Bioresource Technology 100 (2009) 5332–5339

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Bioresource Technology

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Clonal response to cold tolerance in creeping bentgrass and roleof proline-associated pentose phosphate pathway

Dipayan Sarkar a, Prasanta C. Bhowmik a, Young-In Kwon b, Kalidas Shetty b,*

a Dept. of Plant, Soil, and Insect Sciences, Stockbridge Hall, University of Massachusetts, Amherst, MA 01003, United Statesb Dept. of Food Sciences, Chenoweth Laboratory, University of Massachusetts, Amherst, MA 01003, United States

a r t i c l e i n f o

Article history:Received 29 October 2007Received in revised form 12 March 2009Accepted 14 March 2009Available online 2 July 2009

Keywords:Creeping bentgrassClonal lineCold stressAntioxidant activityGlucose-6-phosphate dehydrogenase

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.03.086

* Corresponding author. Tel.: +1 413 545 1022; faxE-mail address: kalidasshetty@yahoo.com (K. Shet

a b s t r a c t

Single seed origin creeping bentgrass (‘Penncross’) clonal lines were screened to find genetic heterogene-ity, which reflected diversity of phenolic production linked to cold stress within a cross-pollinated culti-var. In this study, total soluble phenolic and antioxidant activity varied among 20 creeping bentgrassclonal lines, confirming wide heterogeneity in this cross-pollinated species. Correlations between pheno-lic content and proline-associated pentose phosphate pathway were also found among the clonal lines.The active metabolic role of proline in cellular metabolic adjustment to cold stress and its support forlikely energy synthesis via mitochondrial oxidative phosphorylation was inferred in creeping bentgrassclonal lines based on the activity of proline dehydrogenase. Results of photochemical efficiency of theseclonal lines after cold temperature treatment (4 �C) also indicated a close association between stress tol-erance and proline-associated pentose phosphate pathway regulation for phenolic biosynthesis and anti-oxidant response. This study provides a sound metabolic based rationale to screen bentgrass clonal linesfor enhanced cold stress tolerance.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Creeping bentgrass (Agrostis stolonifera L.) is a fine textured, sto-loniferous turfgrass species used in temperate to warm–humid cli-mate zones for golf course putting greens (Zhang et al., 2002).Turfgrass growing in temperate climates are constantly subjectedto varied climatic conditions (Fry and Huang, 2004). Creeping bent-grass is one of the most hardy cool-season turfgrass that can with-stand temperature extremes. This ability of creeping bentgrassallows better performance to extreme environmental changesthroughout the year. This species has higher ability to toleratelow temperature and maintain good quality turf below freezing,whereas other plant species are not able to sustain their biologicalfunction (Turgeon, 1996). Although creeping bentgrass exhibitssuperior cold hardiness characteristics, winter injury is observedin temperate zones of the United States and in Canada, where win-ter is severe and prolonged.

Environmental conditions that induce or favor photooxidativestress are common events in growth and developmental processesof plants (Paliyath and Droillard, 1992; Polle, 1997; Thompsonet al., 1987) and may have relevance in cold adaptation. As a resultof its oxidizing capacity, O2 acts primarily as an electron acceptor,and leads to the formation of a variety of reactive oxygen intermedi-

ll rights reserved.

: +1 413 545 1262.ty).

ates (Asada, 1993). These reactive oxygen species can attack cellmembranes by a cascade of free radical chain reactions, resultingin extensive damage to cell membrane and other cellular structures(Halliwell and Gutteridge, 1989). As a part of their aerobic existence,plants have developed an effective defensive system to detoxifyreactive intermediates through various antioxidant response mech-anisms and this has relevance in cold stress management.

Phenolic compounds are secondary metabolites distributedwidely in plants (Javanmardi et al., 2003). The antioxidant activityof phenolics is actually governed by their redox properties, whichplay crucial roles in absorbing and neutralizing free radicals,quenching single and triplet oxygen or decomposing peroxides(Rice-Evans et al., 1997; Shetty, 2004). In response to biotic and abi-otic stress, biosynthesis of plant phenolic antioxidants takes placethrough the stimulation of secondary metabolite pathways. Fewstudies have examined the effect of low temperature stress on thelevels of phenylpropanoids in plants (Prasad, 1996; Rice-Evanset al., 1997). The recovery from the chilling stress indicates somekind of antioxidant protection; potentially involving anthocyaninand phenylpropanoid biosynthesis (Christie et al., 1994). Directrole of antioxidants in the induction of low temperature tolerancein cool-season turfgrass has not been investigated. However, effectsof other oxidative stresses, such as high temperature, low light,ultraviolet-B radiation as well as the role of antioxidative systemin stress tolerance of turfgrass have been investigated (Ervinet al., 2004; Jiang et al., 2005; Larkindale and Huang, 2004; Wang

D. Sarkar et al. / Bioresource Technology 100 (2009) 5332–5339 5333

et al., 2003; Xu and Huang, 2004). In cool-season turfgrass, antiox-idant enzymes could either directly enhance low temperaturetolerance or induce defensive systems for tolerance through signal-ing mechanisms. A key regulation of inducible antioxidant enzymeresponse was suggested to be via proline synthesis (Shetty, 2004).

Amino acids play significant roles in cold hardiness in plants.Accumulation of some specific amino acids during cold acclimationis documented in several plant species (Sagisaka and Araki, 1983;Sakai and Larcher, 1987). The studies conducted on Poa annua L.showed higher accumulation of amino acids with cold acclimation(Dionne et al., 2001). After exposure to subfreezing temperature, ahigher level of proline, glutamine and glutamic acid in P. annuacrown were observed (Dionne et al., 2001). Proline is synthesizedfrom glutamate by a series of reduction reactions. In this synthesisprocess, proline and pyrroline-5-carboxylate (P5C) may regulateredox and hydride ion-mediated stimulation of pentose phosphate

Reactions of Phenolic Radical on the membrane----

Mitochondrial matrix

Inner membrane

Inner membrane space

OH-

OHOH

H+

H+

H+

pH gradientelectrochemical gradient

Proline

P-5-C

Glutamate

e-

e-

PDH

(Proline dehydrogenase)Proline

P-5-C

Glutamate

Chloroplast/

N

N

N

N

ShikimaPathwa

ChorismateIndole

Phenylalanine

Cinnamate

Phenolics PhPat

GST/PO/SOwith phenolantioxidant scavenges frNADPH froPentose Pho

Tryptophan IAA

e-

e-

e- 1/2 O2

3 ATP

H2 O

Alternative oxidative phosphorylation under oxidation stress with phenolic antioxidants

ATPase

Proline (Replacing NADH)

O2Reactive oxygen

OrganelleNuclearVacuole aCytosolic

Mitochondria

COO-

-O

ReceptorPhenolic Radicthe Membraneinvolve Peroxipolymerization

SignalingTransport mobilizationC -transport stimulationTransport inhibition etc.

2H+

2H+

2H+

2H+

2H+

2H+

Fig. 1. Model for the role of proline-associated pentose phosphate pathway in regulatingof external phenolic phytochemicals.

pathway (Hagedorn and Phang, 1983; Phang, 1985). During respi-ration, oxidation reactions produce hydride ions, which helpreduction of P5C to proline in the cytosol. Through the reactionof proline dehydrogenase, proline can be oxidized in the mitochon-dria. Within the mitochondria, instead of NADH, proline can beused as a reducing equivalent and can support oxidative phosphor-ylation. The reduction of P5C in cytosol provides NADP+, which is aco-factor for glucose-6-phosphate dehydrogenase (G6PDH).G6PDH plays crucial committed role, by catalyzing the first ratelimiting step of the pentose phosphate pathway. Phang (1985) firstproposed this model and suggested its role in stimulation of purinemetabolism via ribose-5-phosphate in animal cells.

On the basis of the Phang (1985) model, Shetty (1997) proposeda model (Fig. 1) that proline-associated pentose phosphate path-way could stimulate both the shikimate and phenylpropanoidpathways, and therefore, the modulation of this pathway could

CytosolGlucose

Glucose-6-P

6-Phospho-glucose-lactone

G6PDH(glucose-6-phosphate dehydrogenase)

6-phosphogluconate dehydrogenase

Ribulose-5-P

Ribose-5-P

PRPP

Adenine

Guanine

Purine

ADP+

ADPH

ADP+

ADPH

Erythrose-4-Pte y

enylpropanoid hway

D ics initiate response and ee radicals with m proline- linked sphate Pathway

Sugar phosphates for anabolicreactions

(Phosphoribosyl

pyrophosphate)

Phenolic

radicalH+

H+ H+

ndreactions

Proton/hydride ions (hyperacidification)

H+

H+ H+

H+ induced prolin -linked pentose phosphate pathway

al Reactions on and cell wall can dase-induced

Phenolic Antioxidant

DNA/RNA,ATP

Cytokinin

phenolic biosynthesis in plants, which also accommodates the mechanism of action

5334 D. Sarkar et al. / Bioresource Technology 100 (2009) 5332–5339

lead to the stimulation of phenolic phytochemicals. It was also pro-posed that demand for NADPH for proline synthesis during stresstreatments may increase cellular NADP+/NADPH ratio, whichshould activate G6PDH. Therefore, pentose phosphate pathwaymay drive metabolic flux towards erythrose-4-phosphate for thebiosynthesis of shikimate and phenylpropanoid metabolites. Atthe same time, proline serves as a reducing equivalent, instead ofNADH for oxidative phosphorylation (ATP synthesis) in the mito-chondria (Shetty, 2004; Shetty and McCue, 2003).

This model (Shetty and Wahlqvist, 2004) has been proposed forthe mode of action of phenolic metabolites based on the correlationbetween stress stimulated phenolic biosynthesis and stimulation ofantioxidant enzyme response pathways in plants. The synthesis ofphenolic metabolites and stimulation of antioxidant response path-way would be able to minimize the oxidation induced damagewithin tissues. Phenolic antioxidants can behave as antioxidantsby trapping free radicals in direct interactions or scavenge themthrough a series of coupled, antioxidant enzyme defense systemreactions (Shetty and Wahlqvist, 2004). A coupled enzymatic de-fense system could involve low molecular weight antioxidants,such as ascorbate, glutathione (GSH), a-tocopherol, carotenoidsand phenylpropanoids, in conjunction with several enzymes suchas superoxide dismutase (SOD), catalase (CAT), peroxidases, gluta-thione reductase and ascorbate peroxidase (Bowler et al., 1994; Pin-hero et al., 1997; Rao et al., 1996). In this study the hypothesis wasthat proline-associated pentose phosphate pathway may play acrucial role under low temperature stress in cool-season turfgrassand as a result, phenolic phytochemicals can effectively counteroxidative stress within cells during cold stress adaptation.

The major drawback for consistent phenolic phytochemicalsproduction in cross-pollinated plant species is the genetic hetero-geneity (Shetty, 2004). Creeping bentgrass is cross-pollinated spe-cies, and seeded creeping bentgrass cultivars possess a certaindegree of heterogeneity (Beard, 1973). Thus, diversity of phenolicproduction among diverse single seeded originating clonal linesmay be characteristic of this turfgrass species. To overcome sucha problem, plant tissue culture and micro propagation techniqueshave been developed to isolate a clonal pool of plants originatingfrom single heterozygous seeds (Shetty, 2004). With the help ofthis clonal propagation approach using vegetative cuttings fromsingle heterozygous seeds among a heterogenous seed population,different clonal lines (from single seed origin plant) of creepingbentgrass were screened for evaluating their cold tolerance. Themajor hypothesis was that a specific clonal line with a higher phe-nolic profile would contribute to enhanced stress tolerance com-pared to lines having reduced phenolic and antioxidant responsesystem. The clonal lines developed by this approach would be ex-pected to have high antioxidant response pathway by couplingproline-associated pentose phosphate pathway with stimulationof antioxidant enzymes. A stronger biochemical rationale forscreening of single seeded clonal line will also help to developnew superior stress tolerant cultivars of creeping bentgrass.

2. Methods

2.1. Plant material, growth condition and isolation of single seed clonallines

Heterozygous seeds of creeping bentgrass from a heterogenousmixture of ‘Penncross’ cultivar seed source were planted individu-ally in 4.5 cm diameter pots to screen 20 single seed originatingclonal lines. Single seed originating creeping bentgrass clonal lineswere grown in soil–sand mixture. Clonal lines were kept in growthchamber with 25/20 �C, day/night temperature, and with 12-hphotoperiod. Clonal lines were watered every alternate day. Fertil-

ization and mowing were carried out once a week. After sufficientgrowth (three months), leaf clippings were collected for biochem-ical analysis. Each clonal line is a phenotype originating from dif-ferent suspected heterozygous single seed in a heterogeneousseed population.

2.2. Enzyme extraction

Creeping bentgrass leaf tissue (200 mg) was collected and thor-oughly macerated by using a cold pestle and mortar with cold en-zyme extraction buffer [0.5% polyvinylpyrrolidone (PVP), 3 mMEDTA, and 0.1 M potassium phosphate buffer of pH 7.5]. The ex-tracted sample was centrifuged at 13,500 rpm for 10 min at 2–5 �C and stored in ice. The supernatant was used for further bio-chemical and enzyme analysis.

2.3. Glucose-6-phophate dehydrogenase (G6PDH) assay

A modified method originally described by Deutsch (1983) wasused. The enzyme reaction mixture containing 5.88 lmol ß-NADP,88.5 lmol MgCl2, 53.7 lmol glucose-6-phosphate, and 0.77 mmolmaleimide was prepared. This mixture was used to obtain baseline(zero) of the spectrophotometer reading at 340 nm wavelength. To1 mL of this mixture, 100 lL of the extracted sample was added.The rate of change in absorbance per min was used to quantifythe enzyme in the mixture with the help of the extinction coeffi-cient of NADPH (6.22 mM�1 cm�1).

2.4. Proline dehydrogenase (PDH) assay

A modified method described by Costilow and Cooper (1978)was carried out to assay the activity of proline dehydrogenase.The enzyme reaction mixture containing 100 mM sodium carbon-ate buffer (pH 10.3), 20 mM L-proline solution and 10 mM NADwas used. To 1 mL of this reaction mixture, 200 lL of extracted en-zyme sample was added. The increase in absorbance was mea-sured at 340 nm for 3 min, at 32 �C. The absorbance wasrecorded at zero time and then after 3 min. In this spectrophoto-metric assay, one unit of enzyme activity is equal to the amountcausing an increase in absorbance of 0.01 per min at 340 nm(1.0 cm light path).

2.5. Succinate dehydrogenase (SDH) assay

To assay the activity of succinate dehydrogenase a modifiedmethod described by Bregman (1987) was used. Bentgrass tissueextract suspension was diluted with 2.0 mL of enzyme extractionbuffer. Then enzyme sample was assayed at room temperaturefor succinate dehydrogenase activity. The assay mixture containing1.0 mL of 0.4 M potassium phosphate buffer (pH 7.2), 40 lL of0.15 M sodium succinate (pH 7.0), 40 lL of 0.2 M sodium azide,and 10 lL of 6.0 mg/mL 2,6-dichlorophenolindophenol (DCPIP)was prepared. This mixture was used to obtain baseline (zero) ofthe spectrophotometer reading at 600 nm wavelength. To 1.0 mLof this mixture, 200 lL of the enzyme sample was added. The rateof change of absorbance per min was used to quantify the enzymein the mixture using the extinction coefficient of DCPIP(19.1 mM�1 cm�1).

2.6. Guaiacol peroxidase (GPX) assay

A modified method described by Laloue et al. (1997) was usedto assay the activity of guaiacol peroxidase. The enzyme reactionmixture containing 0.1 M potassium phosphate buffer (pH 6.8),56 mM guaiacol solution, and 50 mM hydrogen peroxide was used.

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To 990 lL of this reaction mixture, 10 lL of enzyme sample wasadded. The absorbance was recorded at zero time and then after5 min. The rate of change in absorbance per min was used to quan-tify the enzyme in the mixture by using the extinction coefficientof the oxidized product tetraguaiacol (26.6 mM�1 cm�1).

2.7. Catalase (CAT) assay

A method originally described by Beers and Sizer (1952) wasused to assay the activity of catalase. To 1.9 mL of distilled water1 mL of 0.059 M hydrogen peroxide (Merck’s Superoxol or equiva-lent grade, Merck Co. & Inc., Whitehouse Station, NJ) in 0.05 Mpotassium phosphate, pH 7.0 was added. This mixture was incu-bated in a spectrophotometer for 4–5 min to achieve temperatureequilibration and to establish blank rate. Then 0.1 mL of diluted en-zyme was added and the disappearance of peroxide was followedspectrophotometrically by recording the decrease in absorbanceat 240 nm for 2–3 min. The change in absorbance DA240/min fromthe initial (45 s) linear portion of the curve was calculated. Oneunit of catalase activity was defined as amount that decomposesone micromole of H2O2

Units=mg ¼ ðDA240=minÞ � 100043:6� mg enzyme=mL of reaction mixture

:

2.8. Superoxide dismutase (SOD) assay

A competitive inhibition assay was performed that used xan-thine oxidase generated superoxide to reduce nitroblue tetrazo-lium (NBT) to blue formazan. Spectrophotometric assay of SODactivity was carried out by monitoring the reduction of NBT at560 nm (Oberley and Spitz, 1984). The reaction mixture contained13.8 mL of 50 mM potassium phosphate buffer (pH 7.8) containing1.33 mM diethylenetetraaminepentaacetic acid (DETEPAC); 0.5 mLof 2.45 mM NBT; 1.7 mL of 1.8 mm xanthine and 40 IU/mL catalase.Then 100 lL of phosphate buffer and 100 lL of xanthine oxidasewere added to 0.8 mL of reagent mixture. The change in absor-bance at 560 nm was measured every 20 s for 2 min and the con-centration of xanthine oxidase was adjusted to obtain a linearcurve with a slope of 0.025 absorbance per min. The phosphatebuffer was then replaced by the enzyme sample and the changein absorbance was monitored every 20 s for 2 min. One unit ofSOD was defined as the amount of protein that inhibits NBT reduc-tion to 50% of the maximum.

2.9. Total protein assay

Protein content was measured by the method of Bradford assay(Bradford, 1976). One part of dye reagent (Bio-Rad protein assay kitII, Bio-Rad Laboratory, Hercules, CA) was diluted with four parts ofdistilled water. A volume of 5 mL of diluted dye reagent was addedto 50 lL of the bentgrass tissue extract. After vortexing and incu-bating for 5 min, the absorbance was measured at 595 nm againsta blank (5 mL reagent and 50 lL buffer solution) by using a UV–visGenesys spectrophotometer (Milton Roy Inc., Rochester, NY).

2.10. ABTS [2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)]cation radical and antioxidant activity assay

The total antioxidant activity of creeping bentgrass leaf extractwas measured by the ABTS+ radical cation-decolorization assayinvolving preformed ABTS+ radical cation (Pellegrini et al., 1999).ABTS (Sigma Chemical Co., St. Louis, MO) was dissolved in waterto a 7 mM concentration. ABTS+ radical cation was prepared byreacting 5 mL of 7 mM ABTS stock solution with 88 lL of

140 mM potassium persulphate, and mixture was allowed to standin the dark at room temperature for 12–16 h before use. Prior tothe assay, ABTS+ stock solution was diluted with 95% ethanol (ratio1:88) to give an absorbance of 0.70 ± 0.02 at 734 nm, and wasequilibrated to 30 �C. ABTS (1 mL) was added to glass test tubescontaining 50 lL of each tissue extract, and mixed by vortex mixerfor 30 s. After 2.5 min incubation, mixtures were read at 734 nm.The readings were compared with controls, which contained50 lL of 95% ethanol instead of the extract. The Trolox referencestandard for relative antioxidant activities was prepared with5 mM stock solution of Trolox in ethanol for introduction intothe assay system at concentrations within the activity range ofthe assay (0–20 lM final concentration) for preparing a standardcurve to which all data were referenced. The percent inhibitionwas calculated by

% inhibition ¼Acontrol

734 � Aextract734

h i� �

½Acontrol734 �

� 100:

2.11. Total soluble phenolic assay

The total soluble phenolics were determined by an assay mod-ified from Shetty et al. (1995). A quantity of 50 mg fresh weight(FW) bentgrass leaf tissue was immersed in 2.5 mL of 95% ethanoland kept in the freezer for 48 h. After 48 h the sample was homog-enized and centrifuged at 12,000g for 10 min. Then 0.5 mL of sam-ple supernatant and 0.5 mL of distilled water was mixed andtransferred into a test tube and 1 mL of 95% ethanol and 5 mL ofdistilled water was added. In each sample, 0.5 mL of 50% (v/v) Fo-lin–Ciocalteu reagent was added and mixed. After 5 min, 1 mL of5% Na2CO3 was added to the reaction mixture and allowed to standfor 60 min. A blank was prepared with 0.5 mL distilled water in-stead of sample. After 1 h, absorbance was measured at 725 nm.The absorbance values were converted to total phenolics and wereexpressed in milligrams equivalents of gallic acid per gram FW ofthe sample. Standard curves were established using various con-centrations of gallic acid in 95% ethanol.

2.12. HPLC analysis of proline

High performance liquid chromatography (HPLC) analysis wasperformed using an Agilent 1100 liquid chromatograph (AgilentTechnologies, Santa Clara, CA) equipped with a diode array detec-tor (DAD 1100). The analytical column was a reverse phase Nucle-osil C18, 250 nm � 4.6 mm with a packing material of 5 lmparticle size. The samples were eluted out in an isocratic mannerwith a mobile phase consisting of 20 mM potassium phosphate(pH 2.5 by phosphoric acid) at a flow rate of 1 mL/min and detectedat 210 nm. L-Proline (Sigma chemicals, St. Louis, MO) dissolved inthe 20 mm potassium phosphate solution was used to calibratethe standard curve. The amount of proline in the sample was re-ported as milligram of proline per gram FW.

2.13. Photochemical efficiency

Photochemical efficiency of creeping bentgrass shoot was mea-sured by using a fluorometer (OS1-FL Opti-Sciences, Tyngsboro,MA). The test was carried at the dark adapted mode and Fv/Fm

(Fv/Fm = [Fm � Fo]/Fm is the ratio of variable fluorescence to maxi-mal fluorescence) ratio was calculated. Plants were kept in darkat least 2 h before the measurement. The first measurements weretaken in 25/20 �C day/night temperature. The second observationsof photochemical efficiency were taken after exposing all clonallines to 4 �C for one week.

5336 D. Sarkar et al. / Bioresource Technology 100 (2009) 5332–5339

2.14. Visual color ratings

Turfgrass color was visually estimated before and, after coldtreatment (4 �C) exposure on a scale of 1–9, with 9 indicating thedarkest green color and 1 indicating lowest turf quality. Photo-graphs of 20 creeping bentgrass clonal lines were taken after expos-ing them to cold treatment (4 �C) by using Panasonic digital camera.

2.15. Statistical analysis

Mean, standard error, standard deviations, and correlation coef-ficient (r2) were calculated for all measurements in 20 creepingbentgrass clonal lines and analysis was done using Microsoft ExcelXP.

3. Results

3.1. Glucose-6-phosphate dehydrogenase (G6PDH) and succinatedehydrogenase (SDH) activity

Significantly low G6PDH activity was observed in CB-6, CB-13,CB-15, CB-21, and in CB-22, whereas high G6PDH activity was ob-served in CB-2, CB-5, CB-8, CB-17, CB-19, CB-20, CB-23 and CB-27(Table 1). High G6PDH activity in CB-20 and CB-27 correspondedwith high proline dehydrogenase (PDH) activity and high prolinecontent. Similarly, low G6PDH activity in CB-13, CB-15, and CB-21was associated with low PDH and low proline content, indicatingthe likely effective coupling to pentose phosphate pathway re-sponse. The key enzyme succinate dehydrogenase (SDH) of the tri-carboxylic acid cycle pathway was measured to understand itsrelevance during cold stress adaptation in creeping bentgrass clonallines. Creeping bentgrass clonal lines CB-13 and 15 with low G6PDHactivity showed relatively high SDH activity compared to CB-20 andCB-27, whereas SDH activity was low, but G6PDH activity was high(Table 1). These results indicated differences among creeping bent-grass clonal lines with respect to their dependence on either pen-tose phosphate pathway, or TCA cycle under similar environment.

3.2. Proline content and proline dehydrogenase (PDH) activity

The PDH activity of CB-20 and CB-27 was significantly higherthan CB-13 and CB-15 (Table 1). Higher PDH activity was also

Table 1Glucose-6-phosphate dehydrogenase (G6PDH), proline dehydrogenase (PDH), succi-nate dehydrogenase (SDH), and total proline of 20 creeping bentgrass clonal lines.

CBLine

G6PDH nmol/mgprotein

PDH unit/mgprotein

SDH nmol/mgprotein

Total proline mg/gfresh weight

2 3.02 2.58 0.07 1.04 2.79 24.94 0.05 1.545 3.24 16.31 0.05 1.346 1.99 33.92 0.03 1.937 2.43 25.43 0.06 1.378 2.85 2.04 0.08 1.31

10 2.61 3.26 0.04 0.912 2.19 1.94 0.06 0.8713 1.68 1.61 0.05 1.3215 1.16 1.50 0.06 0.6716 2.39 26.83 0.06 1.2917 2.59 12.80 0.07 1.6519 2.37 17.67 0.07 1.8120 2.47 36.18 0.05 1.7321 0.85 2.69 0.08 0.722 1.93 28.8 0.08 1.6823 3.59 10.29 0.06 1.6624 2.97 17.84 0.08 2.0826 2.35 33.44 0.06 1.9527 3.82 27.19 0.05 2.09SE± 0.16 2.80 0.003 0.098

observed in many other clonal lines (CB-6, CB-16, and CB-26).Higher PDH activity in these clonal lines corresponded with higherG6PDH activity and lower SDH activity. The total proline contentwas also found higher in CB-20, and in CB-27 compared to CB-13and CB-15. Total proline content in various creeping bentgrass clo-nal lines evaluated in this study ranged between 0.67 mg/g FW and2.09 mg/g FW.

3.3. Total soluble phenolics and free radical-linked antioxidant activityin creeping bentgrass clonal lines

Significant heterogeneity in total phenolic content among 20creeping bentgrass clonal lines was observed (Table 2). Total phe-nolic content ranged between 0.72 mg/g FW (CB-21) and 1.51 mg/g FW (CB-22). High total phenolic content was found in CB-22(1.51 mg/g FW), CB-19 (1.49 mg/g FW), CB-27 (1.47 mg/g FW),CB-7 (1.37 mg/g FW), and in CB-20 (1.20 mg/g FW), whereas lowphenolic content was observed in CB-21 (0.72 mg/g FW), CB-24(0.87 mg/g FW), CB-12 (0.84 mg/g FW), and in CB-15 (0.94 mg/g FW). Total antioxidant activity varied significantly among creep-ing bentgrass clonal lines (Table 2). High antioxidant activity wasobserved in CB-20 (42%), CB-19 (35%), CB-27 (33%), CB-22 (32%),and in CB-7 (30%), whereas CB-17 (19%), CB-10 (22%), CB-12(23%), CB-24 (23%) showed comparatively lower antioxidant activ-ity. High antioxidant activity corroborated with high phenolic con-tent in many creeping bentgrass clonal lines, like CB-19, CB-22, CB-27, CB-7, and CB-20. Similarly, positive correlation between lowantioxidant activity, and low phenolic content was also observedamong CB-12, CB-24, and CB-17 clonal lines.

3.4. Superoxide dismutase (SOD), catalase (CAT), and guaiacolperoxidase (GPX) activity

Moderate SDH activity in CB-13 and CB-15 corresponded withhigh SOD and CAT activity. In case of CB-27, activity of SOD, andCAT was found to be lower than that of CB-13 and CB-15. In CB-15, low G6PDH activity corresponded to low GPX activity, althoughthis correlation was not observed in many other clonal lines. But inmany clonal lines, a higher GPX activity correlated with a higherphenolic content, indicating potential for lignification of phenolicsby GPX.

Table 2Superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), totalphenolic, and total antioxidant activity (ABTS) of 20 creeping bentgrass clonal lines.

CBLine

SOD unit/mgprotein

CAT unit/mgprotein

GPX nmol/mgprotein

ABTS(%)

Total phenolic mg/gfresh weight

2 10.64 88.1 13.00 27.23 1.234 9.16 92.3 20.91 28.22 1.085 12.04 78.7 8.81 24.25 1.206 8.42 83.9 15.41 23.54 1.137 8.71 74.8 12.80 30.07 1.378 10.99 81.2 19.02 23.97 1.16

10 11.44 88.4 23.19 21.56 1.2412 13.31 99.9 22.11 22.83 0.8413 13.91 117.8 19.18 27.37 1.0715 11.53 115.3 5.44 25.81 0.9416 10.57 59.9 18.05 27.65 1.1917 15.36 70.1 12.74 19.00 1.0219 7.65 81.0 12.84 34.89 1.4920 7.55 70.1 12.94 41.70 1.2021 12.30 74.7 19.24 27.37 0.7222 11.04 52.7 16.47 31.63 1.5123 14.01 72.6 14.47 24.96 1.0924 11.10 56.6 6.91 22.97 0.8726 7.29 62.5 22.84 27.51 0.9727 7.55 15.2 12.11 32.62 1.47SE± 0.53 5.0 1.15 1.15 0.05

Fig. 2. Black and white image of 20 creeping bentgrass clonal lines after one weekexposure to cold treatment (4 �C).

Table 3Visual quality and photochemical efficiency of creeping bentgrass clonal lines.

CB lines 25/20 �C 4 �C

Visual quality(1–9 scale)

Fv/Fm ratioa Visual quality(1–9 scale)

Fv/Fm ratioa

2 9.0 0.80 8.5 0.744 8.5 0.78 8.5 0.745 8.5 0.76 8.0 0.726 8.5 0.75 8.0 0.747 8.0 0.77 8.0 0.768 8.5 0.77 8.0 0.74

10 8.5 0.74 8.0 0.7212 8.0 0.75 7.0 0.7013 8.5 0.75 7.5 0.7015 8.0 0.77 7.5 0.6616 8.5 0.75 8.5 0.7017 8.5 0.76 8.0 0.6719 8.5 0.75 8.0 0.7320 9.0 0.78 9.0 0.7721 8.0 0.75 7.5 0.6922 8.5 0.77 8.0 0.7423 8.5 0.77 8.0 0.7524 8.5 0.76 8.0 0.6926 8.5 0.74 8.0 0.7227 8.0 0.77 8.0 0.74

a Fv/Fm = [Fm � Fo]/Fm is the ratio of variable fluorescence to maximalfluorescence.

Table 4Correlation coefficient (r2) of some biochemical parameters and cold stress toleranceresponse of 20 creeping bentgrass clonal lines after one week exposure to coldtemperature (4 �C).

Correlation Correlation coefficientvalue (r2)

Phenolic/total antioxidant activity +0.50Phenolic/G6PDH +0.40G6PDH/proline +0.36G6PDH/total antioxidant activity +0.50Proline/PDH +0.73Turf quality after cold stress/phenolic +0.42Turf quality after cold stress/total antioxidant activity +0.47Turf quality after cold stress/G6PDH +0.42Turf quality after cold stress/proline +0.39Turf quality/photochemical efficiency (4 �C, 1 week) +0.56Photochemical efficiency after cold stress/phenolic +0.61Photochemical efficiency after cold stress/total

antioxidant activity+0.56

Photochemical efficiency after cold stress/G6PDH +0.61Photochemical efficiency after cold stress/proline +0.38

D. Sarkar et al. / Bioresource Technology 100 (2009) 5332–5339 5337

3.5. Photochemical efficiency and visual quality

Quality of creeping bentgrass clonal lines at 25/20 �C, day/nighttemperature ranged between 8.0 and 9.0 (good visual quality).Exposure to low temperature (4 �C) did not cause any significantreduction of color or quality in creeping bentgrass clonal lines(Fig. 2 and Table 3). Although, visual quality of all clonal lineswas within the acceptable range (above 6), but discoloration of leaf(light green color), and reduction of leaf growth was observed infew clonal lines (CB-12, CB-13, CB-15 and CB-21).

A decrease in Fv/Fm ratio reflecting photochemical efficiency isinitiated by a decrease in Fm in combination with an increase inFo. Reduction of Fv/Fm ratio was observed in all creeping bentgrassclonal lines after plants were exposed to 4 �C from initial temper-ature (25/20 �C, day/night temperature) (Table 3). The reductionof photochemical efficiency was more pronounced in some creep-ing bentgrass clonal lines (CB-12, CB-13, CB-15, CB-21 and CB-24), while some other clonal lines (CB-8, CB-19 and CB-20)showed lower reduction. Creeping bentgrass clonal lines with

low Fv/Fm indicates a mix of photoprotection and photodamageresponses.

4. Discussion

Consistent phenolic production and proper activity of antioxi-dant response system are necessary to develop higher stress toler-ance response. However, in cross-pollinated species, inconsistentphenolic production among a heterogeneous genetic backgroundand variations of antioxidant response system is a major hindrancefor consistent performance of cultivars (Shetty and McCue, 2003).Therefore, one of the strategies is to understand the role of pro-line-associated pentose phosphate pathway in stress tolerancemechanism of cross-pollinated species, and to overcome the genet-ic heterogeneity through screening of single seed originating coldtolerant clonal lines based on this concept.

Evaluation and screening of single seed origin creeping bent-grass clonal lines is first and foremost step in understanding the

5338 D. Sarkar et al. / Bioresource Technology 100 (2009) 5332–5339

above concept. On the basis of the above rationale, we hypothe-sized that genetic heterogeneity in relation to inconsistency inphenolic phytochemical production is a characteristic in cross-pol-linated creeping bentgrass species, and this variation is closely re-lated with proline-associated pentose phosphate pathway. Resultsfrom this study partially support this concept and further investi-gation is warranted. Significant variations in production of totalsoluble phenolics were observed among 20 creeping bentgrass clo-nal lines (CB-2 to CB-27) evaluated, confirming genetic heteroge-neity in this cross-pollinated species. Total antioxidant activity ofthese creeping bentgrass clonal lines also varied widely, confirm-ing the notion of heterogeneity with relation to antioxidant re-sponse system and phenolic phytochemicals production. Positivecorrelation between phenolic content and antioxidant activitywas found in majority creeping bentgrass clonal lines, indicatingthe relationship of phenolic synthesis and stimulation of antioxi-dant response system through phenylpropanoid pathway (Table4). Variations among these creeping bentgrass clonal lines led toscreening and selection of clonal lines with high and low phenoliccontent and antioxidant activity. This indicates that fitness of thesecreeping bentgrass clonal lines may differ under different stressconditions and this offers the possibility to evaluate them for stressadaptation characteristic. The activity of guaiacol peroxidase (GPX)also showed similar variations in these creeping bentgrass clonallines. GPX as an isoenzyme of peroxidase and, protects plant cellsfrom oxidative damage through its role in antioxidant response(Gaspar et al., 1991). Peroxidases, play multiple roles during thisprocess, by involving in auxin and ethylene metabolism, redoxreactions in plasma membranes, cell wall modifications (lignifica-tion and suberization), and also in defense mechanisms. It is be-lieved that peroxidase is responsible for the cross linking ofphenolic moieties during the biosynthesis of lignins and lignansin the plant cell wall (Morales and Barcelo, 1997). Although, ourstudies compared diversity of GPX activity in specific bentgrassclonal lines under normal condition (without stress), but there isa possibility of inducible alteration of this varied GPX activity un-der stress situations among these creeping bentgrass clonal linesthat may contribute to their fitness.

Activity of glucose-6-phosphate dehydrogenase (G6PDH), thefirst rate limiting enzyme in pentose phosphate pathway signifi-cantly differs among 20 creeping bentgrass clonal lines. G6PDHactivity drives pentose phosphate pathway toward biosynthesisof phenolics through shikimate pathway (Shetty, 1997, 2004).We hypothesized that creeping bentgrass clonal lines with highG6PDH activity (CB-2, CB-19, CB-20, CB-23, and CB-27) drive pri-mary metabolic flux towards phenylpropanoid pathway and betterable to stimulate higher antioxidant response system compared toclonal lines with low G6PDH activity and results in general confirmthis correlation (Table 4). Similar trend was observed with respectto proline dehydrogenase (PDH) activity, which indicates that pro-line oxidation is effective in all these creeping bentgrass clonallines with high G6PDH. Correlation between G6PDH activity andPDH activity in majority of creeping bentgrass clonal lines indi-cates that proline-coupled pentose phosphate pathway was likelystimulated. Total proline content in 20 bentgrass clonal lines alsoconfirms the relevance of pentose phosphate pathway and functionof proline in all these clonal lines. These observations have im-mense importance in terms of understanding the active metabolicrole of proline in cellular process to support energy (ATP) synthesisfrom mitochondrial oxidative phosphorylation. From these initialstudies it supports the hypothesis that under stress induced condi-tion specific creeping bentgrass clonal lines may shift to more en-ergy efficient proline-associated pentose phosphate pathwayprocess to generate proline for energy and at the same time pro-vide NADPH and sugar phosphates from pentose phosphate path-way to drive phenolic synthesis and antioxidant response system

to protect from oxidative damage. Activity of this pathway alsoconserves energy due to reduced activity of the TCA cycle andNADH generation, as proline can be a potential reductant in themitochondria for oxidative phosphorylation under biotic and abi-otic stress. All creeping bentgrass clonal lines showed a significantactivity of succinate dehydrogenase (SDH) under non-stressed con-dition, which is an important enzyme in tricaroboxylic cycle (TCA/Krebs cycle). This indicates that TCA/Krebs cycle is also active in allcreeping bentgrass clonal lines to generate NADH for supportingoxidative phosphorylation under normal conditions. However, un-der stress condition either both processes (proline-associated pen-tose phosphate pathway and TCA cycle) can be modulated and finetuned to generate energy and at the same time protect creepingbentgrass cells from oxidative stress or these processes can switchin between (more dependency towards proline-associated pentosephosphate pathway) to deliver more energy efficient tolerancemechanism to specific creeping bentgrass clonal lines.

Superoxide dismutase (SOD) and catalase (CAT) are two otherimportant antioxidant enzymes that protect cells from oxidativedamage, particularly under stress condition (Larkindale andHuang, 2004; Polle, 1997). From the results of this study, thesetwo antioxidant enzymes were significantly active in all creepingbentgrass clonal lines, which reflect that these two enzymes canscavenge free radicals in creeping bentgrass clonal lines to reducethe cellular damage. Differences in their activity among these bent-grass clonal lines also imply that oxidative pressure and countermeasures to it is not similar in magnitude in different single seededclonal lines. This variation could allow screening of different stresstolerant bentgrass clonal lines that could adapt cold stress. Evenunder high oxidative pressure this activity can be altered to main-tain homeostasis in specific creeping bentgrass clonal lines basedon the diversity of responses among clonal lines. During oxidativephosphorylation, there is always possibility of free radical genera-tion and activity of these antioxidant enzymes can significantlycounter and detoxify free radicals in the cytoplasm and in othercellular compartments (Polle, 1997; Shetty, 1999, 2004). The find-ings of our experiment inferred that proline-associated pentosephosphate pathway and its close association with antioxidant re-sponse system could play a significant role in specific creepingbentgrass clonal lines and contribute to stress tolerance.

Abitotic and biotic stress can sometimes reduce carboxylationefficiency, which causes decrease in the rate of generation of NADPHand ATP in plant cells (Baker and Rosenqvist, 2004). Regeneration ofribulose 1,5-bisphosphate is important for photosynthetic activityand this intermediate metabolite is also a product of pentose phos-phate pathway. Generally, under cold stress, reduction of photosyn-thetic activity has been observed in many plant species (Groom andBaker, 1992; Perks et al., 2004). Although creeping bentgrass is acool-season turfgrass species and it can tolerate sub freezing tem-perature for a significant amount of time, stress induced reductionof photochemical efficiency may be a part of the physiological re-sponse in this species. Our results showed a slight reduction of pho-tochemical efficiency in creeping bentgrass clonal lines at 4 �Ctemperature. As creeping bentgrass can tolerate lower temperaturethan this, our finding is just an indication of the cold stress inducedchanges in this species. The interesting part of this study is the rel-ative low photochemical efficiency and its correlation to low pheno-lic production along with lower antioxidant activity in some specificcreeping bentgrass clonal lines (CB-12, CB-13, CB-15, and CB-21)(Table 4). The above findings refer a strong association betweenthese biochemical characteristics in specific creeping bentgrass clo-nal lines and it reflects ultimately to the stress tolerance mecha-nism. Proline-associated pentose phosphate pathway is alsoassociated with this physiological response as high and low regula-tion of this pathway correlates with their tolerance behavior inmany of these single seeded bentgrass clonal lines.

D. Sarkar et al. / Bioresource Technology 100 (2009) 5332–5339 5339

In summary, this study confirms that genetic heterogeneity ispresent among diverse single seeded creeping bentgrass clonallines originating from a heterogeneous pool of seeds of cross-polli-nated cultivar (‘Penncross’). This genetic heterogeneity among var-ious clonal lines is also reflected in the synthesis of phenolicphytochemicals and the antioxidant response system. Correlationbetween PPP driven synthesis of these secondary metabolitesand proline catabolism in several specific clonal lines opens up abroader and critical metabolic regulation under stress in creepingbentgrass. Further it also dictates their stress tolerance behaviorwhich can determine the fitness criteria for specific clonal linesof this cross-pollinated species. This study has major implicationsto understand and link several redox-linked metabolic pathwaysin regulation of cold stress tolerance in creeping bentgrass. Thiscan provide strategies to develop new stress tolerant creepingbentgrass clonal lines that are consistent in cold stress response.This concept can be used to understand different stress tolerancemechanisms and molecular adjustments of other cool-seasonturfgrasses under abiotic stress. Further extension of this study willalso help to adjust agronomic management practices of turfgrassdepending on the level of stress by evaluating critical biochemicalresponse pathways.

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