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Antioxidant metabolism associated with summer leaf senescence and turf quality decline for creeping bentgrass.

THE GROWTH of cool-season grasses is most active in spring and fall when temperatures range from 15 to 24[degrees]C, but often declines during summer months when temperatures increase above the optimal range. Decline in turf quality during the summer is a major problem for creeping bentgrass in the transition zone and warm climatic areas. Researchers have been investigating the major environmental and physiological factors involved in summer bentgrass decline. However, the causes of summer bentgrass decline are still not completely understood. Previous studies suggest that high temperature is a major factor leading to summer bentgrass decline, although other factors such as high or low soil moisture, high humidity, and disease infection also contribute to this problem (Beard and Daniel, 1965; Huang et al., 1998a, 1998b). Limited carbohydrate availability due to the increase in respiration rate and the reduction in photosynthesis under high temperature conditions has been associated with summer bentgrass decline, particularly under low mowing conditions (Liu and Huang, 2001; Xu and Huang, 2003).

Leaf senescence and damage to cell membranes are typical symptoms of summer bentgrass decline under high temperature conditions, and these have been attributed to lipid peroxidation of cell membranes and the suppression of antioxidant enzymes (Liu and Huang, 2001). Plants exposed to environmental stresses such as heat, cold, drought, and salinity produce active oxygen species such as superoxide radical ([O.sup.-.sub.2]), hydrogen peroxide ([H.sub.2][O.sub.2]), hydroxyl free radical (OH), and singlet oxygen ([sup.1][O.sub.2]) (Scandalios, 1993; Zhang and Kirkham, 1996). Those active oxygen species are highly cytotoxic and can react with unsaturated fatty acids to cause peroxidation of essential membrane lipids in plasmalemma and intercellular organelles (Scandalios, 1993). Malondialdehyde (MDA) is the product of membrane lipid peroxidation. Increasing MDA content in plant cells indicates damage of cell membranes (Scandalios, 1993; Zhang and Kirkham, 1996), which can lead to inhibition of photosynthesis and respiration processes, and thus plant growth. Plant cells are protected against the attack of the species of active oxygen through a complex antioxidant system. Antioxidant enzymes including SOD, CATs, POD, and APX scavenge active oxygen species to protect plant cells. Superoxide dismutase is the key enzyme in the active oxygen scavenger system because it catalyzes superoxide free radical dismutation into [H.sub.2][O.sub.2] and [O.sub.2] (Scandalios, 1993). In turn, CAT, POD, and APX break down [H.sub.2][O.sub.2] in the living cells (Scandalios, 1993). Liu and Huang (2000) and Huang et al. (2001) reported that SOD activity of creeping bentgrass increased during the initial periods of heat stress (35[degrees]C), but both SOD and CAT activities decreased rapidly and lipid peroxidation increased following prolonged periods (2 to 3 wk) of heat stress, suggesting that the scavenging ability declined during prolonged heat stress and could cause oxidative damage.

Despite the importance of the antioxidant system in stress tolerance, few studies have characterized the seasonal changes of antioxidant enzymes under field conditions. It is unclear if summer bentgrass decline, particularly for heat-sensitive cultivars under low mowing conditions, is related to oxidative damage. Knowledge of seasonal changes in antioxidant activities under field conditions would help to better understand the complexity of summer bentgrass decline. The objectives of this study were to investigate the seasonal changes in activities of SOD, CAT, POD, and APX and lipid peroxidation under different mowing heights for cultivars differing in heat tolerance (L-93 and Penncross) and to determine whether such changes could be associated with leaf senescence and turf quality decline during summer. Previous studies reported that L-93 has better heat tolerance than Penncross (Liu and Huang, 2000, 2001).


The experiment was conducted on an USGA-specification putting green at the Turfgrass Research Center, Manhattan, KS, in 1999 and 2000. Two creeping bentgrass cultivars, L-93 and Penncross, were seeded in 205 cm x 318 cm plots in September 1996. Grass was mowed daily except Sunday at 3- or 4-mm heights from early May to early November. During this period, the green was irrigated daily to replace 100% water loss estimated by measuring evapotranspiration rate with minilysimeters. The green received four applications of urea fertilizers for a total of 98 kg N [ha.sup.-1] in 1999 (May, June, September, and October at 28, 25, 22, and 23 kg N [ha.sup.-1], respectively) and 65 kg N [ha.sup.-1] in 2000 (May, June, September, and October at 17, 16, 15, and 17 kg N [ha.sup.-1], respectively).

The experiment was a split-plot randomized design. Cultivars were the main plots and mowing heights were assigned randomly within each cultivar as the subplots. Each treatment had three replications. All measurements were taken on two subsamples in each plot at least once a month from May to November. The average of the subsamples was used as the treatment means. Effects of mowing, cultivars, and time of measurement on various parameters were determined with the standard F test in the ANOVA according to the general linear model procedure of the Statistical Analysis System (SAS Inc., Cary, NC). Differences between mowing heights and cultivar means at a given time of measurement or differences between times for a given cultivar and mowing height were separated by the LSD test at the 0.05 level.

Seasonal changes in air temperature were monitored with thermocouples (Fig. 1). Daily maximum and minimum air temperature data were collected with a datalogger from May to November in both years.


Turf quality was evaluated visually on color, uniformity, and density on a 1-to-9 scale, with 9 being the best and 1 being the worst. Leaf senescence was evaluated at various times by measuring leaf CHL content, EL of cell membranes, and total soluble protein content. Chlorophyll content was measured by soaking 0.05 g of leaves in 20 mL of dimethyl sulfoxide in the dark for 48 h (Hiscox and Israelstam, 1979). Absorbance of extractant at 663 and 645 nm was measured with a spectrophotometer (Spectronic Instruments, Inc., New York). For EL measurements, 0.05-g leaf samples were rinsed and then shaken for 18 h before the conductivity of the incubation solution was determined ([C.sub.initial]); tissues were then killed by autoclaving for 20 min, and the final conductivity of the solution was measured ([C.sub.max]). Relative EL was calculated as the percentage of [C.sub.initial] over [C.sub.max].

For the assays of SOD, CAT, POD, APX, and MDA, 0.5-g samples of leaves were collected from each plot at 0900 h in May, June, July, August, September, and October in 1999 and 2000. Leaf samples were frozen immediately in liquid nitrogen and stored at -70[degrees]C until use. For the extraction of enzymes and MDA, frozen samples were homogenized with 7 mL of 50 mM phosphate buffer solution (pH 7.0), grounded in a mortar in 0[degrees]C ice water, and centrifuged at 20 000 x g for 25 min in a refrigerated centrifuge. The supernatant was collected in a bottle for the determination of soluble protein, enzyme activities, and MDA content. Protein content was determined using bovine serum albumin as a standard (Bradford, 1976).

The SOD activity was measured according to the method of Zhang and Kirkham (1996). Twenty microliters of supernatant was added to a 3-mL reaction solution containing 63 [micro]M nitroblue tetrazolium (NBT; 2,2'-di-p-nitrophenyl-5,5'-diphenyl-[3,3'-dimethoxy-4,4'-diphenylene]), 1.3 [micro]M riboflavin (7,8-dimethyl-10-ribitylisoalloxazine), 13 [micro]M methionine, 0.1 [micro]M ethylenediaminetetraacetic acid (EDTA; ethylenediamine-N,N,N',N'-tetraacetic acid), and 50 mM phosphate buffer (pH 7.8). The solutions were placed under light at 80 [micro]mol photons [m.sup.-2] [s.sup.-1] for 10 min. The absorbance of reacted solutions and nonreacted solutions at 560 nm was determined with a spectrophotometer (Spectronic Instruments, Inc., New York). One unit of SOD activity was defined as the amount of SOD required to cause 50% inhibition of the rate of NBT reduction at 560 nm. The activity of CAT was determined as a decline in absorbance at 240 nm for 1 min (Chance and Maehly, 1955). The 3-mL reaction solution contained 15 mM [H.sub.2][O.sub.2] and 50 mM phosphate buffer (pH 7.0), and 0.1-0.2 mL enzyme extract. The activity of POD was measured as a decline in absorbance at 470 nm for 1 min (Chance and Maehly, 1995). The 5-mL reaction mixture contained 20 mM guaiacol (1-hydroxy-2-methoxybenzene), 10 mM phosphate buffer (pH 7.0), and 0.1 mL enzyme extract. The reaction was started by adding 20 [micro]L of 40 mM [H.sub.2][O.sub.2]. The activity of APX was determined as a decline in absorbance at 290 nm for 1 min. The reaction mixture contained 0.1 mM EDTA, 0.1 mM [H.sub.2][O.sub.2], 0.5 mM aminosalicylates, 50 mM phosphate buffer (pH 7.0), and 0.2 mL enzyme extract.

The MDA content was measured using the method of Dhindsa et al. (1981). A 1-mL MDA extract was added to 4 mL of trichloroacetic acid containing 0.5% thiobarbituric acid [4,6(1H,5H)-pyrimidinedione]. The solution was heated at 95[degrees]C for 30 min and then quickly cooled in running water. The solution was centrifuged at 10 000 x g for 10 min. The absorbance of the supernatant was measured at 532 and 600 nm. The concentration of MDA was calculated by subtraction of A600 from A532 and an extinction coefficient of 155 [mm.sup.-1] [cm.sup.-1] for MDA (Heath and Packer, 1968).


Cultivar Variation and Mowing Effects

L-93 exhibited better turf quality than Penncross during most of the experimental period under both mowing heights, and this difference was more pronounced in 2000 than in 1999 (Fig. 2). The decline of quality for L-93 during summer months was less dramatic than that for Penncross compared with their respective levels in May and June. Low mowing height (3 mm) significantly reduced turf quality for both cultivars during most of the experimental period from May to November (Fig. 2). The mowing effects on turf quality were more pronounced during the months of August in 1999 and July through September in 2000.


Cultivar variations were detected for CHL content (Fig. 3), EL (Fig. 4), and MDA content (Fig. 5). However, no cultivar differences were detected for the other parameters under either mowing height in 1999 or 2000. Specifically, L-93 had significantly higher CHL content (Fig. 3) than Penncross in late July and mid-August in 1999 when both mowed at the 4-mm height. There was no significant difference on CHL content between the two cultivars in 2000. L-93 had lower EL than Penncross in late July and mid-August 1999 and September 2000 under both mowing heights (Fig. 4) and had lower MDA content at the 3-mm height in July and mid-August 1999 (Fig. 5).


Low mowing height reduced CHL content of Penncross during July and August 1999 (Fig. 3). However, mowing heights had no effects on all the other parameters in either 1999 or 2000 (Fig. 4-10).


Seasonal Changes

Air temperature (daily maximum) increased from 25[degrees]C in late May to [approximately equal to] 40[degrees]C in late July, and remained at the highest level in August 1999 (Fig. 1a). Daily maximum air temperature increased from 24[degrees]C in May to the highest level of 43[degrees]C in late August 2000 (Fig. 1b). Air temperature decreased to below 24[degrees]C by the middle of October in both years.

Dramatic seasonal variation was detected for all physiological parameters including turf quality, EL, the content of CHL and MDA, and activities of SOD, CAT, POD, and APX for both cultivars under both mowing heights in both 1999 and 2000 (Fig. 2-10). The seasonal patterns of these parameters were essentially the same for the two cultivars and not affected by mowing heights. Therefore, the following section focuses on the discussion of seasonal changes in all those parameters, regardless of cultivar and mowing height.

Turf quality was the highest in June in both years, declined in July, and reached the lowest level in August in 1999 and in September in 2000 (Fig. 2). Turf quality in October and November increased back to the level of May in both years.

Chlorophyll content was highest in June 1999, decreased to the lowest in August 1999, and then recovered by October (Fig. 3). There was no significant seasonal change on CHL content in 2000. Electrolyte leakage increased from May to reach the maximum in August 1999 and September 2000, and then declined in September 1999 and October 2000 (Fig. 4). Protein content decreased from May to the lowest level in September in 1999, and then increased to the same level of May (Fig. 5). There was no significant seasonal change on leaf protein content in 2000.

The MDA content in leaves increased significantly from May to the maximal level in July and August 1999 and July through September 2000 (Fig. 6). The MDA content returned to the level of May by October in both years.

The SOD activity increased from May to early July, decreased rapidly to the lowest level in late July and August, and then increased in October 1999. In 2000, SOD activity increased from May to mid-July and declined to the lowest level in mid-August, and then recovered in September (Fig. 7).

The CAT activity also increased from May to early July and decreased rapidly to the lowest level in August, but recovered slightly in the fall in 1999 (Fig. 8). In 2000, CAT activity remained unchanged from May to mid-August and decreased to the lowest level in September. The activity of CAT did not recover in October 2000.

The POD activity followed the same seasonal pattern as SOD activity in 1999 (Fig. 9). In 2000, the activity slowly increased from May to July, decreased to the lowest level in August and September, and then increased dramatically from September to October.

The seasonal changes in APX activity were similar to that of POD in both years (Fig. 10). The only difference was that the highest level of APX activity occurred in July and lasted until August while POD activity already decreased by August in 2000.


Decline in turf quality during summer months was observed for both cultivars under both mowing heights in both years. Leaf protein content and CHL content declined to the minimal level during 1999 summer months and then resumed in the fall; however, leaf protein and CHL content did not decline during the summer of 2000. Electrolyte leakage and MDA content, however, exhibited significant increases during the same time period. An increase in EL suggests membrane injury has occurred (Blum and Ebercon, 1981). Furthermore, the extent of lipid peroxidation has been used to assess the level of free radical damage to cell membranes under stress conditions (Scandalios, 1993). Since MDA is the final product of peroxidation of unsaturated fatty acids, this assay has often been used as an indicator of the level of lipid peroxidation (Scandalios, 1993). Taken together, the increase seen in EL and MDA content during the summer months indicates that cell membranes were injured when temperature increased to supraoptimal levels. These results indicated that turf quality decline and leaf senescence during the summer was associated with the damage of cell membranes due to lipid peroxidation.

The activities of SOD, POD, and APX in both years and CAT in 1999 showed a marked peak in early summer (May to early July) as temperature increased from the optimal to the moderately high level, but then declined in August and September when the temperature was reached the highest level. Enzyme levels again increased as temperature decreased in fall. The data indicate that the ability of the enzymatic detoxification system to scavenge active oxygen species was stimulated in response to increasing air temperatures at the beginning of the summer, but decreased when temperature was too high. The level of enzyme activities in 1999 was twice as high as in 2000, even though temperature levels did not change as much over the 2 yr. Antioxidant enzyme activities are affected by other factors, including nutrient and soil moisture status, and maturity or age of plants (Zhang and Kirkham, 1996; Zhang and Schmidt, 2000). The dramatic fluctuations in enzyme activities between growing seasons could be affected by those factors besides temperature. Plants received 33% more N fertilizer in 1999 than in 2000.

Liu and Huang (2000) and Huang et al. (2001) also found the increase in SOD activity during the first 7 d of heat stress and dramatic decline in SOD activity after 14 to 21 d of heat stress under controlled environmental conditions. Transient increases in antioxidant activities with increasing temperatures have been reported in other species (Guo et al., 1998; Jiang and Huang, 2001). The transient increases in the scavenging ability may reflect the enhanced production of active oxygen species in response to increasing temperatures and also indicate heat acclimation of the antioxidant enzymes (Scandalios, 1993). Heat acclimation of the SOD system is most likely due to expression of heat tolerant isozymes. However, the antioxidant ability was impaired as temperature further increased to higher levels, which could cause overaccumulation of active oxygen species and severe oxidative damage.

Seasonal antioxidant variations have been observed in the leaves of both perennial species (Polle and Rennenberg, 1994; Polle et al., 1996) and of annual plants (Gillham and Dodge, 1987). Most of these studies suggested that environmental factors, such as light, temperature, or even airborne photooxidants to be the forces causing seasonal antioxidants fluctuations. Kroniger et al. (1992) suggested that the seasonal variation in antioxidants to be developmentally controlled. In our study, antioxidant enzyme activities decreased as air temperature increased, but increased when temperature dropped, indicating that temperature was involved in the changes in the antioxidant system for creeping bentgrass.

Creeping bentgrass cultivars L-93 and Penncross exhibited a similar pattern of seasonal changes in activities of SOD, CAT, POD, and APX. Liu and Huang (2000) and Huang et al. (2001) found that L-93 had higher activities of SOD and CAT, but a lower activity of POD than Penncross under controlled environmental conditions. Higher antioxidant activity has been reported in heat-tolerant than in sensitive cultivars under the heat stress condition in other species (Kraus et al., 1995).

The overall seasonal pattern and the magnitude of antioxidant activities and lipid peroxidation were not affected by mowing heights, although low mowing heights reduced turf quality during the summer. Turf quality reduction at low mowing height for creeping bentgrass has been reported by others (Carrow, 1996; Beard, 1997; Liu and Huang, 2001), but no research has been documented on mowing effect on antioxidant activities. Low mowing also had minimal effects on cell membrane leakage and protein content of leaves in both cultivars. The lack of significant effects of mowing heights on those cellular parameters and antioxidant activities implies that pronounced summer bentgrass decline under low mowing conditions was not related to the cellular damage and the antioxidant system. The adverse effects of low mowing on turf performance could be mainly due to the removal of large amount of leaves that otherwise are available for photosynthesis (Carrow, 1996). Previous studies suggested that low mowing caused the reduction in photosynthetic capacity, increase in respiration rate, and reduction of carbohydrate availability and allocation to roots, which contributed to summer bentgrass decline (Liu and Huang, 2001; Xu and Huang, 2003).

In summary, summer bentgrass decline, as manifested by decline in turf quality and loss of CHL and protein and increases in EL, was related to oxidative stress, regardless of cultivar and mowing height. Superoxide dismutase, CAT, POD, and APX had the capability to scavenge active oxygen species in the early summer when temperature was at the moderate level. Those antioxidant enzymes lost active oxygen scavenging ability in midsummer when temperatures were extremely high. Our previous research reported that exogenous application of cytokinins delayed the oxidative damage for creeping bentgrass exposed to heat stress and improved heat tolerance (Liu and Huang, 2002). Zhang and Schmidt (2000) reported that application of hormone-containing compounds enhanced antioxidant enzyme activities, leading to better drought tolerance of creeping bentgrass. Therefore, cultural practices or genetic modifications that promote antioxidant activity or prevent its decline could improve plant tolerance to heat stress and alleviate summer decline of creeping bentgrass.

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; CHL, chlorophyll; EDTA, ethylenediaminetetraacetic acid; EL, electrolyte leakage; MDA, malondialdehyde; NBT, nitroblue tetrazolium; POD, hydrogen peroxidase; SOD, superoxide dismutase.


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Qingzhang Xu and Bingru Huang *

Dep. of Plant Biology and Pathology, Rutgers Univ., New Brunswick, NJ 08901. Received 28 Oct. 2002. * Corresponding author (
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Title Annotation:Turfgrass Science
Author:Xu, Qingzhang; Huang, Bingru
Publication:Crop Science
Date:Mar 1, 2004
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