abies (L.) H. Karst.] needles by Schmieden et al. (1993). They reported variation in total glutathione content with the highest levels (up to 300 µg g–1 dry wt.) during the winter and the lowest values (as low as 63 µg g–1 dry wt.) during the summer. The ratio of GSH/GSSG varied as well with a value of 12 in the winter and a lower value of 2 in the summer. They concluded that lower values of the ratio indicated increased oxidative stress in the tissues. The changes in glutathione metabolism that occur during the transition from summer to winter are in response to gradual and long-term changes in environmental conditions. Seasonal changes in oxidative stress are most probably different from those of transient and continuously variable water and temperature stresses seen in this study, thus the failure to see similar changes in response to water stress is understandable.
Glutathione Reductase Activity
Figure 5
demonstrates glutathione reductase activity during the growing season. The activity generally increased during the season. With the exception of the elevated activity in the dryland treatment on the second sampling date, there were no clear differences in glutathione reductase activity that appeared to be associated with the water treatments.
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Stress-related changes in both the amount and form of glutathione, as well as the activity of glutathione reductase, were reported by Sgherri and Navari-Izzo (1995). They found increases in glutathione amount and glutathione reductase activity in the leaves of sunflower following exposure to water stresses in the field. When exposed to severe water deficits, they found that the glutathione pool became more oxidized, and both the total glutathione pool and activity of glutathione reductase declined. In another study that involved stress exposures under field conditions, Gamble and Burke (1984) monitored the activity of glutathione reductase in the leaves of wheat grown under different levels of water and temperature stress. They reported that glutathione reductase activity was increased under water deficits in what they suggested was an adaptive response. With the exception of the studies by Gamble and Burke (1984) and Sgherri and Navari-Izzo (1995), the above-cited studies all involved stresses imposed on relatively young plants grown in pots with limited water, as compared with this study, in which the water deficits developed gradually as the growing plants consumed the available water in much larger soil volumes in the field.
Ascorbate and Ascorbate Peroxidase
Figure 6
shows the activity of ascorbate peroxidase during the growing season. The ascorbate peroxidase activity generally increased as the season progressed with evidence of higher activity in the two water-deficit treatments, as compared with the full-irrigation treatment. The amount of ascorbate across the season is shown in Fig. 7
, which shows relatively high amounts at the initial sampling dates before the onset of water stress. The amount declines in all three treatments by DOY 212, when water stress had developed. Ascorbate was elevated in the water deficit treatments relative to the full irrigation at all subsequent sampling dates, even on DOY 269, when differences in leaf water potential were no longer detected (Fig. 2).
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Malondialdehyde
Malondialdehyde is an indicator of oxidative-stress related peroxidation of membrane lipids, and its concentration has been shown to correlate with peroxides resulting from temperature- and water-stress-related membrane damage (Halliwell and Gutteridge, 1989). Figure 8
shows the seasonal pattern of MDA in leaves from the three water treatments in Year 2. There was no indication of elevated MDA levels related to the water stress and, in fact, the highest levels of MDA that were observed early in the season were found in the full-irrigation treatment. Iturbe-Ormaetxe et al. (1998) reported elevated MDA levels in peas that had been exposed to acute short-term water stress in containers. Huang et al. (2001) reported increased MDA content in response to high soil temperatures in two cultivars of creeping bentgrass that were grown in sand-filled tubes for 42 d. While MDA was elevated by stress in both heat-sensitive and tolerant cultivars, it was higher in the heat-sensitive cultivar. The similar levels of MDA in well-watered plants in their study and both well-watered and water-stressed plants in our study suggests that oxidation of membranes was not enhanced by the water stresses imposed in this study.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The failure to observe water-stress-related glutathione differences in either the first or second year might be explained as a failure to develop water stress of sufficient intensity and duration to result in the increased production of ROS in the plant. In both years of the study, both the fraction of PET replaced by irrigation and rain and the reductions in yield were similar. The PET values and lint yields indicate the development of moderately severe water stresses in both years, and the yield differences between the treatments are sufficiently large to indicate impaired plant performance resulting from environmental stresses. The development of water stresses, coupled with the observed changes in ascorbate metabolic indicators, suggest that the failure to observe changes in glutathione are not related to the failure to develop water deficits in the plants.
Glutathione amount and form, and glutathione reductase activity in cotton did not vary in response to differential water status of the magnitude and timing imposed in this study. If altered glutathione metabolism is a de facto indicator of increased oxidative stress, then the absence of alterations suggests that oxidative stress was the same in the dryland and irrigated treatments. Since very few studies of the response of oxidant metabolism to stress have been performed using field stressed plants, it is possible that some of the glutathione inductions previously reported are the result of oxidative stresses that are different from those induced in this study. An alternative explanation of the results is that the plants in the dryland treatment did indeed experience increased oxidative stress, but that cotton has only limited ability to alter glutathione metabolism in an adaptive manner in response to oxidative stress. Under this scenario, the lack of an increase in the foliar pool of glutathione in cotton under water stress might indicate an avenue for the improvement of cotton performance under field water stresses through the enhancement of glutathione metabolism. However, the failure to detect water-stress-related increases in MDA levels suggests that the oxidative stress damage that occurred was mitigated before membrane damage could occur.
Several studies have already demonstrated that glutathione metabolism can be altered, and that such alterations can improve plant performance under certain types of stress (Foyer et al., 1994; May et al., 1998; Noctor et al., 1998; Foyer and Noctor, 2001). The more than two-fold seasonal changes in the glutathione pool found in this study indicate that there is a mechanism for altering the pool size. The absence of variation in glutathione in response to water stress suggests that it may not be a vital component of the cotton plant's response to water stress. In light of the relative constancy of at least some degree of oxidative stress in plants even under optimal water conditions, there would be a strong advantage for the plant to have antioxidant activity in excess of the level needed for protection under normal conditions. It is possible that the amount of glutathione present in the leaves was adequate for the oxidative compounds present in both the irrigated and dryland treatments. If indeed there was sufficient reduced glutathione present in the leaves, it is possible that transgenic approaches to increasing glutathione levels may not be very productive. This possibility is supported by the failure to improve plant protection by transgenic modification of glutathione metabolism reported in some studies.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication February 12, 2004.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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-glutamylcysteine synthase by non-allosteric feedback inhibition feedback inhibition by glutathione. J. Biol. Chem. 250:1422–1426.Related articles in Crop Science:
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