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a Dep. of Plant Biology and Pathology, Rutgers Univ., New Brunswick, NJ 08901
b College of Agricultural and Biological Sci., Shanghai Jiao Tong Univ., Shanghai 201101, China
* Corresponding author (huang{at}aesop.rutgers.edu).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: CAT, catalase • EL, electrolyte leakage • Fv/Fm, photochemical efficiency • MDA, malondialdehyde • NBT, nitro blue tetrazolium • SOD, superoxide dismutase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Much is known about the adverse impact of drought or heat stress on plant growth and physiological activities, but much less about the recovery of physiological functions following stress relief. For perennial turfgrasses, the most important strategy is not the maintenance of growth or production during stress, but the ability to survive and recover rapidly from the combined summer stress after rainfall or irrigation or after a temperature drop. Previous research in turfgrass recovery has mainly focused on the relief of drought stress. Several studies reported that turfgrass species and cultivars vary in their recuperative ability from drought stress alone (Hook et al., 1992; Qian and Fry, 1997; Huang et al., 1998). After rewatering following drought stress, leaf water status and turf quality of drought-tolerant tall fescue cultivars returned to the prestress levels, but Fv/Fm did not recover completely, indicating permanent damage of the photosynthetic apparatus in existing leaf tissues (Huang et al., 1998). White et al. (1992) reported that recovery of tall fescue from drought stress was associated with low basal osmotic potential before stress, prolonged positive turgor maintenance, and delayed leaf rolling during stress. Qian and Fry (1997) also reported that osmotic adjustment contributed to plant regrowth from drought stress following rewatering in warm-season turfgrass species.
While physiological recovery from drought stress alone has been addressed previously in several turfgrass species, recuperative capacity of various physiological processes for cool-season turfgrasses from combined drought and heat stress have not been well understood. Understanding the underlying mechanisms of recovery is essential for improving the survival of cool-season grasses in dry and hot environments. Selecting traits for improved recovery may be of more economic importance than selecting for improved growth during drought (Norris and Thomas, 1982). Therefore, the objective of this study was to examine physiological factors associated with the persistence and recovery of Kentucky bluegrass exposed to combined drought and heat stress following rewatering and/or temperature drop. We compared two cultivars, Midnight and Brilliant, which were chosen based on their contrasting tolerance to drought and heat stress. Our previous studies have shown that Midnight is more tolerant to drought (Wang et al., 2003a, 2003b) or heat stress alone (2003, unpublished data) compared with Brilliant.
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MATERIALS AND METHODS |
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Treatments and Experimental Design
Plants were first grown in well-watered condition (watered every other day until drainage occurred at the bottom of pot) at optimum temperature (20/15°C) for 14 d in the growth chamber, and measurements were made as the initial control. Simultaneous drought and heat stress was then imposed by withholding irrigation and exposure of plants to 35/30°C (day/night). The treatment was repeated in four growth chambers. Plants of two cultivars were randomly placed in each growth chamber. The growth chambers had 75% relative humidity, a 14-h photoperiod, and photosynthetically active radiation of 600 µmol m–2 s–1. Volumetric soil water content in a 0- to 20-cm soil layer of each pot was monitored by time domain reflectometry (Soil Moisture Equipment Corp., Santa Barbara, CA). At the initiation of the treatment, volumetric soil moisture was at the field capacity (28%). By 14 d of combined stress, soil moisture dropped to 5% and most plants were desiccated and brown.
Plants were then exposed to the following treatments to examine for recovery following 14 d of combined drought and heat stress: (i) continually maintained unwatered and heat stressed (stress control); (ii) rewatered, but still exposed to heat stress (rewatering); (iii) unwatered, but returned to optimum temperature (20/15°C) (cooling); and (iv) rewatered and exposed to optimum temperature (rewatering and cooling). For the rewatering treatments, plants were watered every other day until water drained from the bottom of pots. Each treatment and cultivar consisted of four replicates (pots) which were randomly arranged in four growth chambers. Four growth chambers were set at 20/15°C (day/night) and four were set at 35/30°C (day/night). One replicate of Treatments i and ii was maintained in each of the four heat-stress chambers while one replicate of Treatments iii and iv was maintained at the optimum-temperature chambers.
The experiment was a completely randomized plot design. Cultivar difference during combined stress, duration of stress treatment, recovery treatment, and the interaction of cultivar with stress treatment or with recovery treatment were determined by ANOVA according to the general linear model procedure of SAS (SAS Institute, 1992). Means of stress durations, recovery treatments, and cultivars were tested with LSD at a probability level of 0.05.
Measurements
During the stress period, all measurements except turf quality were made on the top three fully expanded leaves from multiple plants in each pot. During the recovery, measurements were taken only for leaves existing before rewatering or cooling to examine the recuperative ability of stressed tissues to avoid the confounding effects of newly regenerated tissues.
Turf quality was visually rated on 1-to-9 scale (1 = brown, dead turf, 9 = green and dense) based on shoot color and density.
Cell membrane stability was estimated by measuring EL from leaf tissues. Samples of fresh leaves (0.1 g) were rinsed and immersed in 20 mL of deionized water. The conductivity of the solution (Cinitial) was measured after the leaves were shaken for 24 h using a conductivity meter (YSI Incorporated, Yellow Springs, OH). Leaves then were killed by autoclaving at 140°C for 20 min. The conductivity of killed tissues (Cmax) was measured after samples were cooled down to room temperature. Relative EL was calculated as the percentage of Cinitial over Cmax.
Leaf Fv/Fm was estimated by measuring chlorophyll fluorescence (a ratio of variable to maximum photo yield to Fv/Fm) with a fluorescence induction monitor (BioScientific Ltd., Herts, UK). Leaf chlorophyll was extracted by soaking 0.05 g of leaf sample in 20 mL of dimethyl sulfoxide in the dark for 72 h following the procedure described by (Hiscox and G.F. Israelstam, 1979). Absorbance of the extractant at 663 and 645 nm was measured with a spectrophotometer (Spectronic Instruments, Inc., Rochester, NY) and converted to chlorophyll content using the formulas described by Arnon (1949).
For the analysis of antioxidant enzymes, 0.5 g fresh leaves were ground with a tissue grinder in 8 mL of 50 mM ice-cold phosphate buffer (pH 7.0) containing 1% (w/v) polyvinylpyrrolidone and 0.2 g of white quartz sand, which were placed in a ice bath. The homogenate was centrifuged at 15000 x g for 20 min at 4°C. The supernatant was used for assay of enzyme activity and the level of lipid peroxidation.
The activity of SOD was determined by measuring its ability to inhibit the photoreduction of nitro blue tetrazolium (NBT; 2,2'-di-p-nitrophenyl-5,5'-diphenyl-[3,3'-dimethoxy-4,4'-diphenylene] ditetrazolium chloride) following the method of Giannopolitis and Ries (1977). The reaction solution (3 mL) contained 50 µM NBT, 1.3 µM riboflavin (7,8-dimethyl-10-ribitylisoalloxazine), 13 mM methionine [2-amino-4-(methylthio)-butyric acid], 75 nM EDTA (ethylenediaminetetraacetic acid), 50 mM phosphate buffer (pH 7.8), and 20 µL of enzyme extract, with nonenzyme solution as control. Test tubes containing the reaction solution were irradiated under a set of six fluorescent light tubes at 78 µmol m–2 s–1 for 15 min. The absorbance of the irradiated and nonirradiated solution at 560 nm was determined with a spectrophotometer (Spectronic Instruments, Inc., Rochester, NY). One unit of SOD activity was defined as the amount of enzyme that would inhibit 50% of NBT photoreduction.
Catalase activity was measured using the method of Chance and Maehly (1955). The reaction solution (3 mL) contained 50 mM phosphate buffer (pH 7.0), 15 mM H2O2, and 0.1 mL of enzyme extract. Reaction was initiated by adding the enzyme extract. Because of the linear decline of absorbance at 240 nm within the first 3 min, changes of the absorbance were read every minute. One-unit CAT activity was defined as the absorbance change of 0.01 units per minute. Both SOD and CAT activities were expressed on a protein basis. Protein content was determined using the method described by Bradford (1976).
Lipid peroxidation is the symptom most easily ascribed to oxidative damage and often used as an indicator of oxidative stress (Zhang and Kirkham, 1994). The content of malondialdehyde (MDA), a final product of lipid peroxidation, was determined using the method described by Dhindsa et al. (1981). A 0.5-mL aliquot of extract was added to a tube containing 1 mL of 20% (v/v) trichloroacetic acid and 0.5% (v/v) thiobarbituric acid. The mixture was heated in a water bath at 95°C for 30 min. After cooled to room temperature and centrifuged at 10000 x g for 10 min, the supernatant was read for absorbance at 532 and 600 nm. The absorbance for nonspecific absorption at 600 nm was subtracted from the value at 532 nm. The amount of MDA (red pigment) was calculated using the adjusted absorbance and the extinction coefficient 155 mM–1 cm–1 (Heath and Packer, 1968).
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RESULTS |
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Superoxide dismutase activity declined across time with combined stress for both cultivars (Fig. 2A). Superoxide dismutase activity dropped to below the prestress level at 4 d for Brilliant and at 6 d for Midnight. Midnight had higher SOD activity than Brilliant at 4, 6, and 8 d of stress. Superoxide dismutase activity declined to only 12% of the prestress level for both cultivars at 14 d of treatment. Catalase activity decreased to below the initial level at 2 d of combined stress for Brilliant and 6 d for Midnight (Fig. 2B). Midnight had a higher CAT activity than Brilliant at 4 and 8 d of stress. Catalase activity dropped to 23 and 6% of the prestress level for Midnight and Brilliant, respectively, by 14 d of treatment. The MDA content increased with the duration of the stress (Fig. 2C). Significant increases in MDA content above the initial level were observed at 6 d for Brilliant and at 8 d for Midnight. Brilliant had a higher MDA content than Midnight after 4 d of treatment.
Recovery following Rewatering and Cooling
Turf quality for Midnight resumed, but did not reach the prestress levels following 12 d of rewatering alone or with cooling (Fig. 3A) . No recovery in turf quality for Midnight occurred when plants remained unirrigated at optimum temperature. Turf quality for Brilliant recovered partially with rewatering and cooling, but did not change with cooling or rewatering alone (Fig. 3B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rapid recovery from stress is an important aspect of the persistence of perennial grasses. Recovery following rewatering alone or together with cooling occurred for all physiological parameters measured in Midnight previously exposed to the combined stress, suggesting that this drought-tolerant Kentucky bluegrass cultivar had high environmental plasticity. However, both rewatering and cooling were required for the recovery of cell membrane stability and Fv/Fm for Brilliant, and the recovery in those parameters were less pronounced for Brilliant than for Midnight. Chlorophyll content, SOD and CAT activities, and lipid peroxidation did not change for Brilliant with rewatering alone or with cooling. The variation in the recuperative ability between the two cultivars could be because of the difference in the severity of stress damage, as Brilliant generally maintained lower physiological activities than Midnight during the combined drought and heat stress. Qian and Fry (1997) reported that recovery from drought stress alone was positively correlated with the level of drought tolerance (in terms of osmotic adjustment) in several turfgrass species. A drought-tolerant tall fescue cultivar recovered faster and more dramatically from drought stress following rewatering compared with a sensitive cultivar (Huang et al., 1998).
Returning plants to the optimum temperature while remaining unirrigated had no effects on the recovery of any of the parameters for either cultivar. However, rewatering only resulted in rapid restoration of physiological functions, to the same extent as rewatering in combination with cooling. These results indicate that irrigation is essential in order for Kentucky bluegrass to fully recover from the combined drought and heat stress, regardless of temperature, suggesting that water stress is the major factor controlling plant survival in dry and hot environments. Lack of water causes stomatal closure and reduces transpirational cooling under heat stress, which is found to be the major cause of plant mortality during summer for tree seedlings (Kolb and Robberecht, 1996).
Cell membrane stability and Fv/Fm resumed fully to the prestress level by 12 d of rewatering alone or together with cooling for Midnight, but chlorophyll content recovered only partially for Midnight, and never recovered for Brilliant. Similar to chlorophyll content, turf quality, which was rated partially on the basis of leaf greenness, also recovered to a lesser extent than Fv/Fm. These results suggest that combined drought and heat stress could have caused more severe damage to chlorophyll due to either enhancement of degradation or inhibition of synthesis than to cell membrane and Fv/Fm. Furthermore, the results suggest that resumption of cell membrane integrity and Fv/Fm could be more important for rapid regrowth or recovery in turf quality than resynthesis of chlorophyll upon rewatering. Sanchez et al. (1983) reported that recovery of photosynthesis preceded that of chlorophyll synthesis in response to rewatering for maize (Zea mays L.). Rapid recovery in Fv/Fm suggests that the functional forms of photosystem II for Kentucky bluegrass were restored quickly upon relief of drought or combined stress. Zhang et al. (2003) reported that Kentucky bluegrass with higher Fv/Fm during heat stress regrows faster after being transplanted to low temperature conditions. However, Huang et al. (1998) found that 21 d of soil drying caused long-term negative effects on Fv/Fm for tall fescue, which never returned to the prestress level following rewatering.
Superoxide dismutase and CAT activities also increased back to the prestress level for Midnight by rewatering alone or together with cooling, which could lead to the rapid decline in MDA content, indicating that antioxidant enzymes or recovery from the oxidative damage was involved in the rapid repair or recovery from the combined stress for drought-tolerant plants. However, drought and heat stress caused irreversible oxidative damage for Brilliant, which could be a factor, at least in part, for the partial recovery of physiological activities for the drought-sensitive cultivar. No previous data are available on oxidative recovery in response to rewatering from drought stress or cooling from heat stress or in combination for perennial grass species. In other species such as pea (Pisum sativum L.) the recovery in shoot growth from drought following rewatering was accompanied by the increase in SOD content and activity (Mittler and Zilinskas, 1994). Chilling-induced oxidative stress is associated with irreversible damage in maize (Prasad et al., 1994). The increase in SOD activity and the decrease in MDA content contribute to the recovery of pea plants from short-term salinity stress (Hernandez and Almansa, 2002).
Taken together, the results suggest that simultaneous drought and heat stress was detrimental for Kentucky bluegrass, particularly the drought-susceptible cultivar. In order for plants to fully recover from the combined drought stress, rewatering or rewatering in association with temperature drop was essential. Rewatering was more important than temperature decline for Kentucky bluegrass to recover from the combined stress. Therefore, timely irrigation is important for rapid recovery of Kentucky bluegrass following summer stress. Rapid increases in membrane stability, Fv/Fm, and antioxidant enzyme activity following rewatering or together with cooling could contribute to the recovery from simultaneous drought and heat stress. These physiological parameters could be used to select Kentucky bluegrass germplasms for the improvement of summer stress survival and recovery.
Received for publication August 7, 2003.
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This article has been cited by other articles:
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  Y. He and B. Huang Protein Changes during Heat Stress in Three Kentucky Bluegrass Cultivars Differing in Heat Tolerance Crop Sci., November 7, 2007; 47(6): 2513 - 2520. [Abstract] [Full Text] [PDF]
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K. Su, D. J. Bremer, S. J. Keeley, and J. D. Fry Effects of High Temperature and Drought on a Hybrid Bluegrass Compared with Kentucky Bluegrass and Tall Fescue Crop Sci., September 1, 2007; 47(5): 2152 - 2161. [Abstract] [Full Text] [PDF]
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