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Dep. Crop and Soil Environ. Sci., Virginia Polytechnic Institute and State Univ., Blacksburg, VA 24061-0404
* Corresponding author (xuzhang{at}vt.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: APX, ascorbate peroxidase • BCA, bicinchoninic acid • CAT, catalase • d, day • DTPA, diethylenetriaminepentaacetic acid • EL, electrolyte leakage • EU, enzyme unit • PAR, photosynthetically active radiation • PEc, canopy photochemical efficiency • ROS, reactive oxygen species • SOD, superoxide dismutase • TNC, total nonstructural carbohydrate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plants possess various mechanisms to survive environmental stresses. Damage due to freezing temperatures results from the formation of ice crystals causing rupture of cellular membranes. Dehydration is a major contributor to freeze damage. Injury during exposure to chilling temperatures also results from reduced or defective metabolic defense functions (Dionne et al., 2001a; Karpinski et al., 2002; Thomashow, 1999).
It has been well documented that increased cold acclimation could improve freeze tolerance of plants, including turfgrasses (Anderson et al., 1993). Plants possess various adaptive mechanisms for surviving freezing temperatures, such as increases in certain sugars or amino acids, synthesis of novel proteins, and the degree of unsaturation of membrane lipid fatty acids and antioxidant capacity (Karpinski et al., 2002; Munshaw et al., 2006).
Carbohydrate reserve levels have been considered to be one of the major factors for bermudagrass winter survival (Beard, 1973). Starch and sucrose are the primary storage carbohydrates in crowns and stolons of bermudagrass. Soluble sugars such as sucrose also function as cryoprotectants. Increases in nonstructural carbohydrates during cold acclimation have been reported in bermudagrass and other warm-season turfgrass species (Ball et al., 2002; Fry et al., 1993; Fry and Huang, 2004; Rogers et al., 1975). Shahba et al. (2003) noted that water soluble sugars such as fructose and glucose were correlated with freezing tolerance of saltgrass [Distichlis spicata (L.) Greene]. Several other studies have indicated no correlation between freezing tolerance and nonstructural carbohydrate content in bermudagrass (Dunn and Nelson, 1974) and other warm-season species (Bush et al., 2000; Fry et al., 1991; Maier et al., 1994). It appears that the role of carbohydrates in freezing tolerance is still not well defined. Zhang et al. (2004) noted that N-rich compounds such as proline may be more closely associated with bermudagrass cold tolerance compared to carbohydrates.
Plants possess various N-rich defensive metabolites and enzymes to cope with low temperature stress. Increase in some proteins in ‘Midiron’ bermudagrass during cold acclimation was correlated with its superior freezing tolerance compared to ‘Tifway’ (Gatschet et al., 1996). Bermudagrass cultivars with greater stolon proline content exhibited greater freezing tolerance than those with less proline during the winter (Munshaw et al., 2006; Zhang et al., 2004).
Rapid increases in light intensity and/or chilling may create an imbalance, so that the energy absorbed through the light harvesting complex exceeds what can be dissipated or transduced by photosystem II (PSII) (Karpinski et al., 2002). Excess energy may be directed to O2 and result in accumulation of toxic reactive oxygen species (ROS). To protect from oxidative stress, plants have evolved efficient antioxidant defense systems to scavenge ROS such as superoxide radicals (O2–·), hydrogen peroxide (H2O2), and hydroxyl radicals (HO) (McKersie and Bowley, 1997). Superoxide dismutases (SOD) are metalloenzymes which convert O2–· to H2O2, and are considered as the "primary defense" against ROS (Perl-Treves and Perl, 2002). Hydrogen peroxide is further reduced to water by APX. Catalase, localized in peroxisomes, scavenges H2O2 produced by glycolate oxidase in the C2 photorespiratory cycle (Perl-Treves and Perl, 2002). Antioxidant enzymes have been shown to condition tolerance to low temperature stress. Overexpression of a chloroplast Cu/Zn SOD gene increased resistance to chilling stress in tobacco (Gupta et al., 1993).
The electrolyte leakage technique is commonly used to assess the level of cell injury caused by low temperatures and to test the relative freezing tolerance of turfgrasses (Anderson et al., 1988, 2002; Cardona et al., 1997; Fry et al., 1991; Miller and Dickens, 1996; Shashikumar and Nus, 1993). The concept of LT50 has been used as a measure of cold hardiness and is defined as the predicted test temperature resulting in 
50% loss of total electrolyte (Shashikumar and Nus, 1993). When interpreting these results, it has been assumed that an EL of 50% or more is lethal (Fry et al., 1993). Predicted EL killing temperature (LT50) and glasshouse regrowth evaluation were in close agreement when bermudagrass was tested (Anderson et al., 1988; Miller and Dickens, 1996). Maier et al. (1994) indicated that a significant positive correlation (r = 0.81) existed between EL-predicted and regrowth lethal temperature for ‘Raleigh’ St. Augustinegrass [Stenotaphrum secundatun (Walt.) Kuntze.]. Miller and Dickens (1996) found that predicted killing temperature from EL tests and glasshouse regrowth evaluation were in close agreement for ‘Tifdwarf’ and ‘Tifway’ bermudagrass cultivars.
Great variation exists between bermudagrass cultivars in freezing tolerance (Anderson et al., 2003; Taliaferro et al., 2004). Lowest killing temperatures reported for bermudagrasses range from –17.1 to –4°C (Fry and Huang, 2004). Enhancement of cultivar freezing tolerance as a means of reducing risk of winterkill has been a major goal of many bermudagrass improvement programs (Anderson et al., 2003). Riviera, licensed for commercial production in 2001, was a top performer among all varieties in the 1997 National Turfgrass Evaluation Program (NTEP) bermudagrass test. Excellent cold tolerance and high turf quality ratings of Riviera were reported across environments. Plantings of Riviera as far north as Warrensburg, MO, and Evansville, IN, have had minimal or no winter injury, greened up early, and produced high quality turf (Taliaferro et al., 2004). Princess-77 is a widely used cultivar with highly rated visual quality but relatively poor cold tolerance. However, there are few reports on the physiological mechanisms of cultivar variation in freezing tolerance. Investigations concerning the physiological bases of cultivar differences in freezing tolerance would provide valuable selection information for turfgrass breeders and practitioners, especially in the transition zone. The objectives of this study were to examine changes in the levels of carbohydrates, proline, proteins, and antioxidant enzymes during cold acclimation and to investigate relationships of C- and N- rich defense compounds with bermudagrass freezing tolerance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three months after transplanting, four plugs from each cultivar were transferred to a growth chamber and subjected to cold acclimation at 8/4°C (day/night) with a photosynthetically active radiation (PAR) intensity of 200 µmol m–2 s–1 over a 10-h photoperiod for a period of 21 d. The remaining plugs (four from each cultivar) were grown in a temperature-controlled glasshouse at 25/23°C (day/night) with natural sunlight (average 
350 µmol m–2 s–1 PAR; 11-h photoperiod). The grass was mowed to 1.25 cm weekly until acclimation was initiated. The grass plugs were watered once a week but not fertilized during the acclimation period.
Measurements
At 0, 7, 14, and 21 d after acclimation initiation, canopy photochemical efficiency (PEc) was measured according to Zhang et al. (2005). Leaf samples were collected for analysis of protein and antioxidant enzymes (SOD, CAT, and APX), and stolon samples were collected for analysis of carbohydrates and proline content from both acclimated and nonacclimated plants. The samples for proline and protein analysis were immediately frozen with liquid N, and stored at –80°C. The stolon samples for carbohydrate analysis were dried at 100°C for 1 h, at 60°C for another 24 h, and then ground to pass a 1-mm mesh screen. The ground samples were then stored at –80°C.
At the end of acclimation, the soil was washed from the plants and the roots were removed. The turf plugs from acclimated and nonacclimated treatments were then divided into six subsamples. Each subsample was wrapped in a wet paper towel and subjected to a series of freezing temperatures. A freezing chamber was programmed to cool to –2, –4, –6, –8, and –10°C with a 2°C decline every 2 h at which time a subsample was removed for analyses. After thawing overnight at 4°C, stolons from each subsample were used for EL measurement as described later in this section.
Carbohydrates
Nonstructural carbohydrates including glucose, fructose, sucrose, and starch in stolons were extracted and analyzed according to the procedures of Hendrix (1993) with modifications. Sugars were extracted from ground samples (20 mg) in 2 mL of 80% ethanol in an 80°C water bath for 15 min, and then centrifuged at 3000 g for 10 min. The supernatant was collected. The residue was re-extracted and the supernatant was collected. The supernatants from two extractions were pooled and brought up to 5 mL with 80% ethanol. To 1.5 mL supernatant, 20 mg active charcoal was added. The extract was shaken, centrifuged at 2200 g for 15 min, and the supernatant was stored at –80°C for later analysis.
To the residue after sugar extraction, 1.0 mL 0.1 M KOH was added. The sample in the tube was heated in boiling water for 60 min and cooled. To each tube, 0.2 mL of 1 M acetic acid was added followed by 100 µL 1.0 M Tris buffer (pH 7.2). After adding 200 µL alpha-amylase, the tube was kept in an 85°C waterbath for 30 min. After reducing pH to less than 5 with 1.0 M acetic acid, 1.0 mL amyloglucosidase was added and then the samples kept in a 55°C waterbath for 60 min. Enzyme action was stopped by heating at 100°C for 4 min, with the extract brought up to 6 mL with distilled H2O, mixed, and centrifuged at 3000 gn for 10 min. The extract was stored at –80°C until analysis for the sugars.
For the sugar analysis, an extract (20 µL) was transferred to a microtitration plate and dried at 50°C for 1.5 h. After drying, 20 µL deionized-distilled H2O was added to each well and the plate was covered for 1 h. A series of standard solutions (a mixture of glucose, fructose, and sucrose; 0, 0.005, 0.025, 0.05, 0.125, 0.25 g L–1) were added to separate wells for standard curve development. To each well, 100 µL glucose reagent solution (Sigma GAHK-20, St. Louis, MO) was added and the plate was kept at room temperature (18–25°C) for 30 min. The glucose was measured on a microplate reader (SpectroMax plus 386, Molecular Devices Corp., Sunnyvale, CA) at 340 nm (under reduced light). Next, 10 µL of 0.25 enzyme units (EU) phosphoglucose isomerase was added to each well and incubated at room temperature for 30 min. After measurement of reducing sugars (glucose + fructose) at 340 nm, 10 µL of 83 EU invertase solution was added and incubated for 30 min before measurement at 340 nm for total sugars (glucose + fructose + sucrose).
The starch in the residue following sugar extraction was degraded into glucose under action of alpha-amylase and amyloglucosidase enzymes according to the procedure of Hendrix (1993). Glucose was measured as described previously and its content calculated based on the standard curve. Starch content was determined based on the glucose content.
Leaf Protein and Stolon Proline Contents
Leaf protein concentration was analyzed by the bicinchoninic acid (BCA) method, with bovine serum albumin serving as the standard (Smith, 1985). Proline content of the stolon tissues was estimated according to the colorimetric procedure of Bates (1973).
Leaf Antioxidant Enzyme Activity
Antioxidant enzymes in leaf tissue (100 mg fresh weight) were extracted according to Zhang et al. (2005). Superoxide dismutase activity was determined according to the method of Banowetz et al. (2004). Briefly, to each well, 125 µL reaction solution containing 50 mM Pipes buffers (pH 7.5), 0.4 mM O-dianisidine, 0.5 mM diethylenetriaminepentaacetic acid (DTPA), and 26 µM riboflavin, and then 20 µL enzyme extract were added. The absorbance at 560 nm of solution mixture was read immediately after addition of enzyme extract on a microplate reader (Opsys MR; Thermo Labsystems, Chantilly, VA). The reaction was initiated by switching on the light and slowly rotating sample tubes under the light for 30 min under one circular fluorescent lamp (irradiance = 60 µmol m–2 s–1) at room temperature (25°C). Absorbance at 560 nm was measured again. The SOD activity was calculated based on changes in absorbance and from the standard curve. Catalase and APX activity were analyzed according to Zhang et al. (2005).
Electrolyte Leakage
Stolons were collected from each subsample (0.15 g) after freezing treatment, rinsed with distilled water, and transferred into a 50-mL centrifuge tube and deionized distilled water (30 mL) was added. The samples were placed in a shaker for 12 h at 25°C. After an initial electrical conductivity (EC1) reading using a conductivity meter, the samples were autoclaved at 120°C for 30 min. After cooling, a second EC (EC2) reading was taken. The EL (%) is expressed as (EC1/EC2) x 100.
Determination of LT50
Freezing tolerance was evaluated based on predicted killing temperature from EL measurements in this study. Predicted LT50 and glasshouse regrowth evaluation after freezing treatment of bermudagrasses have been shown to be in close agreement (Anderson et al., 1988; Miller and Dickens, 1996). The response curve between test temperature and EL was fitted to a sigmoidal response using Sigmaplot (Systat Software Inc., 2000). Response curves were developed for each treatment and each LT50 was determined from the inflection point of the curve (Tmid) based on the sigmoidal response equation (Anderson et al., 1988; Cardona et al., 1997).
Experimental Design and Data Analysis
A randomized complete block design was used with four replications. The data were analyzed using ANOVA and mean separations were performed using a Fisher's Protected LSD test (SAS Institute Inc., 2001). Since the sample sizes for the freezing tests were small, no attempt was made to examine growth recovery from nodes after freezing.
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RESULTS |
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Total Sugar (Glucose + Fructose + Sucrose) Content
Total sugar content was increased 7 d after acclimation initiation in Princess-77, but not Riviera, when compared to the nonacclimated (Table 1). At the end of acclimation, total sugars were increased by 1.8-fold in Riviera and 1.2-fold in Princess-77, respectively, when compared to the levels at initiation. Total sugar content of nonacclimated stolons did not change over time for either cultivar.
Starch Content
Starch content was similar in both cultivars when measured at the beginning and 7 d after cold acclimation (Table 1). Cold acclimation significantly increased starch accumulation in Riviera at 21 d, but not in Princess-77. Further, starch content was greater in Riviera than Princess-77 when measured at 14 and 21 d after acclimation initiation.
Total Nonstructural Carbohydrates
Increased TNC content of 85% in Riviera due to acclimation was measured at 21 d after acclimation initiation, but no TNC increase was measured for Princess-77 (Table 1). At the end of cold acclimation, Riviera had a TNC content 86% greater than Princess-77.
Nitrogen-rich Defense Compounds
Proline Content
Stolon tissue proline content increased in response to cold acclimation in both cultivars (Table 2). When measured at the end of cold acclimation, proline content was increased by 120% in Riviera and 59% in Princess-77 relative to the nonacclimated tissues. Riviera had greater proline accumulation than Princess-77 during cold acclimation. When measured at 7 d after acclimation initiation, proline content in Riviera was 33% greater than that of Princess-77. At the end of acclimation, proline content in Riviera was 43% greater than that of Princess-77.
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Antioxidant Enzyme Activities
The activity of SOD increased in the first 7 d and then declined in both cultivars (Table 2). Following 14 and 21 d of acclimation, Riviera SOD activity was greater than that of Princess-77.
Catalase activity was decreased in response to cold acclimation in both cultivars (Table 2). At the end of acclimation, CAT activity was reduced by 66% in Riviera and 76% in Princess-77.
Ascorbate peroxidase activity declined in response to cold acclimation, especially in Princess-77. At the end of cold acclimation, APX activity was reduced by 45% in Riviera and 81% in Princess-77. After acclimation, Riviera had an APX activity 1.7-fold greater than Princess-77.
Photochemical Efficiency
From 7 to 14 d after acclimation initiation PEc declined quickly (Fig. 1
). Cold acclimated cultivars had lower PEc than nonacclimated at 14 and 21 d. Cold acclimation resulted in equivalent cultivar decreases in PEc at Days 14 and 21.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been suggested that greater starch and TNC accumulation during cold acclimation for ‘Meyer’ zoysiagrass [Zoysia japonica (Steud) Meyer] relative to bermudagrass may partially account for its improved winter survival (DiPaola and Beard, 1992). Our results support this suggestion at the intraspecific level as cold-tolerant Riviera's starch and TNC levels were almost double those of cold-sensitive Princess-77 following 21 d of acclimation (Table 1). Cold acclimation also resulted in a significant increase of soluble carbohydrate contents in both cultivars. However, greatest increases were noted in Riviera where accumulation of glucose and sucrose were significantly correlated with increased freezing tolerance. Although Dunn and Nelson (1974) found that sucrose levels rose in stolons of three bermudagrass cultivars during fall acclimation, differences among cultivars were slight and not related to increased winter survival. Our data indicate almost double the sucrose accumulation in stolons in Riviera relative to Princess-77 during cold acclimation. Greater stolon sucrose levels have also been associated with improved freezing tolerance in centipedegrass [Eremochloa ophiuroides (Munro) Hackel] (Fry et al., 1993), buffalograss [Buchloe dactyloides (Nutt.) Engelm.] (Ball et al., 2002), and zoysiagrass (Rogers et al., 1975).
Karpinski et al. (2002) noted that a major plant response to low temperature is synthesis of metabolites that function as osmolytes or compatible solutes that increase osmotic potential, decreasing the cellular freezing point, and enhancing freezing tolerance. Osmolytes such as soluble carbohydrates and proline are also thought to interact with membrane phospholipids and proteins to stabilize their structures as water is removed during freezing (Koster and Leopold, 1988). Stolon proline levels increased in response to cold acclimation in both cultivars, but accumulated to a greater extent in Riviera (Table 2). Munshaw et al. (2006) similarly reported that greater stolon proline levels were associated with improved freezing tolerance in Midiron and Riviera bermudagrass cultivars relative to Princess-77. Proline also functions as an antioxidant and is involved in reducing photodamage in the thylakoid membrane by scavenging and/or reducing the production of singlet oxygen (Alia and Mohanty, 1997). Proline stabilization of proteins during cold acclimation may have been related to our measured increases in soluble leaf protein at the end of cold acclimation for both cultivars.
Our data indicate that leaf protein content increased to a greater extent following acclimation in cold-tolerant Riviera relative to cold-sensitive Princess-77. This is consistent with previous results by Zhang et al. (2004). Gatschet et al. (1996) reported that increased protein synthesis in Midiron bermudagrass crowns was correlated with its greater freezing tolerance as compared to Tifway. Plants may increase their capacity for protein synthesis in during cold acclimation (Cloutier, 1983). Although the role of protein alteration during cold acclimation is unclear, it has been demonstrated that protein accumulation plays a determinant role in freezing tolerance of certain cool- and warm-season species (Dionne et al., 2001b; Gatschet et al., 1996; Hughes and Dunn, 1996).
Chilling temperatures increase oxidative stress and have been shown to stimulate synthesis of antioxidant metabolites and enhance antioxidant enzyme activities (Karpinski et al., 2002). Tolerant or acclimated plants have been shown to have increased activities of SOD, CAT, and APX that can delay photooxidative damage during chilling stress (Karpinski et al., 2002). One result may be a greater capacity to maintain active photosynthesis at low temperatures and develop larger reserves of carbohydrate and N-rich compounds with protective functions (proline and proteins). Since the photosynthetic system, rich in fatty acids, is sensitive to oxidative damage, and antioxidant enzymes such as SOD are located in leaf tissues, changes of antioxidant enzymes and soluble proteins in the leaves were examined in this study. Acclimating Riviera increased SOD activity to a greater extent and maintained greater SOD and APX activities relative to Princess-77 (Table 2). Increased Riviera antioxidant activity may have been related to a slower rate of PEc decline relative to Princess-77 as acclimation proceeded (Fig. 1).
The possibility that less cold-hardy cultivars do not increase N-rich metabolites (proline, proteins, and antioxidants) during acclimation as effectively as cold-hardy cultivars may partially explain the inconsistency of results from several past studies which focused on nonstructural carbohydrates only (Bush et al., 2000; Dunn and Nelson, 1974; Fry et al., 1993; Gatschet et al., 1996; Shahba et al., 2003). Zhang et al. (2004) found that Riviera accumulated N-rich metabolites (proline and protein) in leaves more effectively than Princess-77 during field-based cold acclimation. Dionne et al. (2001b) indicated that increases in amino acids, specific soluble polypeptides, and proteins of crowns in response to subzero temperature are associated with greater freezing tolerance of annual bluegrass (Poa annua L.). Similar results have been reported in other plant species (McKenzie et al., 1988; Perras and Sarhan, 1989).
The results of this study suggest that both nonstructural carbohydrates and N-rich compounds play important roles in freezing tolerance of bermudagrass. This alteration of defensive metabolism during cold acclimation may condition bermudagrass to better survive freezing temperatures during a harsh winter. Selection of bermudagrass cultivars that have the ability to rapidly accumulate defensive compounds (nonstructural carbohydrates and N-rich metabolites) during cold acclimation may be an important approach for increasing winter survival.
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ACKNOWLEDGMENTS |
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Received for publication February 17, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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