ANTIOXIDANT ENZYME RESPONSES OF ...

International Turfgrass Society Research Journal Volume 12, 2013

ANTIOXIDANT ENZYME RESPONSES OF PERENNIAL RYEGRASS ACCESSIONS DURING COLD ACCLIMATION D. Sarkar, P. C. Bhowmik*, M. DaCosta, K. Shetty and E. Watkins ABSTRACT Cold acclimation responses of two perennial ryegrass (Lolium perenne L.) accessions were investigated to determine the role of proline-associated pentose phosphate pathway (PPP) for phenolic biosynthesis and stimulation of antioxidant response systems. Perennial ryegrass accessions PI 223178 (PR-1) and PI 598433(PR-2) were subjected to a cold acclimation treatment that consisted of a constant 2 °C for 21 d. Leaves were harvested weekly for assessment of phenolic content, proline content, and antioxidant enzyme activities. After 21 d of cold acclimation, PR-2 exhibited higher leaf phenolic content, proline accumulation, and total antioxidant activity compared to PR-1. Higher glucose-6-phosphate dehydrogenase (G6PDH) and proline dehydrogenase (PDH) activities were also observed in PR-2, which suggested an increased role of proline-associated PPP in this accession during cold acclimation. Higher catalase (CAT) activity in PR-2 suggested a counter mechanism of this accession against low temperature-induced cellular peroxidation. This study indicated that fitness of perennial ryegrass accessions at low temperature may be due to a combination of many biochemical properties including, antioxidant stimulation, proline accumulation, and phenolic biosynthesis through stimulation of PPP to support these anabolic responses. INTRODUCTION Perennial ryegrass (Lolium perenne L.) is one of the most widely utilized cool-season grasses for turf and forage. Although many breeding improvements have been made in terms of agronomic and turf-type characteristics (e.g. color, density, disease resistance, traffic tolerance), perennial ryegrass is one of the most susceptible cool-season species to low temperature injury. As a result, the persistence of perennial ryegrass has been found to be limited in northern climatic regions due to winter injury (Taylor et al., 1997). Differences in freezing tolerance have been observed among cultivars or genotypes of perennial ryegrass in field and controlled environment experiments, which suggest good D. Sarkar, P.C. Bhowmik and M. DaCosta, Dept. of Plant, Soil, and Insect Sciences, Stockbridge Hall, University of Massachusetts, Amherst, MA 01003 USA; K. Shetty, Dept. of Food Sciences, Chenoweth Lab, University of Massachusetts, Amherst, MA 01003 USA; E. Watkins, Dept. of Horticultural Science, Alderman Hall, University of Minnesota, St. Paul, MN 55108 USA. *Corresponding author: ([email protected]). Keywords: CAT, catalase; G6PDH, glucose-6-phosphate dehydrogenase; GPX, guaiacol peroxidase; PLPPP, prolineassociated pentose phosphate pathway; PDH, proline dehydrogenase; SDH, succinate dehydrogenase; SOD, superoxide dismutase.

potential for improvements in perennial ryegrass winter hardiness characteristics (Ebdon et al., 2002; Hulke et al., 2008; Shinozuka et al., 2006). Based on earlier research conducted in perennial ryegrass and other plant species, the physiological and cellular changes induced by low, non-freezing temperatures (cold acclimation) play important roles in the acquisition of freezing tolerance (Steponkus, 1990). Cool-season grasses generally require temperatures between 0 to 8 °C for approximately 2 to 3 weeks to initiate cold hardening. During the cold acclimation period, plants accumulate protective compounds such as carbohydrates, amino acids, proteins, and antioxidants, which help to improve the stability of membranes and other cellular structures at low temperatures (Sakai and Larcher, 1987). Most investigations conducted in relation to perennial ryegrass freezing tolerance have focused on compatible solute accumulation, such as carbohydrates, during cold acclimation (Harrison et al., 1997). Limited research has been conducted to evaluate the contribution of the antioxidant defense system during cold acclimation of perennial ryegrass. The combined effects of cold-induced decreases in photosynthetic capacity and continued light absorption have been associated with enhanced generation of reactive oxygen species (ROS). These compounds, such as superoxide radicals, singlet oxygen, hydrogen peroxide, and hydroxyl radicals, can cause significant oxidative damage to plant cells (Paliyath and Droillard, 1992; Polle, 1997; Wise and Naylor, 1987). Plants have evolved a complex antioxidant defense system that is comprised of various hydrophilic and lipophilic metabolites, enzymes, and secondary metabolites like phenolics, which scavenge free radicals and reduce the levels of ROS in cells (Beckman and Ames, 1997; Christie et al., 1994). The regulation and activity of the antioxidant response systems has been shown to be important for plant tolerance to low temperature stress as it can counter low temperature– induced free radicals. Zhou and Zhao (2004) found a strong association between antioxidant enzyme accumulation and cold tolerance in perennial forage grasses, reporting that most antioxidant enzymes accumulated in grasses during early acclimation period of autumn. A few studies have also examined accumulation of phenolics related to low temperature stress in plants (Prasad, 1996; Rice-Evans et al., 1997). Phenolics can act as an antioxidant by scavenging free radicals and thus can counter oxidative stress, induced by freezing temperature in plants. In addition to induction of antioxidants and phenolics, amino acids also play an important role in cells under stress conditions. Proline is the most abundant amino acid in the cytosol and can act either as an osmotic regulator or as a metabolic regulator during abiotic stress response. Proline is known to accumulate in plants prior to the development of winter dormancy or during the onset of frost

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in the autumn. Draper (1975) observed 15% higher accumulation of proline in perennial ryegrass during the coldhardening process. Although significant research has been conducted on the role of proline as osmotic regulator, there have been less investigations on proline as a metabolic regulator in plant's abiotic stress tolerance. Proline is synthesized from glutamate by series of reduction reactions. In this synthesis process, proline and pyrroline-5-carboxylate (P5C) may regulate redox and hydride ion-mediated stimulation of the pentose phosphate pathway (PPP) (Hagedorn and Phang, 1983). During respiration, oxidation reactions could produce hydride ions, which may help reduction of P5C to proline in the cytosol. Within the mitochondria, proline dehydrogenase (PDH) can oxidize proline and produce flavin adenine dinucleotide (FADH2), which can be used for phosphorylation in the electron transport chain (Hare and Cress, 1997; Rayapati and Stewart, 1991). The reduction of P5C in the cytosol provides nicotinamide adenine dinucleotide phosphate (NADP+), which is a co-factor for glucose-6-phosphate dehydrogenase (G6PDH). Glucose-6-phosphate dehydrogenase (G6PDH) plays a crucial role, by catalyzing first rate limiting step of the PPP, which is essential for many anabolic needs under stress and developmental response. Phang (1985) first proposed this model and stated the role of proline-associated PPP in the stimulation of purine metabolism via ribose-5-phosphate in animal cells. On the basis of this hypothesis, Shetty (1997) proposed a model that proline-mediated PPP could stimulate both the shikimate and phenylpropanoid pathways in plants, and that the modulation of this pathway could lead to the stimulation of phenolic phytochemicals relevant for stress response (UV-B stress, temperature stress, and biotic stress). The role of the PPP and its key enzyme G6PDH during cold hardening of plants have been previously studied (Sagisaka, 1974; Sadakane et al., 1980). Bredemeijer and Esselink (1995) observed increasing G6PDH activity in a cold-tolerant cultivar of perennial ryegrass during cold acclimation. Stimulation of the PPP may generate nicotinamide adenine dinucleotide phosphate (NADPH) and building blocks required for cold hardening (Guy and Carter, 1984) and it may also counteract cold stress by promoting the degradation of sugars via a metabolic route that avoids cold-sensitive glycolysis. The synthesis of phenolic metabolites and stimulation of antioxidant response pathways would be able to minimize the oxidation-induced damage within tissues where it occurs under low temperature. In our previous study (Sarkar et al., 2009a), it was observed that cool-season grasses stimulated phenolic biosynthesis and antioxidant enzyme response through up regulation of PPP during cold acclimation. Therefore, we hypothesized that the prolineassociated PPP may play a crucial role in the fitness of perennial ryegrass accession during cold acclimation by stimulating phenolics and antioxidant enzyme response. The specific objectives of this study were to examine two accessions of perennial ryegrass (PR-1 and PR-2) during cold acclimation and to investigate the biochemical mechanism linked to phenolic biosynthesis which may be associated with cold acclimation in this species.

International Turfgrass Society Research Journal

MATERIALS AND METHODS Plant Material and Growth Conditions. Plant material consisted of accession PI 223178 (PR-1) and accession PI 598433 (PR-2), were selected based on evaluations of contrasting winter survival in field studies (Hulke et al., 2006, 2007) and contrasting freezing tolerance from controlled freezing studies (Hulke et al., 2008). The freezing tolerances (lethal temperature resulting in 50% mortality, LT50) of PR-2 and PR-1 were previously reported as approximately of -14.0 °C and -10.7 °C, respectively (Hulke et al., 2008). Single seeds of each accession were transplanted into pots (10 cm diameter, 10 cm depth) filled with a commercial potting medium (Pro-Mix, Griffin Greenhouse and Nursery Supplies, Tewksbury, MA). Plants were hand clipped and fertilized weekly with full strength Hoagland solution. After three months of establishment in a greenhouse, plants were transferred to a controlled environment growth chamber (Conviron, Winnipeg, Canada) and maintained at 15/10 °C day/night temperatures, 10-h photoperiod, and photosynthetic photon flux density (PPFD) of 300 µmol m-2 s-1. The cold acclimation regime used in our study was performed according to the methods previously described by Hulke et al., 2008. After 15 d, plants were subjected to constant 2 °C day/night temperatures for 21 d with a 10-h photoperiod and PPFD of 250 μmol m-2s-1 to induce cold acclimation. Leaf samples were collected at 0, 7, 14, and 21 d of acclimation for biochemical analysis. Enzyme Extraction. Leaf tissues (200 mg fresh weight) were collected and thoroughly macerated by using a cold mortar and pestle with cold enzyme extraction buffer [0.5% polyvinylpyrrolidone (PVP), 3 mmol L-1 ethylenediaminetetraacetic acid (EDTA), and 0.1 mol L-1 potassium phosphate buffer of pH 7.5]. The extracted samples were centrifuged at 10,188 gn for 10 min at 2 to 5 °C and stored on ice. The collected supernatant was used for further biochemical study. Total Soluble Phenolic (TSP) Assay. Total soluble phenolics were determined by an assay modified from Shetty et al. (1995). Approximately 50 mg fresh weight (FW) of perennial ryegrass leaf tissue was immersed in 2.5 mL of 95% ethanol and kept in a freezer for 48 h. After 48 h the sample was homogenized and centrifuged at 12,000 x g for 10 min. Then 0.5 mL of the supernatant and 0.5 mL of distilled water was mixed and transferred into a test tube to which 1 mL of 95% ethanol and 5 mL of distilled water was added. In each sample, 0.5 mL of 50% (v/v) Folin-Ciocalteu reagent was added and mixed. After 5 min, 1 mL of 5% Na2CO3 was added to the reaction mixture and allowed to stand for 60 min. A blank was prepared with 0.5 mL distilled water instead of the leaf extract. Absorbance was recorded at 725 nm after 1 h using a spectrophotometer (Genesys Spectronic 5, Milton Roy, Ivyland, PA). The absorbance values were converted to total phenolics and were expressed in milligram equivalents of gallic acid per gram fresh weight (F.W.) of the sample. Standard curves were established using various concentrations of gallic acid in 95% ethanol. ABTS [2, 2'-azino-bis(3-ethylbenzthiazoline-6sulphonic acid)] Cation Radical and Total Antioxidant Activity (TAA) Assay. The total antioxidant activity of leaf

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extract was measured using the ABTS+ radical cationdecolorization assay involving preformed ABTS+ radical cation (Pellegrini et al., 1999). A 7 mmol L-1 stock solution of ABTS (Sigma Chemical Co., St. Louis, MO) was made, and then the ABTS+ radical cation was prepared by reacting 5 mL of 7 mmol L-1 ABTS stock solution with 88 µL of 140 mmol L-1 potassium persulphate. The mixture was allowed to stand in the dark at room temperature (25 °C) for 12-16 h before use. Prior to assay, the ABTS+ stock solution was diluted with 95% ethanol (ratio 1:88) to give an absorbance at 734 nm of 0.70 ± 0.02, and was equilibrated to 30 °C. A 1 mL aliquot of ABTS was added to glass test tubes containing 50 µL of each tissue extract, and was vortexed for 30 s. After 2.5 min incubation, the absorbance of the solution was read at 734 nm using a spectrophotometer. The readings were compared with controls, which contained 50 µL of 95% ethanol instead of the extract. The Trolox reference standard for relative antioxidant activities was prepared with 5 mmol L-1 stock solution of Trolox in ethanol for introduction into the assay system at concentrations within the activity range of the assay (0-20 µmol L-1 final concentration) for preparing a standard curve to which all data were referenced. The percent inhibition was calculated by: % inhibition = ([ A734control – A734 extract]) × 100 [A734control ] Glucose-6-phosphate Dehydrogenase (G6PDH) Assay. Activity of G6PDH was quantified using the method originally described by Deutsch (1983) with modifications. An enzyme reaction mixture containing 5.88 µmol L-1 ßNADP, 88.5 µmol L-1 MgCl2, 53.7 µmol L-1 glucose-6phosphate, and 0.77 mmol L-1 maleimide was prepared. This mixture was used to obtain baseline (zero) of the spectrophotometer reading at a wavelength of 340 nm. To 1 mL of this mixture, 100 µL of the extracted enzyme sample was added. The rate of change in absorbance per min was used to quantify the enzyme activity using extinction coefficient of NADPH (6.22 mmol L-1 cm-1). Succinate Dehydrogenase (SDH) Assay. SDH activity was measured using the method described by Bregman (1987) with modifications. Enzyme sample was assayed at room temperature (25 °C) for SDH. The assay mixture containing 1.0 mL of 0.4 mol L-1 potassium phosphate buffer (pH 7.2), 40 µL of 0.15 mol L-1 sodium succinate (pH 7.0), 40 µL of 0.2 mol L-1 sodium azide, and 10 µL of 6.0 mg/mL 2,6-dichlorophenolindophenol (DCPIP) was prepared. This mixture was used to obtain baseline (zero) of the spectrophotometer reading at 600 nm. To 1.0 mL of this mixture, 200 µL of the enzyme sample was added. The rate of change of absorbance per minute was used to quantify the enzyme in the mixture using the extinction coefficient of DCPIP (19.1 mmol L-1 cm-1). Proline dehydrogenase (PDH) Assay. PDH activity was measured using the method previously described by Costilow and Cooper (1978) with modifications. The enzyme reaction mixture contained 100 mmol L-1 sodium carbonate buffer (pH 10.3), 20 mmol L-1 L-proline solution and 10 mmol L-1 nicotinamide adenine nucleotide (NAD). To 1 mL of this reaction mixture, 200 µL of extracted enzyme sample was added. The increase in absorbance was measured

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at 340 nm for 3 min, at 32 °C. The absorbance was recorded at time zero and then after 3 min. One unit of enzyme activity is equal to the amount causing an increase in absorbance of 0.01 per min at 340 nm (1.0 cm light path). Proline Assay. Proline was quantified using high performance liquid chromatography (HPLC) analysis with a liquid chromatograph (model 1100; Agilent Technologies, Santa Clara, CA) equipped with a diode array detector (DAD 1100, Agilent Technologies, Santa Clara, CA). The analytical column was reverse phase Nucleosil C18 (250 nm x 4.6 mm with a packing material of 5 µm particle size; Agilent Technologies, Santa Clara, CA). The extract samples were eluted out in an isocratic manner with a mobile phase consisting of 20 mmol L-1 potassium phosphate (pH 2.5 by phosphoric acid) at a flow rate of 1 mL min-1 and detected at 210 nm. To construct the standard curve, L-Proline (Sigma chemicals, St. Louis, MO) was dissolved in the 20 mmol L-1 potassium phosphate buffer. The amount of proline in the sample was reported as mg of proline per milliliter and converted to mg per g fresh weight (F.W). Total Protein (TP) Assay. Protein content was determined by the method described by Bradford (1976). One part dye reagent (Bio-Rad protein assay kit II, Bio-Rad Laboratory, Hercules, CA) was diluted with 4 parts distilled water. A volume of 5 mL of diluted dye reagent was added to 50 µL of the leaf tissue extract. The sample was then vortexed and incubated for 5 min, and absorbance was measured at 595 nm against a blank (5 mL reagent and 50 µL phosphate buffer) by using a UV-VIS Genesys spectrophotometer (Milton Roy, Inc., Rochester, NY). Superoxide Dismutase (SOD) Assay. A competitive inhibition assay was performed that used xanthine-xanthine oxidase-generated superoxide to reduce nitroblue tetrazolium (NBT) to blue formazan. Spectrophotometric assay of SOD activity was carried out by monitoring the reduction of NBT at 560 nm (Oberley and Spitz, 1984). The reaction mixture contained 13.8 mL of 50 mmol L-1 potassium phosphate buffer (pH 7.8) containing 1.33 mmol L-1 diethylenetetraaminepentaacetic acid (DETEPAC); 0.5 mL of 2.45 mmol L-1 NBT; 1.7 mL of 1.8 mmol L-1 xanthine and 40 IU mL-1 catalase. To 0.8 mL of reagent mixture 100 µL of phosphate buffer and 100 µL of xanthine oxidase was added. The change in absorbance at 560 nm was measured every 20 sec for 2 min and the concentration of xanthine oxidase was adjusted to obtain a linear curve with a slope of 0.025 absorbance min-1. The phosphate buffer was then replaced by the enzyme sample and the change in absorbance was monitored every 20 sec for 2 min. One unit of SOD was defined as the amount of protein that inhibits NBT reduction to 50% of the maximum. Catalase (CAT) Assay. Catalase activity was quantified using the method originally described by Beers and Sizer (1952) with modifications. To 1.9 mL of distilled water, 1 mL of 0.059 mol L-1 hydrogen peroxide (Merck's Superoxol or equivalent grade) in 0.05 mol L-1 potassium phosphate (pH 7.0) was added. This mixture was incubated in a spectrophotometer for approximately 5 min to achieve temperature equilibration and to establish the blank rate. To this mixture, 0.1 mL of diluted enzyme sample was added and

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the disappearance of peroxide was measured spectrophotometrically by recording the decrease in absorbance at 240 nm for approximately 3 min. The change in absorbance ΔA240/min from the initial (45 sec) linear portion of the curve was calculated. One unit of CAT activity was defined as amount that decomposes one µmole of H2O2 Units /mg =

PR-2 and PR-1; however, reduction of SDH activity was found in PR-1 at 14 and 21 d of cold acclimation. Significantly higher SDH activity was observed in PR-2 during entire cold acclimation period.

ΔA 240 /min × 1000

43 . 6× mg enzyme/ mL of reaction mixture Guaiacol Peroxidase (GPX) Assay. Guaiacol peroxidase activity was determined using the method described by Laloue et al. (1997) with modifications. The enzyme reaction mixture containing 0.1 mol L-1 potassium phosphate buffer (pH 6.8), 56 mmol L-1 guaiacol solution, and 50 mmol L-1 hydrogen peroxide was used. To 990 µL of this reaction mixture, 10 µL of enzyme sample was added. The absorbance was recorded at 0 and 5 min. The rate of change in absorbance per minute was used to quantify the enzyme in the mixture by using the extinction coefficient of the oxidized product tetraguaiacol (26.6 mmol L-1 cm-1). Statistical Analysis. Experiments were carried out in randomized complete block design with four replications per accession for each sampling date (0, 7, 14, and 21 d acclimation). The effects of accession and cold acclimation duration were determined using the analysis of variance (ANOVA) according to the general linear model procedure for the Statistical Analysis System (version 8.2; SAS Institute, Cary, NC). Means were separated using Fisher’s protected least significant difference (LSD) test at the 0.05 probability level. RESULTS Changes in Total Soluble Phenolics and Free RadicalLinked Antioxidant Activity of Perennial Ryegrass during Acclimation. Total soluble phenolic content varied significantly among PR-1 and PR-2 under prolonged cold acclimation (Fig. 1). During initial cold acclimation (7 d acclimation), there was no significant difference in total soluble phenolic content between PR-1 and PR-2. But with the further progression of cold acclimation treatment (14 and 21 d acclimation), higher total soluble phenolic content was observed in shoots of PR-2 compared to the PR-1. Total antioxidant activity of perennial ryegrass leaves was also significantly lower in PR-1 accession compared to (7, 14 and 21 d), PR-2 accession during cold acclimation (Fig. 2). Like total phenolic content, significantly higher antioxidant activity was observed in shoots of PR-2 at the end of cold acclimation treatment. G6PDH and SDH Activity during Acclimation. The G6PDH activity of PR accessions was assayed in order to determine the stimulation of PPP in response to cold acclimation. The G6PDH activity was increased after cold acclimation treatment (7, 14 and 21 d) in both PR accessions (Fig. 3). The higher G6PDH activity was observed in PR-2 after 14 and 21 d of cold acclimation. In general, SDH activity was significantly higher in PR-2 compared to the PR-1 (Fig. 4). With the first initiation of cold acclimation treatment (7 d), a significant increase in SDH activity was observed in both

Figure 1. Total soluble phenolic content (mg g-1 FW) of two perennial ryegrass accessions, PR-1 and PR-2, during cold acclimation (0, 7, 14, and 21 d). Vertical bars indicate least significant difference (P ≤ 0.05) for susceptible and tolerant accessions comparisons for each day.

Figure 2. Total antioxidant activity (ABTS%) of two perennial ryegrass accessions, PR-1 and PR-2, during cold acclimation (0, 7, 14, and 21 d). Vertical bars indicate least significant difference (P ≤ 0.05) for susceptible and tolerant accessions comparisons for each day.

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total proline content gradually increased in PR-2 (Fig. 6). At the end of the acclimation treatment, significantly higher proline content was found in PR-2 when compared to PR-1.

Figure 3. Glucose-6-phosphate dehydrogenase activity (µmol mg-1 protein) of two perennial ryegrass accessions, PR-1 and PR-2, during cold acclimation (0, 7, 14, and 21 d). Vertical bars indicate least significant difference (P ≤ 0.05) for susceptible and tolerant accessions comparisons for each day.

Figure 4. Succinate dehydrogenase activity (µmol mg-1 protein) of two perennial ryegrass accessions, PR-1 and PR-2, during cold acclimation (0, 7, 14, and 21 d). Vertical bars indicate least significant difference (P ≤ 0.05) for susceptible and tolerant accessions comparisons for each day.

Changes in PDH Activity and Proline Content during cold acclimation. Activity of PDH was investigated to confirm the role of proline oxidation as a potential energy source via oxidative phosphorylation under cold acclimation in PR accessions. The PDH activity decreased in PR-1 with continuous exposure to cold temperature at 14 and 21 d acclimation (Fig. 5). Higher PDH activity was observed in PR-2 when compared to PR-1 during 7, 14 and 21 d of acclimation. A significant stimulation of PDH activity was observed in PR-2 after 7 d of acclimation. In order to understand the role of proline and its coupling with pentose phosphate pathway in low temperatureinduced conditions, total proline content of cold-acclimated PR accessions was measured. Initially, higher proline content was observed in PR-1 when compared to PR-2, but with continuous exposure to cold temperature (2 °C) for 21 days,

Figure 5. Proline dehydrogenase activity (Units mg-1 protein) of two perennial ryegrass accessions, PR-1 and PR-2, during cold acclimation (0, 7, 14, and 21 d). Vertical bars indicate least significant difference (P ≤ 0.05) for susceptible and tolerant accessions comparisons for each day.

Figure 6. Total proline content (mg g-1 FW) of two perennial ryegrass accessions, PR-1 and PR-2, during cold acclimation (0, 7, 14, and 21 d). Vertical bars indicate least significant difference (P ≤ 0.05) for susceptible and tolerant accessions comparisons for each day.

Changes in SOD, CAT and GPX Activity. In order to understand whether cold acclimation was able to modulate antioxidant enzymes; with PPP in low temperature, the activities of three important antioxidant enzymes (SOD, CAT, and GPX) were analyzed. Superoxide dismutase (SOD) activity did not vary significantly between PR-1 and PR-2 (Fig. 7). Significant reduction of SOD activity was observed for both PR-2 and PR-1 after exposure to cold temperature (2 °C) for 7 d, but after 21 d of cold acclimation, SOD activity slightly increased in both accessions when compared to earlier cold acclimation period. Catalase (CAT) activity increased in PR-2 during cold acclimation, and at the end of cold acclimation (21 d) it was significantly higher in PR-2

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compared to PR-1 (Fig. 8). The highest CAT activity was found in PR-2 after 21 d of cold acclimation treatment. The GPX did not change significantly in PR accessions during cold acclimation (Fig. 9). In case of PR-1, GPX activity decreased slightly with continuous exposure to cold temperature (2 °C) for 21 d, while the activity of GPX was gradually increased in PR-2 throughout the cold acclimation period.

Figure 9. Guaiacol peroxidase activity (µmol mg-1 protein) of two perennial ryegrass accessions, PR-1 and PR-2, during cold acclimation (0, 7, 14, and 21 d). Vertical bars indicate least significant difference (P ≤ 0.05) for susceptible and tolerant accessions comparisons for each day.

Figure 7. Superoxide dismutase activity (Units mg-1 protein) of two perennial ryegrass accessions, PR-1 and PR-2, during cold acclimation (0, 7, 14, and 21 d). Vertical bars indicate least significant difference (P ≤ 0.05) for susceptible and tolerant accessions comparisons for each day.

Figure 8. Catalase activity (Units mg-1 protein) of two perennial ryegrass accessions, PR-1 and PR-2, during cold acclimation (0, 7, 14, and 21 d). Vertical bars indicate least significant difference (P ≤ 0.05) for susceptible and tolerant accessions comparisons for each day.

DISCUSSION In general, results showed that PR-2 accession of perennial ryegrass accumulated higher phenolics and higher antioxidant enzymes response compared to PR-1 during cold acclimation. During prolonged cold acclimation, higher leaf phenolic content was observed for PR-2. This may indicate enhanced capacity of this accession to minimize oxidative stress at low temperatures, since phenolics have been shown to act as antioxidants that protect cellular components and membranes against cold stress-induced generation of ROS (Christie et al., 1994; Rice-Evans et al., 1997). Pennycooke et al. (2005) also observed higher total phenolics in cold acclimated Petunia (Petunia spp. L) and found a positive correlation between higher phenolic content and chilling tolerance. We also previously reported that differential accumulation of phenolics was associated with cold tolerance among creeping bentgrass (Agrostis stolonifera L.) clonal lines (Sarkar et al., 2009b). Results of total antioxidant activity indicated a close association between plant defense stimulated by antioxidant activity and low temperature-induced oxidative stress tolerance in perennial ryegrass. At 14 and 21 d of cold acclimation, total antioxidant activity was higher for PR-2 compared to PR-1. Anderson et al. (1995) observed that in non-acclimated maize (Zea mays L.) seedlings, chilling injury was partly due to the buildup of ROS, while in coldacclimated seedlings, chilling tolerance developed due to an increased antioxidant system that protect against the accumulation of ROS. The lower total antioxidant activity and total phenolic content in PR-1 during cold acclimation suggested a low potential to counter low temperature-induced oxidative stress compared to PR-2. Three different antioxidant enzymes (SOD, CAT, and GPX) showed different trends among PR-1 and PR-2 accessions during cold acclimation. Concentration and relative composition of several isozymes of SOD, CAT, GPX, and ascorbate peroxidase (APX) were significantly altered in plant cells during exposure to several biotic and abiotic

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stresses (Pinhero et al., 1997). In our study, SOD activity did not change significantly in between PR-1 and PR-2 during cold acclimation. Low temperature-induced slightly higher trend of SOD activity may indicate a higher amount of oxidative stress in PR-1. Perennial ryegrass accession may be able to counter cold induced oxidative pressure without inducing higher SOD activity, or through an alternative mechanism such as NADPH oxidase (Davicino et al., 2008). Like SOD, GPX activity also did not vary significantly between PR-2 and PR-1 during cold acclimation. But CAT activity was significantly higher in PR-2 at the end of cold acclimation treatments indicating a probable defense mechanism of this accession under cold induced peroxidation of cell. Instead of SOD and GPX, PR-2 probably counter cold-induced oxidative stress through up regulation of CAT activity and can maintain cellular redox balance more efficiently than PR-1 under cold stress. Previous studies indicated the metabolic changes in the primary metabolism supported phenolic-linked antioxidant response in plants during abiotic stress (Kwon et al, 2009; Sarkar et al., 2009 a, b). The interesting observation of this study was higher G6PDH activity in PR-2 when compared to PR-1. A short term reduction in temperature (7 d) induced higher G6PDH activity in both PR accessions, but with further exposure to low temperature, G6PDH activity declined in PR-1. Increased G6PDH activity has been also observed in perennial ryegrass during cold hardening process in earlier studies (Bredemeijer and Esselink, 1995). Higher G6PDH activity in PR-2 suggests higher carbon diversion through PPP in this line during cold acclimation. This result provides an insight on the role of PPP in PR accessions during cold acclimation and could play a role in their cold tolerance characteristic. PPP regulation may generate NADPH and building blocks required for cold-hardening in plants (Guy and Carter, 1984; Hatano and Kabata, 1982), which may supports several anabolic pathways in PR-2 during cold acclimation and help to up regulate antioxidant defense systems at low temperature (Shetty, 2004). Succinate dehydrogenase (SDH) is another key enzyme that provides NADH for mitochondrial oxidative phosphorylation. Our results showed higher SDH activity for PR-2 which may indicate a higher demand for energy in PR-2 during cold acclimation. The combination of higher G6PDH and SDH activity may provide a balance between the anabolic and catabolic needs in this PR accession and subsequently help to drive phenolic biosynthesis and antioxidant enzyme response during cold acclimation. Taken together, better energy balance along with more efficient oxidative defense mechanisms may explain better stress tolerance for PR-2 compared to PR-1. The activity of another important enzyme, PDH also partially supports these findings. As PR accessions were exposed to prolonged cold temperature from 14 to 21 d, PR-2 exhibited higher PDH activity, suggesting increased proline oxidation to support mitochondrial oxidative phosphorylation in this accession. The high PDH activity also correlated with higher proline accumulation in PR-2 after 21 d of cold acclimation treatment and thus indicated the role of proline as a metabolic regulator in this accession during cold

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acclimation. Higher proline accumulation in the PR-1 may indicate an important role for this free amino acid in low temperature-induced stress tolerance in perennial ryegrass. Higher accumulation of several amino acids, including proline, also previously observed during cold hardening in PR plants (Draper, 1975). In addition, higher proline accumulation was correlated with higher G6PDH activity, suggesting the coupling of proline synthesis in the cytosol with the PPP. Under stress conditions, synthesis of proline may be directly coupled with PPP, driving synthesis of NADPH and sugar phosphates for supporting anabolic pathways, including phenolic biosynthesis and antioxidant response pathways (Shetty and Wahlqvist, 2004). Proline synthesis may drive the PPP in the cytosol while simultaneously supporting the oxidation of proline in the mitochondria, which may provide protons to drive oxidative phosphorylation in PR-2 (Hare and Cress, 1997). This type of energy partitioning system could play a critical role in the fitness mechanisms of perennial ryegrass during low temperature stress. CONCLUSION In summary, PR-1 and PR-2 accessions exhibited a partial variability in their physiological changes during cold acclimation period. Under short term cold acclimation, PR-1 and PR-2 exhibited similar phenolic content and proline accumulation, whereas after prolonged cold acclimation treatment higher phenolic content, higher total antioxidant activity and higher proline accumulation were observed in PR-2. At the end of the cold acclimation treatment PR-2 showed higher PPP regulation coupled with higher proline accumulation suggesting a more energy-efficient strategy in this PR accession at low temperature stress. Although differences in freezing tolerance among the two accessions were previously reported based on field studies and controlled freeze tests (Hulke et al., 2006, 2007, 2008), we did not conduct freeze tests in the current experiment to determine lethal temperature resulting in 50% mortality (LT50). As a result, we cannot make any direct correlations between the observed changes in antioxidant capacity during cold acclimation and differences in freezing tolerance between the two accessions. However, the data provide useful information on the biochemical regulations and shift of important antioxidant enzymes during cold acclimation in perennial ryegrass that can be used in future studies to test these antioxidant parameters with intraspecific differences in freezing tolerance. The ultimate goal will be to utilize these results to aid in the development of novel perennial ryegrass cultivars with improved persistence in low temperature environments. REFERENCES Anderson, M.D., T.K. Prasad, and C.R. Stewart. 1995. Changes in isozyme profiles of catalase, peroxidase and glutathione reductase during acclimation to chilling in mesocotyles of maize seedlings. Plant Physiol. 109:12471257.

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Rayapati, P.J., and S.R. Stewart. 1991. Solubilization of a proline dehydrogenase from maize (Zea mays L.). Plant Physiol. 95:787-791. Rice-Evans, C.A., N.J. Miller, and G. Paganga. 1997. Antioxidant property of phenolic compounds. Trends Plant Sci. 2:152-159. Sadakane, H., K. Kabata, K. Ishibashi, T. Watanabe, and S. Hatano, 1980. Studies on frost hardiness in Chlorella ellipsoidea. V. The role of glucose and related compounds. Environ. Exp. Bot. 20:297-305. Sagisaka, S. 1974. Transition of metabolisms in living poplar bark from growing to wintering stages and vice versa. Plant Physiol. 54:544-549. Sakai, A., and W. Larcher. 1987. Frost survival of plants: Response and adaptation to freezing stress. SpringerVerlag, Berlin. Sarkar, D., P. C. Bhowmik, Y-K. Kwon, and K. Shetty. 2009a. Cold acclimation responses of three cool-season turfgrasses and role of proline-linked pentose phosphate pathway. J. Amer. Soc. Hort. Sci. 134:210-220. Sarkar, D., P. C. Bhowmik, Y-K. Kwon, and K. Shetty. 2009b. Clonal response to cold tolerance in creeping bentgrass and role of proline-associated pentose phosphate pathway. Bioresource Technol. 100:53325339. Shetty, K. 1997. Biotechnology to harness the benefits of dietary phenolics; focus on Lamiaceae. Asia Pacific J. Clinical Nutr. 6:162-171. Shetty, K. 2004. Role of proline-linked pentose phosphate pathway in biosynthesis of plant phenolics for functional food and environmental applications: a review. Process Biochem. 39:789-803.

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