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Crop Science 41:436-442 (2001)
© 2001 Crop Science Society of America

TURFGRASS SCIENCE

Drought and Heat Stress Injury to Two Cool-Season Turfgrasses in Relation to Antioxidant Metabolism and Lipid Peroxidation

Yiwei Jianga and Bingru Huangb

a Dep. Horticulture, Forestry, and Recreation Resources, Kansas State Univ., Manhattan, KS 66506-5506
b Dep. of Plant Science, Foran Hall, 59 Dudley Rd., Rutgers Univ., New Brunswick, NJ 08901

Corresponding author (Huang{at}aesop.rutgers.edu)


    ABSTRACT
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Drought and high temperature are two major factors limiting the growth of cool-season turfgrasses during summer in many areas. The objective of the study was to examine whether the adverse effects of drought and heat alone or in combination on tall fescue (Festuca arundinacea L.) and Kentucky bluegrass (Poa pratensis L.) involve oxidative stress. Grasses were exposed to drought (withholding irrigation), heat (35°C/30°C), and the combined stresses for 30 d in growth chambers. Turf quality (TQ), leaf relative water content (RWC), and chlorophyll content (Chl) decreased with prolonged drought, heat, and combined stresses for both species, but the severity of decline varied with stress type and duration. Transient increases in superoxide dismutase (SOD), ascorbate peroxidase (AP), and glutathione reductase (GR) activities occurred at 6 or 12 d of drought and the combined stresses in both species; however, the activities of all three enzymes decreased with extended periods of drought and the combined stresses. The SOD activity was not affected by heat stress alone. The activities of AP and GR were reduced after 18 d of heat stress for both species, but reductions were less than under the combined stresses. The catalase (CAT) activity continued to decrease to below the control level, beginning at 12 d for drought-stressed or heat-stressed plants and 6 d for plants exposed to the combined stresses. Lipid peroxidation occurred after 18 d of stresses in both species, as indicated by the increase in malondialdehyde (MDA) content. The results suggested that injuries of drought, heat, or the combined stresses to both tall fescue and Kentucky bluegrass, as manifested by declines in TQ, RWC, and Chl, could be associated with a decrease in antioxidant enzyme activities and an increase in membrane lipid peroxidation.

Abbreviations: TQ, turf quality • RWC, relative water content • Chl, chlorophyll content • ASA, ascorbic acid • SOD • superoxide dismutase • CAT, catalase • AP, ascorbate peroxidase • GR, glutathione reductase • GSH, reduced glutathione • GSSG, oxidized glutathione • MDA, malondialdehyde


    INTRODUCTION
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
COOL-SEASON TURFGRASSES often suffer from extended periods of drought stress, heat stress, or both during summer months in the transitional zone. Drought and heat stresses cause declines in turf quality that has been associated with reductions in root growth, leaf water potential, cell membrane stability, photosynthetic rate, photochemical efficiency, and carbohydrate accumulation (Howard and Watschke, 1991; Carrow, 1996; Perdomo et al., 1996; Huang et al., 1998a,b; Huang and Gao, 1999; Jiang and Huang, 2000). Simultaneous drought and heat stresses are more detrimental than either stress alone for various plant species. Drought and heat stress have caused significant decrease in the rate of CO2 uptake in bean (Phaseolus vulgaris L.) (Yordanov et al., 1997); leaf growth in sorghum [Sorghum bicolor (L.) Moench] (Kaigama, 1986); and leaf water content and water potential in wheat (Triticum aestivum L.) (Shah, 1992).

Many physiological factors could be involved in the drought or heat stress injury to cool-season turfgrasses. In some species, drought or heat injury induces oxidative stress, resulting from the production and accumulation of toxic oxygen species such as superoxide radicals , hydrogen peroxide (H2O2), and hydroxyl radicals (OH·) (Bowler et al., 1992; Foyer et al., 1994; Inze and Van Montagu, 1995). The active oxygen species produced during stress can damage many cellular components including lipids, proteins, carbohydrates, and nucleic acids (Monk et al., 1989). Oxidative stress can lead to inhibition of the photosynthesis and respiration processes and, thus, plant growth. Plants have evolved enzymatic and nonenzymatic systems to scavenge active oxygen species. In enzymatic systems, for example, superoxide dismutase (SOD) catalyses the dismutation of O-2 to H2O2 and O2. Catalase (CAT) and ascorbate peroxidase (AP) can break down H2O2. Glutathione reductase (GR) also can remove H2O2 via the ascorbate-glutathione cycle to maintain a high level of reduced ascorbate within chloroplasts. However, the function of the scavenging enzyme systems can be interrupted by drought or heat stress, which can result in increases in lipid peroxidation and consequent membrane damage (Chowdhury and Choudhuri 1985; Zhang and Kirkham, 1994; Jagtap and Bhargava, 1995, Dat et al., 1998).

Drought or heat alone or together often limits growth of cool-season turfgrasses, whether the adverse effects of drought or/and heat on cool-season turfgrasses involve oxidative stress that are related to changes in antioxidant enzyme activity have not been examined. Therefore, the present study was designed to examine the effects of drought, heat, and the combined stresses on antioxidant enzyme activities and lipid peroxidation in ‘Montauk’ tall fescue and ‘Livingston’ Kentucky bluegrass. Both cultivars have good drought tolerance (NTEP, 1996a,b).


    MATERIALS AND METHODS
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials
Sods of tall fescue (cv. Montauk) and Kentucky bluegrass (cv. Livingston) were collected from field plots at the Rocky Ford Turfgrass Research Center, Kansas State University. Grasses were grown in polyvinylchloride (PVC) tubes (10 cm in diam., 60 cm long) filled with silt loam soil (fine, montmorillonitic, mesic, aquic arquidolls) in the greenhouse for 60 d and then transferred to growth chambers with a temperature of 20°C/15°C (day/night), a 14-h photoperiod, and a photosynthetically active radiation of 600 µmol m-2 s-1 at the canopy level. Grasses were maintained at 20°C/15°C and well-watered in growth chambers for 14 d to allow them to adjust to the environment before drought and heat treatments were imposed. Grasses were mowed at 6 cm height for both species before and during stress treatments.

Stress Treatments
The experiment included two temperatures and two soil moisture regimes. Each treatment consisted of four replicates. Temperature treatments were optimum temperature (20°C/15°C, day/night) and high temperature (35°C/30°C). Heat-stressed plants were watered daily. Soil moisture treatments were (i) well-watered, irrigating every other day until free drainage occurred from the bottom of tubes and (ii) drought stress, withholding irrigation for plants grown in both temperature regimes. The treatments were defined as follows: (i) control: high soil moisture and optimum temperature; (ii) drought: low soil moisture and optimum temperature; (iii) heat: high soil moisture and high temperature; (iv) drought + heat: low soil moisture and high temperature. Soil moisture was monitored with time domain reflectometry (TDR, Soil moisture Equipment Corp., CA). Soil volumetric moisture content at the end of the experimental period was 50 g kg-1, which was 17% of the field capacity (290 g kg-1).

Measurements
Turf quality as an integral of color, uniformity, and density was rated visually on the scale of 0 (worst) to 9 (best). Leaf water status was determined by measuring relative water content (RWC) calculated as follows (Barrs and Weatherley, 1962): RWC = (FW - DW)/(SW - DW)100, where FW is the leaf fresh weight, DW is leaf dry weight, and SW is turgid weight of leaves after being soaked in water for 4 h at room temperature. Leaf chlorophyll content was measured according to the methods of Hiscox and Israeltem (1979) and Arnon (1949). Leaf chlorophyll was extracted by soaking 0.05 to 0.1 g of leaf sample in 20 mL dimethyl sulfoxide in the dark for 72 h. All the above measurements were made every 6 d.

For enzyme extracts and assays, leaves were sampled every 6 d. 0.2 g of leaves were frozen in liquid nitrogen and then ground in 4 mL solution containing 50 mM phosphate buffer (pH 7.0), 1% (w/v) polyvinylpolypyrrolidone, and 0.2 mM ascorbic acid (ASA). The homogenate was centrifuged at 15000 g for 30 min, and supernatant was collected and used for enzyme assays.

The activity of SOD was measured according to the method of Giannopolities and Ries (1977). The assay medium contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 mm p-nitro blue tetrazolium chloride (NBT), 2 mm riboflavin, 0.1 mM EDTA, and 20 to 50 mL enzyme extract. One unit of enzyme activity was determined as the amount of the enzyme to reach an inhibition of 50% NBT reduction rate by monitoring the absorbance at 560 nm with a spectrophotometer (Spectronic instrument, Inc., Rochester, NY).

The activity of CAT was determined as a decrease in absorbance at 240 nm for 1 min following the decomposition of H2O2 (Change and Maehly, 1955). The reaction mixture contained 50 mM phosphate buffer (pH 7.0) and 15 mM H2O2. The activity of AP was measured as a decrease in aborsbance at 290 nm for 1 min (Nakano and Asada, 1981). The assay mixture consisted of 0.5 mM ASA, 0.1 mM H2O2, 0.1 mM EDTA, 50 mM sodium phosphate buffer (pH 7.0), and 0.15 mL enzyme extract. The activity of GR was determined by following the decrease in absorbance at 340 nm due to the glutathione-dependence of NADPH for 1 min (Cakmak et al., 1993). The reaction mixture contained 1 mM EDTA, 0.5 mM GSSG, 0.15 mM NADPH, 100 mM sodium phosphate buffer (pH 7.8), and 0.15 mL enzyme extract. Enzyme activities were expressed on the basis of per unit protein weight. Protein content was determined by using bovine serum albumin as a standard according to Bradford (1976).

Lipid peroxidation was measured in terms of malondialdehyde (MDA) content described by Dhindsa et al. (1981). A 1-mL aliquot of supernatant of leaf extracts was mixed with 4 mL of 20% (v/v) trichloroacteic acid containing 0.5% (v/v) 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 of the supernatant was read at 532 nm and 600 nm. The concentration of MDA was calculated by means of an extinction coefficient of 155 mM-1 cm-1 (Health and Packer, 1968).

The experimental design was a series of experiments repeated over temperature and time. Each experiment involved a specific temperature with four plants per datum rested within temperature as described by Kempthorne (1952). Soil moisture treatments were arranged randomly within each temperature regime. Analysis of variance was based on the general linear model procedure of the Statistical Analysis System (SAS) (SAS Institute Inc., Cary, NC). Interactions among temperatures, soil moisture, and treatment duration were statistically significant for all parameters examined. Effects of temperature and soil moisture treatments were analyzed by comparing them with the control at a given measurement time. Least significance difference (LSD) at a 0.05 probability level was used to detect the differences between treatment means.


    RESULTS
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Turf Quality
Drought and heat alone significantly reduced turf quality, starting at 12 d of treatment for both tall fescue and Kentucky bluegrass (Fig. 1) . The combined stresses caused an earlier and more severe decline in turf quality than either drought or heat alone for both species.



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Fig. 1. Turf quality as effected by drought (D), heat (H), and the combined stresses (D+H) in tall fescue and Kentucky bluegrass. Vertical bars indicate LSD values (P = 0.05) for treatment comparison at a given day of treatment

 
Relative Water Content (RWC)
Leaf RWC started to decrease below the control level at 18 d under drought alone for both species (Fig. 2) . The reduction in RWC occurred at 18 d for tall fescue and 24 d for Kentucky bluegrass under heat alone. A more rapid and greater decrease in RWC was observed with the combined stresses than either stress alone for both species.



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Fig. 2. Relative water content (RWC) as effect by drought (D), heat (H), and the combined stresses (D+H) in tall fescue and Kentucky bluegrass. Vertical bars indicate LSD values (P = 0.05) for treatment comparison at a given day of treatment

 
Chlorophyll Content (Chl)
Leaf Chl content of drought-stressed and combined-stressed plants increased to above the control level within 12 d for tall fescue and 6 d for Kentucky bluegrass (Fig. 3) . The Chl content was lower than that of the control under drought after 18 d for tall fescue and after 24 d for Kentucky bluegrass and under heat after 18 d for Kentucky bluegrass and after 24 d for tall fescue. The combined stress caused a more severe reduction in Chl than either stress alone, beginning at 18 d of treatment.



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Fig. 3. Chlorophyll content (Chl) as affected by drought (D), heat (H), and the combined stresses (D+H) in tall fescue and Kentucky bluegrass. Vertical bars indicate LSD values (P = 0.05) for treatment comparison at a given day of treatment

 
Enzyme Activities
The activities of all enzymes remained stable in control plants but exhibited significant changes under stress conditions. A pronounced increase in SOD activities was observed at 12 d of drought, heat, or the combined stresses for both species (Fig. 4) . The activity of SOD then decreased rapidly to a level lower than the control at 18 d of the combined stress and 24 d of drought alone for both tall fescue and Kentucky bluegrass. Heat stress alone did not affect SOD activity after 18 d for either species.



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Fig. 4. Changes in SOD activity in tall fescue and Kentucky bluegrass in responses to drought (D), heat (H), and the combined stresses (D+H). Vertical bars indicate LSD values (P = 0.05) for treatment comparison at a given day of treatment

 
The activity of AP increased at 6 d of drought or the combined stresses and then significantly decreased to below the control level at 18 d in both species (Fig. 5) . Heat stress had no effect on AP activity until 18 d of treatment. The reduction in AP activity after 18 d was more pronounced for the combined stress than either stress alone and for drought than heat stress.



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Fig. 5. Changes in AP activity in tall fescue and Kentucky bluegrass in responses to drought (D), heat (H), and the combined stresses (D+H). Vertical bars indicate LSD values (P = 0.05) for treatment comparison at a given day of treatment

 
Increases in GR activity under drought and the combined stresses were observed at 12 d for tall fescue and 6 and 12 d for Kentucky bluegrass (Fig. 6) . However, the activity of GR decreased after 18 d of drought, heat, or the combined stresses. The reduction in GR activity was greater for the combined stresses than either stress alone and for drought than heat stress in both species.



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Fig. 6. Changes in GR activity in tall fescue and Kentucky bluegrass in responses to drought (D), heat (H), and the combined stresses (D+H). Vertical bars indicate LSD values (P = 0.05) for treatment comparison at a given day of treatment

 
The response of CAT activity to the stresses was different from those of SOD, AP, and GR. A continuous decline in CAT activity was observed in both species during the entire experimental period in all stress treatments (Fig. 7) . Drought or heat stress alone caused a significant reduction in CAT activities, starting at 12 d. The combined stresses led to a more severe reduction in CAT activity of both species than either stress alone, beginning at 6 d.



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Fig. 7. Changes in CAT activity in tall fescue and Kentucky bluegrass in responses to drought (D), heat (H), and the combined stresses (D+H). Vertical bars indicate LSD values (P = 0.05) for treatment comparison at a given day of treatment

 
Lipid Peroxidation (MDA)
Drought and heat stresses induced lipid peroxidation, beginning at 18 d of treatment in both species (Fig. 8) . Lipid peroxidation was more pronounced under the combined stresses than either stress alone, beginning at 12 d for tall fescue and 18 d for Kentucky bluegrass, except at 30 d when the effect of the combined stresses was similar to that of drought alone for both species.



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Fig. 8. Lipid perioxidation indicated by MDA content as affected by drought (D), heat (H), and the combined stresses (D+H) in tall fescue and Kentucky bluegrass. Vertical bars indicate LSD values (P = 0.05) for treatment comparison at a given day of treatment

 

    DISCUSSION
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Drought, heat, or the combined stresses caused significant declines in TQ, RWC, and leaf Chl content in both tall fescue and Kentucky bluegrass. The detrimental effects were more pronounced for the combined stresses than either stress alone. The dramatic decline in TQ under drought and the combined stresses was related closely to a decrease in RWC during the initial periods of stress but not chlorophyll content. Dry and hot environments induce abnormal transpirational water loss which has a cooling effect but also can cause rapid cell desiccation (Turner et al., 1966; Nobel, 1988a,b). Prolonged periods of drought, heat, and the combined stresses caused loss of chlorophyll and lipid peroxidation, which could lead to further quality decline.

Chlorophyll content in live plants is an important factor in determining photosynthetic capacity. Decreased or unchanged Chl level during drought stress has been observed in other species, depending on drought duration and severity (Rensburg and Kruger, 1994; Kyparissis et al., 1995; Zhang and Kirkham, 1996; Jagtap et al., 1998). In the present study, leaf Chl content increased transiently during the initial periods of stresses. However, leaf Chl decreased dramatically with prolonged stresses. Changes in leaf Chl content with drought, heat, or the combined stresses showed the same pattern as the responses of the activities of SOD, AP, and GR to these stresses, suggesting that drought and heat injury may involve a severe Chl photooxidation mediated by oxy-radicals (Wise and Naylor, 1987).

The activity of SOD, which is responsible for scavenging O-2 to produce H2O2 (Smirnoff, 1993), increased transiently at 12 d of drought, heat, or the combined stresses. This increased activity may reflect the enhanced amount of O-2 production and also indicate the possible role for SOD's dismutation effects on O-2 and protection of the photosynthetic apparatus (Foster and Hess, 1982). The initial increases in SOD activity was not related to the rapid decline in leaf RWC. When leaf RWC dropped to about 20% under both drought and the combined stresses, the activity of SOD was inhibited significantly; however, it was not affected under extended heat stress when RWC was above 65%. The results suggested that severe water deficit in plant tissue could impair O-2 scavenging in the cell and favor accumulation of O-2 (Castillo, 1996). The reduction in SOD activity under drought and the combined stresses may have been due to either reduced synthesis or enhanced degradation of the enzyme. The unchanged SOD activity under heat stress could be a response to heat acclimation and could contribute to heat tolerance.

Catalase, mostly localized in peroxisomes, breaks down and detoxifies H2O2. Reports on the effects of stresses on CAT activities vary. Increased, decreased, or unchanged CAT activities under drought stress have been observed (Quartacci and Navari-Izzo, 1992; Smirnoff, 1993; Zhang and Kirkham, 1994; Castillo, 1996). Our data showed that CAT activity continued to decline under all three stresses, and to a greater extent for plants exposed to the combined stresses than either stress alone. A reduction in CAT activity also has occurred during short-time heat shock (Willekens et al., 1995; Foyer et al., 1997; Dat et al., 1998). The results suggested that CAT is sensitive to both drought and heat stress. The decline in CAT activity could be attributed to CAT photoinactivation (Feierabend and Engel, 1986; Polle, 1997) and inhibition of synthesis of new enzyme in the dark (Dat et al., 1998), which may favor the accumulation of H2O2 and cause damage to cell membranes (Dhindsa et al., 1981).

The H2O2 scavenging enzyme, AP, located in both cytosol and chloroplasts, can remove H2O2 efficiently, especially in the chloroplast where CAT is absent (Groden and Beck, 1979). The activity of AP increased during initial periods of drought, heat, and combined stresses and decreased as stresses prolonged. This reduction in AP activity could be associated with the decrease in CAT activity, which results in H2O2 accumulation (Salin, 1988; Chen and Asada, 1992; Castillo, 1996). Glutathione reductase also may remove H2O2 within chloroplasts by maintaining more favorable levels of reduced glutathione (GSH) and oxidized glutathione (GSSG). The activity of GR continued to decrease under heat stress, which could result in H2O2 accumulation during heat stress. Under drought and the combined stresses, GR seems to be crucial in the protection against oxidative stress because its higher level was induced at 12 d of drought and the combined stresses. Smirnoff and Colombe (1988) suggested that the increased in the capacity of the hydrogen peroxide scavenging system may indicate the enhanced rate of hydrogen peroxide formation. The increased GR could protect the chloroplastic component against oxidation by H2O2 and minimize potential inactivation of SOD within chloroplasts (Foster and Hess, 1980). During a long-term drought stress, Castillo (1996) found that GR activity continued to increase even when severe water deficit occurred (at 50% RWC) in a CAM plant, Sedum album L. Our results showed that GR activity decreased when leaf RWC dropped below 50% under drought and the combined stresses. This discrepancy could be due to species variation in drought tolerance.

The accumulation of MDA often is used as an indicator of lipid peroxidation (Smirnoff 1995). Drought and heat stress increased MDA content in both tall fescue and Kentucky bluegrass, similar to what has been found in other species (Irigoyen, et al., 1992; Rensburg and Kruger, 1994; Gong et al., 1997). The dramatic increase in MDA content was related to reductions in SOD, CAT, AP, and GR activities and decreases in turf quality, RWC, and Chl in both species under prolonged drought and the combined stresses. The results indicated that membrane lipid peroxidation occurred from the malfunction of the scavenging system, which could lead to damage to main cellular components (Monk et al., 1989). Less MDA content accumulated under heat stress alone than the combined stresses, suggesting that less lipid peroxidation developed because more stable SOD, higher CAT, AP, and GR activities; and higher RWC were maintained in heat-stressed plants.

Our results demonstrated that soil moisture content had a major impact on the activities of antioxidant enzyme when grasses exposed to combined drought and heat stresses. The addition of heat stress may indirectly influence the rapid loss of antioxidant content by causing rapid loss of soil moisture. Beppu and Kataoka (1999) found that high temperature rather than drought is responsible for the occurrence of double pistils in ‘Satohnishiki’ sweet cherry (Prunus avium L.). However, different high temperatures were used and antioxidant activities were not investigated in their study.

In summary, drought, heat, and the combined stresses induced oxidative injury in both species, as demonstrated by the reduction in antioxidant enzymes and increase in lipid peroxidation. The combined stresses caused earlier and more severe oxidative damage. Both species exhibited a defensive mechanism to protect against free radicals in the early periods of stress treatments, as shown by the transient increases in SOD, AP, and GR activities. Clearly, loss of turf quality in dry, hot environments was associated closely with leaf water deficit and loss of Chl content, as well as a decline in antioxidant enzyme activity and an increase in lipid peroxidation.


    NOTES
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Contribution No. 00-227-J from Kansas Agric. Exp. Stn.

Received for publication December 27, 1999.


    REFERENCES
 TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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