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Environmental and Experimental Botany 79 (2012) 11– 20

Contents lists available at SciVerse ScienceDirect

Environmental and Experimental Botany

journa l h omepa g e: www.elsev ier .com/ locate /envexpbot

ffects of drought preconditioning on freezing tolerance of perennial ryegrass

indsey Hoffmana, Michelle DaCostaa,∗, J. Scott Ebdona, Jiuzhou Zhaob

Department of Plant, Soil, and Insect Sciences, University of Massachusetts, Amherst, MA 01003, United StatesDepartment of Landscape Architecture, Jiangxi University of Finance and Economics, Nanchang, China

r t i c l e i n f o

rticle history:eceived 30 June 2011eceived in revised form 4 January 2012ccepted 8 January 2012

eywords:old acclimationrought preconditioningreezing toleranceolium perenne

a b s t r a c t

Predicted increases in winter temperatures may negatively impact winter survival by preventing maxi-mal cold acclimation prior to freezing temperatures. Accordingly, research is needed to identify strategiesthat may help promote cold acclimation and increase freezing tolerance. Therefore, the objectives of thisresearch were to (i) examine the effects of drought preconditioning (DP) on freezing tolerance of twoperennial ryegrass (Lolium perenne L.) cultivars (‘Buccaneer’ and ‘Sunkissed’) under both non-cold accli-mating (20 ◦C) and cold acclimating (2 ◦C) conditions; and (ii) examine the physiological and biochemicalchanges in leaves and crowns of perennial ryegrass in response to DP. Plants of ‘Buccaneer’ and ‘Sunkissed’perennial ryegrass were subjected to the following treatments in a controlled environment chamber: (i)well-watered at 20 ◦C, (ii) DP at 20 ◦C, (iii) well-watered at 2 ◦C, and (iv) DP at 2 ◦C. Leaf and crown tissues

were harvested for analysis of freezing tolerance (lethal temperature resulting in 50% mortality, LT50),nonstructural carbohydrates, proline, soluble protein, and antioxidant enzyme activities. Drought pre-conditioning resulted in an improvement in freezing tolerance (lower LT50) for Buccaneer, but had nosignificant effect on freezing tolerance of Sunkissed. Furthermore, DP resulted in increases in carbohy-drate, proline, and soluble protein contents, but this response was dependent upon cultivar, tissue, andtemperature regime.

. Introduction

Recent reports by the Intergovernmental Panel on Climatehange (IPCC) indicate that global temperatures could increase by.1–6.4 ◦C within this century, accompanied by an increase in therequency and/or severity of superaoptimal temperatures (IPCC,001, 2007; Meehl and Tebaldi, 2004). During the last 30 years,he greatest warming trends have been observed in winter monthsnd at northern latitudes (IPCC, 2007), and significant increases inoth the occurrence and duration of winter warming episodes havelready been reported in Canada based on the period of 1950–1998Shabbar and Bonsal, 2003). Consequently, elevated temperatures

ay negatively impact winter survival by preventing maximal cold

cclimation prior to freezing temperatures, while more frequentemperature fluctuations throughout winter months may resultn premature losses in freezing tolerance (Bélanger et al., 2002;

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; DP, drought precon-itioning; LT50, lethal temperature resulting in 50% mortality; MDS, total mono-nd disaccharides; POD, guaiacol peroxidase; SC, total storage carbohydrates; SOD,uperoxide dismutase; WW, well-watered.∗ Corresponding author at: 17 Stockbridge Hall, 80 Campus Center Way, Univer-

ity of Massachusetts-Amherst, MA 01003, United States. Tel.: +1 413 545 2547;ax: +1 413 545 3958.

E-mail address: mdacosta@psis.umass.edu (M. DaCosta).

098-8472/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2012.01.002

© 2012 Elsevier B.V. All rights reserved.

Thorsen and Höglind, 2010a). Therefore, research is needed toidentify strategies that may help to promote cold acclimation andincrease plant freezing tolerance.

One strategy to improve freezing tolerance could involve expos-ing plants to a mild stress, other than cold temperatures, to inducecellular changes similar to those associated with cold acclimation.This phenomenon of ‘cross-adaptation’ is based on the overlap ofabiotic stress signaling pathways, and may allow plants to developtolerance to multiple stresses through exposure to a single abi-otic stress (Knight and Knight, 2001; Chinnusamy et al., 2004).For example, Jiang and Huang (2001) found that exposing Ken-tucky bluegrass (Poa pratensis L.) plants to mild drought stress (ordrought preconditioning, DP) led to an enhancement in heat toler-ance, which was attributed to higher root production and higherosmotic adjustment in DP plants. In a different study, salt toler-ance of a salt-sensitive maize (Zea mays L.) genotype was increasedfollowing preconditioning with hydrogen peroxide (H2O2), whichwas associated with higher antioxidant scavenging capacity andimproved growth rates during salt stress (Azevedo Neto et al.,2005). Based on these previous investigations, there seems to bepotential for the use of cross-adaptation as a tool to enhance plant

abiotic stress tolerance.

Freezing stress at the cellular level can be manifested as osmoticand oxidative stresses, which are also common responses todrought. Both freezing and drought rely on similar mechanisms

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hat allow plants to maximize cellular stability and minimizenjury associated with osmotic and oxidative stresses, and there-ore significant overlap in gene regulation has been observeduring exposure to both these stresses (Shinozaki and Yamaguchi-hinozaki, 2000; Puhakainen et al., 2004; Peng et al., 2008;ommasini et al., 2008). For example, increases in both droughtnd freezing tolerance have been associated with the capacity toccumulate similar protective compounds that minimize the neg-tive effects of desiccation, including carbohydrates (e.g. sucrose,ructans), amino acids (e.g. proline), and proteins (e.g. dehydrins,ntioxidant enzymes) (Ingram and Bartels, 1996; Bray, 1997;oekstra et al., 2001). As a result, exposing plants to DP may help tonhance freezing tolerance in some plant species (Gusta et al., 1980;homas and James, 1993; Medeiros and Pockman, 2011). Freez-ng tolerance of winter wheat (Triticum aestivum L.) and winter ryeSecale cereal L.) epicotyls increased following exposure to a 24-hrought period, resulting in a similar degree of freezing toleranceetween drought stressed and cold acclimated plants (Cloutier andiminovitch, 1982). In a different study, Guy et al. (1992) foundhat freezing tolerance of spinach seedlings (Spinacia oleracea L.)ncreased following exposure to water stress and was associated

ith the accumulation of several high-molecular-weight proteins.dditional research is necessary, however, in order to understand

he underlying factors associated with drought-induced enhance-ent of freezing tolerance.Temperate perennial grasses represent an economically impor-

ant group of plants that are used for forage and turf production,ith the expanding use of some perennial grasses as bioenergy

rops. In regards to low temperature stress, winter injury of someemperate perennial grasses can be a significant problem in north-rn climatic regions due to insufficient freezing tolerance, whichan be attributed to genetic variability among species and culti-ars (Gusta et al., 1980; Bélanger et al., 2002; Hulke et al., 2008).or example, perennial ryegrass (Lolium perenne L.) is one of theost widely utilized grasses worldwide for forage and turf due

o rapid establishment rates, excellent forage quality, high yield,nd good tolerance to grazing and traffic; however, low temper-tures and lack of snow cover can limit the persistence of thisrass in northern climates. Predicted future increases in temper-ture and occurrence of winter warming episodes may increaseotential for winter injury by negatively impacting the capacity tochieve and/or maintain freezing tolerance. Thorsen and Höglind2010b) reported that under the warmest climate change scenario,reezing tolerance of perennial grasses may decrease by up to 3.9 ◦Cn certain regions. Additional research is needed to identify strate-ies that may enhance cold acclimation and maximize freezingolerance in perennial grasses. Therefore, the primary objectivesor this research were to (i) examine the effects of DP on freezingolerance of two perennial ryegrass cultivars under both non-coldcclimating (20 ◦C) and cold acclimating (2 ◦C) conditions; and (ii)xamine the physiological and biochemical changes in leaves androwns of perennial ryegrass in response to DP. We hypothesizedhat DP may improve freezing tolerance of perennial ryegrass inhe absence of cold acclimation by increasing the production ofrotective compounds, including compatible solutes and antioxi-ant enzymes. Furthermore, we wanted to address whether DP mayave a synergistic effect when conducted during a cold acclimationeriod.

. Materials and methods

.1. Plant material and growth conditions

Plants of ‘Buccaneer’ (poor spring quality) and ‘Sunkissed’good spring quality) perennial ryegrass were selected based on

perimental Botany 79 (2012) 11– 20

contrasting performance and survival data from the National Tur-fgrass Evaluation Program (NTEP) perennial ryegrass test at theOrono, Maine location (NTEP, 2003). Orono, Maine is the northern-most NTEP test site in New England and among the northernmost inthe contiguous United States, and we hypothesized that contrastingspring survival and quality at this site could be related to intraspe-cific differences in freezing tolerance based on previous research(Ebdon et al., 2002). The two cultivars were seeded into pots (10 cmwidth, 10 cm length, and 36 cm depth) at a rate of 4 mg cm−2.Following a three-week germination period, plants were clippedweekly to a height of approximately 5 cm, watered three times perweek, and fertilized weekly with full strength Hoagland solution(Hoagland and Arnon, 1950). After four months in a greenhouse,plants were transferred to a controlled-environment growth cham-ber (Conviron, Winnipeg, Canada) maintained at 20/15 ◦C day/nighttemperatures with a 10-h photoperiod and photosynthetic photonflux density (PPFD) of 500 �mol m−2 s−1 at plant height. Plants weremaintained under these growth chamber conditions for 7 d prior totreatment initiation.

2.2. Treatments

In order to determine whether DP could enhance freezingtolerance under non-cold acclimating and/or cold acclimating con-ditions, we designed four treatments as follows: (i) well-watered(WW) under a non-cold acclimating temperature regime (20 ◦C)(WW 20 ◦C), (ii) drought preconditioned (DP) under a non-coldacclimating temperature regime (20 ◦C) (DP 20 ◦C), (iii) WW undera cold acclimating temperature regime (2 ◦C) (WW 2 ◦C), and (iv)DP under a cold acclimating temperature regime (2 ◦C) (DP 2 ◦C).Well-watered pots were irrigated four times per week to maintainsoil moisture at pot capacity, which corresponded to approximately20% volumetric moisture content in the top 20 cm of soil (v/v). Inour study, DP was conducted using wilt-based irrigation, which hasbeen shown to induce mild drought stress resulting in osmoticadjustment in grass species (Jiang and Huang, 2001; Qian andFry, 1996). Water was withheld until visual signs of leaf foldingoccurred, at which point plants were re-watered to pot capacity. Inaddition, measurements of leaf relative water (RWC) content andelectrolyte leakage (EL) were conducted at the completion of eachwilt cycle at 20 ◦C and 2 ◦C to assess any detrimental effects of DP ongrass quality. Overall, leaf RWC did not fall below 87% for DP plants(compared to approximately 94% for WW plants), and EL was notsignificantly different between DP and WW treatments (data notshown). This suggested that DP caused mild drought stress with nosignificant damage to plants.

Plants maintained under non-cold acclimating conditions con-sisted of 20/15 ◦C day/night temperatures, 10-h photoperiod, andphotosynthetic photon flux density of 500 �mol m−2 s−1. At 20 ◦C,plants maintained under the DP irrigation regime were subjected tofive wilt cycles, each of which lasted approximately seven days, fora total duration of 35 d of treatment. The number of wilt cycles wasselected based on previous research in turfgrass that demonstratedthat a total of five wilt cycles was sufficient to induce physiolog-ical changes associated with osmotic adjustment (Qian and Fry,1996). At the completion of each wilt cycle, all plants were re-watered to pot capacity prior to starting the next wilt cycle. Anadditional group of plants also were subjected to five wilt events at20 ◦C as described above. These plants were then exposed to a coldacclimation regime, which consisted of a constant 2 ◦C (day/nighttemperatures), 10-h photoperiod, and PPFD of 250 �mol m−2 s−1.

During the cold acclimation period, DP treatments were maintainedusing two additional wilt cycles. Due to the lower evaporativedemand at low temperature, each wilt event lasted approximately15 d for a total of 30 d at 2 ◦C.

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Prior to destructive harvesting, all plants were subjected to a8-h re-watering period to ensure that leaf and crown material ofW and DP treatments were at similar water status. This methodas based on previous research by Morgan (1984) and Blum

1989) which indicated that rehydration of plant tissue followingrought stress is necessary to differentiate between constitutivend induced osmotic adjustment. One set of plants was harvestedt the end of five wilt cycles at 20 ◦C, and another set of plantsas harvested after five wilt cycles at 20 ◦C followed by two wilt

ycles at 2 ◦C. Leaves and crowns (approximately 5 mm above andelow the apex) were collected for carbohydrate, proline, solublerotein, and antioxidant enzyme activity determination. In addi-ion, whole plants (leaves, crowns, and roots) were sampled forreezing tolerance assessment.

.3. Freezing tolerance assessment

Freezing tolerance was determined based on whole-plant sur-ival following controlled freezing tests according to the methodsreviously described by Ebdon et al. (2002). Briefly, ten individuallants were wrapped in a pre-moistened paper towel to ensure iceucleation, placed in freezer bags, and stored at 5 ◦C until sam-le preparation was complete. For each test temperature thereere four replicates containing ten plants, for a total of 40 plantser test temperature per treatment. Following preparation, freezerags containing plants were placed in a programmable freezinghamber (Scientemp Corp., Adrian, MI) and subjected to a rangef eight temperatures consisting of a non-frozen control (5 ◦C)nd seven freeze-test temperatures: −1, −3, −5, −7, −9, −11, and13 ◦C. The freezer was cooled in a stepwise fashion at a rate of◦C h−1 to the desired temperature and held at each test tempera-

ure for 1 h. After each target temperature was reached, plants wereemoved from the freezer and allowed to thaw for at least 12 ht 5 ◦C. After thawing, plants were replanted into cell trays filledith commercial potting media (Pro-Mix; Griffin Greenhouse andursery Supplies, Tewksbury MA) and then placed in a greenhouset approximately 20 ◦C. Following a three-week regrowth period,hole plant survival (%) was calculated for each replicate as: (no.lants survived/total no. plants) × 100. The lethal temperature athich 50% of plants were killed (LT50) was determined by curvetting percent survival to temperature using a four-parameter sig-oid model (Sigma Plot, SPSS, Chicago, IL).

.4. Carbohydrate assay

Leaf and crown materials were initially dried at 100 ◦C and thenubjected to 70 ◦C for at least 72 h before being ground. This pro-ess was utilized to prevent large scale changes in carbohydratesue to degradation or enzymatic conversion that may occur wheneat-drying tissues. Total non-structural carbohydrates (includinglucose, fructose, sucrose, fructans, and starch) were determinedsing the method described by Fu and Dernoeden (2009). Briefly,0 mg of dried ground tissues were placed in a 2 mL microcentrifugeube containing 1 mL of 92% ethanol, vortexed to ensure that tis-ues were dispersed evenly, and then centrifuged at 14,000 rpmor 10 min. The supernatant was collected and transferred to 15 mLubes and the extraction procedure was repeated an additional

times for a total of 3 mL of supernatant per sample. Followingxtraction, uncapped microcentrifuge tubes were placed in an ovent 70 ◦C overnight to evaporate any remaining ethanol and then theesidue was used for determination of starch and fructan concen-ration.

For determination of reducing sugars (glucose and fructose), thextracted solution (3 mL) was diluted with distilled water to a totalolume of 10 mL. A 0.2 mL aliquot of the diluted extraction solu-ion was transferred into a 20 mL volumetric test tube containing

perimental Botany 79 (2012) 11– 20 13

0.8 mL distilled water and 1.25 mL alkaline ferricyanide reagent.The mixture was heated at 100 ◦C in a water bath for 10 min, quicklycooled in an ice bath, and then 2.5 mL of 2 N H2SO4 was addedand tubes were vigorously shaken. Lastly, 1.0 mL arsenomolybdatesolution was added and the total volume was adjusted to 25 mLwith distilled water. The absorbance of the solution was mea-sured at 515 nm using a spectrophotometer (Spectronic Genesys2, Thermo Electron Corporation, Madison, WI). The reducing sugarcontent of the solution was calculated based on reference to a glu-cose standard curve and expressed on a milligram per gram dryweight basis.

For sucrose hydrolysis, a 2 mL aliquot of diluted supernatantwas transferred to glass tubes containing 2 mL 4% H2SO4 (w/v),vortexed, and then boiled at 100 ◦C in a water bath for 15 min.After cooling to room temperature, the samples were neutralizedwith 1 mL 1 N NaOH, and 0.2 mL of this mixture was transferredinto a 20 mL volumetric test tube containing 0.8 mL distilled waterand 1.25 mL alkaline ferricyanide reagent. The mixture was heatedin a water bath at 100 ◦C for 10 min, quickly cooled in an icebath, and then 2.5 mL of 2 N H2SO4 was added. A 1.0 mL aliquotof arsenomolybdate solution was added and the total volumewas adjusted to 25 mL with distilled water. The absorbance ofthe solution was measured at 515 nm using a spectrophotometer(Spectronic Genesys 2, Thermo Electron Corporation, Madison, WI).This solution was used for quantification of total mono- and disac-charides (MDS), which consisted of the sum of glucose, fructose andsucrose. The sucrose content was subsequently calculated as thedifference between MDS and reducing sugar content and expressedon a milligram per gram dry weight basis.

For determination of total storage carbohydrates (SC) (fructansand starch), 0.5 mL of distilled water was added to the microcen-trifuge tubes containing tissue residues and heated for 10 min at100 ◦C in a water bath. Samples were allowed to cool to room tem-perature (approximately 25 ◦C) then 0.4 mL 200 mM acetate buffer(pH 5.1) and 0.1 mL enzyme solution containing amyloglucosidase(A1602 from Aspergillus niger, Sigma–Aldrich Co., St. Louis, MO)and �-amylase (A6255, from porcine pancreas, Sigma–Aldrich Co.,St. Louis, MO) was added. Tubes were sealed, vortexed, and thenincubated at 55 ◦C for 16 h in a water bath. Samples were vortexedseveral additional times during incubation. Following incubation,samples were centrifuged at 14,000 rpm for at least 20 min or untiltissue residue was pelletized. Supernatant was poured into 15 mLcentrifuge tubes and diluted to a total volume of 10 mL with dis-tilled water (1:10 v/v). For determination of starch content, a 0.2 mLaliquot was removed and quantified using the methods describedabove for reducing sugar determination. Fructans were hydrolyzedto fructose by combining 0.1 mL 1 N H2SO4 with a 0.9 mL aliquotof extract. The solution was vortexed, boiled at 100 ◦C for 15 minthen allowed to cool to room temperature before adding 0.1 mL 1 NNaOH. A 0.2 mL aliquot was used to determine SC content by themethods used to determine reducing sugar content, as describedabove. The content of fructans was calculated as the differencebetween SC and starch and expressed on a milligram per gram dryweight basis.

2.5. Proline assay

Leaf and crown tissues were harvested, submerged in liquidnitrogen, and stored at −80 ◦C until proline content was mea-sured according to the method of Bates et al. (1973). A sampleweighing 300 mg (fresh weight) was homogenized in 3% sulfos-alicylic acid and filtered through Whatman #2 paper. Following

filtration, 2 mL of the extracted solution was combined with 2 mLacid–ninhydrin and 2 mL glacial acetic acid and heated at 100 ◦C ina water bath for 1 h. The reaction was terminated in an ice bath andthen 4 mL of toluene was added to the mixture. Following phase

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disaccharides (MDS) in Fig. 2. In most cases, the MDS content inleaves and crowns did not change in response to DP 20 ◦C; how-ever, leaf MDS content for Buccaneer was lower in response to DP20 ◦C (54.0 mg g−1 DW) compared to WW 20 ◦C (72.1 mg g−1 DW).

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4 L. Hoffman et al. / Environmental a

eparation, the upper chromophore containing toluene was trans-erred to a cuvette and measured at 520 nm in a spectrophotometerSpectronic Genesys 2, Thermo Electron Corporation, Madison, WI).roline concentration was determined based on reference to a stan-ard curve using l(−)-proline (Acros Organics, Atlanta, Georgia)nd calculated on a �mol per gram fresh weight basis.

.6. Soluble protein and antioxidant enzyme extraction

Approximately 300 mg of leaf and crown tissues (fresh weight)ere harvested, frozen in liquid nitrogen, and stored at −80 ◦C foretermination of soluble protein and antioxidant enzyme activities.xtraction was based on methods previously described by DaCostand Huang (2007). Tissues were homogenized in 4 mL of 150 mMold phosphate buffer (pH 7.0) and centrifuged at 12,000 rpm for0 min at 4 ◦C. The supernatant was transferred to 15 mL tubes andsed for soluble protein and enzyme activity determination.

.7. Total soluble protein assay

Soluble protein determination was based on the Bradford assay1976). A 3 mL aliquot of solution containing Coomassie blue G-250nd 95% ethanol was added to a tube containing 100 �L of pro-ein extract. Samples were vortexed and absorbances were readt 595 nm following a 5 min color development period. Proteinoncentration was determined using bovine serum albumin as atandard and expressed on a milligram per gram fresh weight basis.

.8. Antioxidant enzyme activity assays

Ascorbate peroxidase (APX) activity was determined using theethods of Nakano and Asada (1981) with modifications. The oxi-

ation of ascorbate was initiated by adding a 100 �L aliquot ofxtract to a solution containing 100 mM sodium acetate bufferH 5.8, 3 �M EDTA (ethylenediaminetetraacetic acid) and 5 mM2O2. The enzyme activity was determined by measuring changes

n absorbances every 10 s for 60 s using a spectrophotometerGenesys 2, Thermo Electron Corporation, Madison, WI). One unitf APX activity was defined as the change in absorbance of 0.01er min and calculated on a units per milligram soluble proteinasis.

Activity of catalase (CAT) was determined using the methodsf Chance and Maehly (1955) with modifications. The oxidation of2O2 was initiated by adding a 100 �L aliquot of extract to a solu-

ion containing 50 mM phosphate buffer (pH 7.0) and 45 mM H2O2.he enzyme activity was determined by measuring changes inbsorbances every 10 s for 60 s using a spectrophotometer. One unitf CAT activity was defined as the change in absorbance of 0.01 perin and calculated on a units per milligram soluble protein basis.Superoxide dismutase (SOD) activity was determined using the

ethods of Giannopolitis and Ries (1977) with modifications. A00 �L aliquot of extractant was added to a solution containing0 mM phosphate buffer (pH 7.8), 60 �M riboflavin, 195 mM methi-nine, 3 �M EDTA, and 1.125 mM nitro blue tetrazolium (NBT) dite-razolium chloride. A solution using no enzyme extract was useds a control. Test tubes were irradiated under fluorescent lights atpproximately 300 �mol−2 m−2 s−1 for 30 min and then placed inhe dark for 10 min to stop the reaction. The absorbance was mea-ured at 560 nm and one unit of enzyme activity was defined as themount of enzyme that would inhibit 50% of NBT photoreductionnd calculated on a units per milligram soluble protein basis.

Guaiacol peroxidase (POD) activity was determined using the

ethods of Chance and Maehly (1955) with modifications. The

xidation of guaiacol was initiated by adding a 50 �L aliquot ofxtractant to a solution containing 0.1 mM sodium acetate bufferpH 5.0), 0.25% guaiacol, and 0.75% H2O2. The enzyme activity

perimental Botany 79 (2012) 11– 20

was determined by measuring changes in absorbances every 10 sfor 60 s using a spectrophotometer. One unit of POD activity wasdefined as the change in absorbance of 0.01 per min and calculatedon a units per milligram soluble protein basis.

2.9. Experimental design and statistical analysis

Plants were arranged as a completely randomized blockdesign. The experiment consisted of two cultivars (Buccaneer andSunkissed), four treatments (WW 20 ◦C, DP 20 ◦C, WW 2 ◦C, DP 2 ◦C),and four replicates per cultivar-treatment combination, for a totalof 32 experimental units. All data were subjected to analysis of vari-ance (ANOVA) according to the general linear model procedure forthe Statistical Analysis System v. 9.1 (SAS Institute, Inc. Cary, NC)and means were separated by Fisher’s protected least significantdifference (LSD) test at the 0.05 probability level.

3. Results

3.1. Changes in freezing tolerance (LT50)

Based on estimation of LT50, Sunkissed exhibited higher base-line freezing tolerance (LT50 of −11.1 ◦C) compared to Buccaneer(LT50 of −9.0 ◦C) when grown under well-watered and non-coldacclimating conditions (WW 20 ◦C) (p < 0.05) (Fig. 1). Following coldacclimation, a similar trend was observed where Sunkissed exhib-ited higher freezing tolerance compared to Buccaneer (statisticallysignificant at the p < 0.10 level). Although the freezing tolerance ofboth Sunkissed and Buccaneer increased in response to cold accli-mation at 2 ◦C, the cultivars varied in their response to DP. Thefreezing tolerance of Buccaneer significantly increased from −9.0 ◦Cat WW 20 ◦C to −11.0 ◦C in response to DP 20 ◦C, and achieved thegreatest level of freezing tolerance in response to DP 2 ◦C (LT50 of−14.0 ◦C). In contrast, exposing Sunkissed to DP did not result inany improvement in freezing tolerance compared to WW plants,regardless of temperature regime.

3.2. Changes in carbohydrate concentrations

The sum of the glucose, fructose, and sucrose carbohydratefractions in leaves and crowns are presented as total mono- and

mortality (LT50), of Buccaneer and Sunkissed perennial ryegrass following expo-sure to WW 20 ◦C, DP 20 ◦C, WW 2 ◦C, and DP 2 ◦C treatments. Freezing tests wereconducted following five wilt events at 20 ◦C and two wilt events at 2 ◦C. For each cul-tivar, letters are LSD values (p ≤ 0.05) indicating statistically significant differencesamong treatments.

L. Hoffman et al. / Environmental and Experimental Botany 79 (2012) 11– 20 15

Table 1Changes in sucrose and fructan concentrations in leaf and crown tissue of Buccaneer and Sunkissed. Treatments consisted of exposure to well-watered 20 ◦C (WW 20 ◦C),drought preconditioning 20 ◦C (DP 20 ◦C), well-watered 2 ◦C (WW 2 ◦C), and drought preconditioning 2 ◦C (DP 2 ◦C).

Cultivar Treatments Leaf Crown

Sucrose (mg g−1 DW) Fructan (mg g−1 DW) Sucrose (mg g−1 DW) Fructan (mg g−1 DW)

Buccaneer

WW 20 ◦C 49.1 ba 11.8 b 21.5 c 23.5 dDP 20 ◦C 34.2 c 12.3 b 25.1 c 30.4 cWW 2 ◦C 82.9 a 21.6 a 56.5 a 55.8 bDP 2 ◦C 83.2 a 24.7 a 40.2 b 65.4 a

Sunkissed

WW 20 ◦C 43.9 b 10.5 d 24.4 b 25.3 cDP 20 ◦C 49.9 b 15.3 c 25.9 b 27.1 cWW 2 ◦C 77.2 a 30.8 a 55.8 a 63.9 b

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a For each cultivar, means followed by the same letter within a column are not si

verall, cold acclimation at 2 ◦C resulted in an increase in leaf andrown MDS accumulation compared to that at 20 ◦C; however,xposing plants to DP 2 ◦C did not result in any additional increasesn MDS for Buccaneer and Sunkissed. Among the carbohydrates

aking up the MDS fraction, sucrose accounted for approximately4% and 79% of leaf and crown MDS, respectively, when averagedver cultivar and treatments (Table 1). As a result, the accumulationf sucrose followed similar trends to that observed for MDS. In leafissues, sucrose content increased in response to cold acclimationt 2 ◦C for both cultivars; however, leaf sucrose content for Bucca-eer was lower in response to DP 20 ◦C (34.2 mg g−1 DW) comparedo WW 20 ◦C (49.1 mg g−1 DW). In crowns, similar responses werebserved with the exception that Buccaneer plants exposed to DP◦C had lower sucrose levels (40.2 mg g−1 DW) compared to plants

◦ −1

t WW 2 C (56.5 mg g DW). In general, crown tissue of bothultivars accumulated lower amounts of sucrose compared to leafissues.

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ig. 2. Total mono- and disaccharides (MDS) in leaf and crown tissues of Bucca-eer and Sunkissed perennial ryegrass following exposure to WW 20 ◦C, DP 20 ◦C,W 2 ◦C, and DP 2 ◦C. For each cultivar, different letters are LSD values (p ≤ 0.05)

ndicating statistically significant differences among treatments.

55.8 a 76.1 a

antly different based on Fisher’s protected LSD (p ≤ 0.05).

The sum of the fructan and starch carbohydrate fractions (SC)in leaves and crowns are presented as total storage carbohy-drates in Fig. 3. Buccaneer did not exhibit any differences in leafSC accumulation at DP 20 ◦C compared to WW 20 ◦C; however,Sunkissed accumulated higher levels of leaf SC in response to DP20 ◦C (31.6 mg g−1 DW) compared to WW 20 ◦C (24.9 mg g−1 DW)(Fig. 3). Exposure to 2 ◦C resulted in a significant increase in leafSC content, but DP 2 ◦C did not result in any further changes inleaf SC content for either cultivar. There was a significantly higheraccumulation of SC in crowns compared to leaf tissues, regardlessof cultivar or treatment. In crowns of Buccaneer, DP 20 ◦C resultedin higher SC content compared to that at WW 20 ◦C. In addition,DP 2 ◦C resulted in higher SC content for Buccaneer and Sunkissed(78.1 and 87.1 mg g−1 DW, respectively) compared to plants at WW2 ◦C (69.4 and 76.1 mg g−1 DW, respectively). Fructans accounted

for approximately 59% of SC in leaves and 82% of SC in crowns,averaged over both cultivars and all treatments (Table 1). Leaffructan content for Buccaneer did not change in response to DP

0

20

40

60

80

100

0

20

40

60

80

100

Buccaneer Sun kissed

WW 20 °C

DP 20 °C

WW 2°C

DP 2°C

WW 20 °C

DP 20 °C

WW 2°C

DP 2°C

mg g

-1D

W

b b

aa

d

c

b

a

bc

a a

cc

b

a

Leaf

Crown

mg g

-1D

W

Fig. 3. Total storage carbohydrates (SC) in leaf and crown tissue of Buccaneer andSunkissed perennial ryegrass following exposure to WW 20 ◦C, DP 20 ◦C, WW 2 ◦C,and DP 2 ◦C. For each cultivar, different letters are LSD values (p ≤ 0.05) indicatingstatistically significant differences among treatments.

16 L. Hoffman et al. / Environmental and Experimental Botany 79 (2012) 11– 20

0

2

4

6

8

10

12

14

0

2

4

6

8

10

12

14

Buccaneer Sunkissed

WW 20°C

DP 20°C

WW 2°C

DP 2°C

WW 20°C

DP 20°C

WW 2°C

DP 2°C

µm

ol g

-1F

W

Leaf

Crown

µm

ol g

-1F

W

c b

a a

c c

b a

c c

b

a

cc

b

a

Fig. 4. Proline levels in leaf and crown tissue of Buccaneer and Sunkissed perennialrcd

2t2DfDct2fsc

3

FNirtaetap

3

bhbep

0

2

4

6

8

10

12

14

0

2

4

6

8

10

12

14

Buccaneer Sunkissed

WW 20°C

DP 20°C

WW 2°C

DP 2°C

WW 20°C

DP 20°C

WW 2°C

DP 2°C

mg

g-1

FW

Leaf

Crown

mg

g-1

FW

cbc

b

a

a

a

a a

cb b

a ab

cd

Fig. 5. Soluble protein levels in leaf and crown tissue of Buccaneer and Sunkissed

yegrass following exposure to WW 20 ◦C, DP 20 ◦C, WW 2 ◦C, and DP 2 ◦C. For eachultivar, different letters are LSD values (p ≤ 0.05) indicating statistically significantifferences among treatments.

0 ◦C, whereas Sunkissed exhibited a significant increase in fruc-ans from 10.5 mg g−1 DW at WW 20 ◦C to 15.3 mg g−1 DW at DP0 ◦C. Sunkissed crown fructan levels did not change in response toP 20 ◦C compared to WW 20 ◦C; however, a significant increase in

ructans was detected in Buccaneer crown tissue from 23.5 mg g−1

W for WW 20 ◦C to 30.4 mg g−1 DW for DP 20 ◦C. Cold acclimationaused a significant increase in leaf fructan content for both cul-ivars compared to non-cold acclimating conditions; however DP◦C resulted in a lower fructan concentration compared to WW 2 ◦C

or Sunkissed. Buccaneer and Sunkissed crown tissue respondedimilarly to DP 2 ◦C with a significant increase in fructan levelsompared to WW 2 ◦C.

.3. Changes in proline content

Leaf proline content for Buccaneer increased from 0.3 �mol g−1

W for WW 20 ◦C to 0.7 �mol g−1 FW in response to DP 20 ◦C (Fig. 4).o differences in leaf proline content were detected for Buccaneer

n response to DP 2 ◦C; however, leaf proline content for Sunkissedeached its highest concentration (4.7 �mol g−1 FW) in responseo DP 2 ◦C compared to all other treatments. For both Buccaneernd Sunkissed, crown proline levels were not significantly differ-nt between DP 20 ◦C and WW 20 ◦C; however, DP 2 ◦C resulted inhe highest crown proline accumulation for both cultivars. Over-ll, exposure to 2 ◦C resulted in a 9-fold increase in leaf and crownroline levels for both Buccaneer and Sunkissed.

.4. Changes in soluble protein

There were no differences in leaf soluble protein contentetween DP 20 ◦C and WW 20 ◦C for either Buccaneer or Sunkissed;

owever, DP 20 ◦C resulted in significantly higher crown solu-le protein content for both cultivars (Fig. 5). When plants werexposed to 2 ◦C, DP caused a significant increase in leaf solublerotein content of Buccaneer (total of 12.6 mg g−1 FW) compared

perennial ryegrass following exposure to WW 20 ◦C, DP 20 ◦C, WW 2 ◦C, and DP 2 ◦C.For each cultivar, different letters are LSD values (p ≤ 0.05) indicating statisticallysignificant differences among treatments.

to WW plants, but no significant differences were observed forSunkissed (average of 10.2 mg g−1 FW across all treatments). Sim-ilar to that observed for crowns at 20 ◦C, DP 2 ◦C resulted insignificantly higher crown soluble protein content for both Buc-caneer (3.3 mg g−1 FW) and Sunkissed (3.5 mg g−1 FW).

3.5. Changes in antioxidant enzyme activity

Buccaneer exhibited higher leaf SOD and POD activities inresponse to DP 20 ◦C compared to WW 20 ◦C; however, therewere no additional differences in antioxidant enzyme activitiesfor Buccaneer in response to DP, regardless of temperature or tis-sue type (Table 2). For Sunkissed, exposure to DP 20 ◦C resultedin significantly higher leaf APX and CAT activities, and highercrown CAT activity compared to WW 20 ◦C. In general, cold accli-mation resulted in lower antioxidant enzyme activities in leavesand crowns of both cultivars compared to that observed at 20 ◦C(Table 2). For Buccaneer, there were no differences in leaf or crownantioxidant enzyme activities in response to DP 2 ◦C. In contrast,Sunkissed exhibited significantly higher leaf SOD and APX activitiesin response to DP 2 ◦C compared to WW 2 ◦C. The only additionalobserved change in crowns of Sunkissed was a decrease in POD inresponse to DP 2 ◦C compared to WW 2 ◦C.

4. Discussion

Information published by the IPCC indicates that global meansurface air temperatures will continue to increase in the future,with a projected winter warming exceeding 4 ◦C in northernregions of North America (IPCC, 2007). In general, predicted future

climate change scenarios will result in less than optimal cold accli-mation conditions, leading to decreases in freezing tolerance andpredisposition of plants to winter injury. In a study examining theNormalized Difference Vegetation Index from 1982 to 1999, Zhou

L. Hoffman et al. / Environmental and Ex

Tab

le

2C

han

ges

in

asco

rbat

e

per

oxid

ase

(APX

),

cata

lase

(CA

T), s

up

erox

ide

dis

mu

tase

(SO

D),

and

guai

acol

per

oxid

ase

(PO

D) a

ctiv

itie

s

in

leaf

and

crow

n

tiss

ue

of

Bu

ccan

eer

and

Sun

kiss

ed. T

reat

men

ts

con

sist

ed

of

exp

osu

re

to

wel

l-w

ater

ed20

◦ C

(WW

20◦ C

),

dro

ugh

t

pre

con

dit

ion

ing

20◦ C

(DP

20◦ C

),

wel

l-w

ater

ed

2◦ C

(WW

2◦ C

),

and

dro

ugh

t

pre

con

dit

ion

ing

2◦ C

(DP

2◦ C

).

Cu

ltiv

ar

Trea

tmen

ts

Leaf

Cro

wn

APX

(un

its

mg−1

SP)

CA

T

(un

its

mg−1

SP)

SOD

(un

its

mg−1

SP)

POD

(un

its

mg−1

SP)

APX

(un

its

mg−1

SP)

CA

T

(un

its

mg−1

SP)

SOD

(un

its

mg−1

SP)

POD

(un

its

mg−1

SP)

Bu

ccan

eer

WW

20◦ C

4.1

aa5.

0

a

53.1

b

40.9

b

19.9

a

1.8

a

284.

9

a

87.1

aD

P

20◦ C

5.0

a

5.5

a

64.1

a

51.8

a

17.6

ab

1.7

ab

248.

5

a 93

.9

aW

W

2◦ C

4.3

a

2.6

b

28.4

c

25.6

c

14.1

b

1.4

b

154.

5 b

51.2

bD

P

2◦ C

4.3

a

2.5

b

24.0

c

30.7

c

14.5

b

1.3

b

153.

8 b

67.9

b

Sun

kiss

ed

WW

20◦ C

3.9

c

4.4

b

44.1

a

31.7

ab

19.0

ab

1.6

b

345.

0

a

82.5

aD

P

20◦ C

4.7

ab

5.2

a

52.3

a

39.9

a

19.2

a

2.4

a

219.

1

b

97.5

aW

W

2◦ C

4.1

bc

2.8

c

22.8

c

31.6

b

16.3

bc

1.4

b

146.

2

c

95.8

aD

P

2◦ C

4.9

a

2.7

c

34.0

b

38.9

ab

14.7

c

1.3

b

135.

3

c

51.1

b

aFo

r

each

cult

ivar

, mea

ns

foll

owed

by

the

sam

e

lett

er

wit

hin

a

colu

mn

are

not

sign

ifica

ntl

y

dif

fere

nt

base

d

on

Fish

er’s

pro

tect

ed

LSD

(p

≤

0.05

).

perimental Botany 79 (2012) 11– 20 17

et al. (2001) reported an increase in the growing season by 4–12 din North America and 19–24 d in Europe. Overall, longer autumngrowing periods may prevent plants from attaining sufficient freez-ing tolerance prior to winter months. For example, Höglind et al.(2008) found that a 2 ◦C increase in autumn temperatures post-poned cold acclimation and in turn resulted in a decrease in freezingtolerance for both timothy (Phleum pratense L.) and perennial rye-grass. Due to increased likelihood of freezing injury, it has becomeincreasingly important to identify traits necessary for winter sur-vival under current and future winter climate scenarios.

Drought preconditioning has been shown to be effective inimproving tolerance to different abiotic stresses, including lowtemperature. Cultivars of winter wheat and winter rye were shownto achieve a similar degree of freezing tolerance whether exposedto DP or a conventional cold acclimation period (Cloutier andSiminovitch, 1982). Thomas and James (1993) also reported a sim-ilar increase in freezing tolerance due to a six-week drought periodin perennial ryegrass compared to a cold acclimation period. In thecurrent study, we found that the greatest gain in freezing toler-ance for both cultivars was associated with cold acclimation (2 ◦Cfor 30 d), and that the effects of DP on freezing tolerance var-ied with the cultivar. Specifically, DP resulted in an increase infreezing tolerance for Buccaneer in the absence of cold acclima-tion (20 ◦C), and DP also had a synergistic effect at 2 ◦C to improvefreezing tolerance (as indicated by the lowest LT50). In contrast,DP did not improve freezing tolerance of Sunkissed at either tem-perature regime. These cultivars were originally selected based ontheir contrasting spring survival and performance in test locationsin the northern U.S., with Sunkissed consistently performing bet-ter than Buccaneer. Overall, these intraspecific differences could beattributed to differences in freezing tolerance or other importantoverwintering traits. When comparing the cultivars in controlledfreeze tests in the current study, Sunkissed exhibited better freez-ing tolerance compared to Buccaneer under non-cold acclimatingconditions, and a similar trend was observed under cold acclimat-ing conditions. Consequently, DP seemed to have a greater effect onimproving freezing tolerance of the less freezing-tolerant cultivar.In fact, exposing Buccaneer to DP allowed this cultivar to achievelevels of freezing tolerance that were comparable to Sunkissed.

The lack of significant effect on freezing tolerance of Sunkissedmay be related to the manner in which the DP treatment was con-ducted. Drought preconditioning was simulated by exposing plantsto multiple wilt cycles under both non-cold acclimating (20 ◦C)and cold acclimating (2 ◦C) conditions, with the purpose of exploit-ing the cumulative effects from plant metabolic changes followingexposure to repeated wilt events. It is possible that the DP treat-ment did not impose sufficient drought stress in terms of frequencyor severity to cause detectible changes in freezing tolerance of themore freezing-tolerant cultivar. Gusta et al. (1980) also reportedthat gains in cold hardiness induced by drought were highly depen-dent on the degree of tissue dehydration in Kentucky bluegrass.Although overall drought resistance of the cultivars was unknown,both cultivars exhibited similar wilting tendency in response todrought. The wilt cycles were considered to impose mild droughtstress on the plants, as evidenced by maintenance of relatively highleaf RWC and low leaf electrolyte leakage. It has been previouslydemonstrated that the severity of drought stress may determinethe extent of allocation and partitioning of assimilates and othermetabolites throughout the plant (Thomas and James, 1999; Xuand Zhou, 2005; DaCosta and Huang, 2006). Since crowns are theprinciple overwintering structures of perennial grasses, the extentof metabolite accumulation in crowns induced by drought stress

may impact overall levels of freezing tolerance. Therefore, addi-tional research is necessary to evaluate the effects of droughtseverity on intraspecific differences in drought-induced freezingtolerance.

1 nd Ex

rbpb2iskfpewidIifhDctciacdfiVopeaf2

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8 L. Hoffman et al. / Environmental a

Several studies have identified a number of genes induced inesponse to both drought and low temperature that are regulatedy abscisic acid-(ABA)-dependent and ABA-independent pathways,roviding evidence for potential mechanisms of cross-adaptationetween these two stresses (Thomashow, 1999; Seki et al., 2001,002a,b). The stress-inducible gene products have been classified

nto two general groups, including those that regulate gene expres-ion and signal transduction (e.g. transcription factors and proteininases), and those that are directly involved in protecting plantsrom dehydration and oxidative stresses (e.g. osmoprotectants, LEAroteins, and detoxification enzymes) (Seki et al., 2003; Shinozakit al., 2003). Therefore, one of the major objectives for our studyas to evaluate changes in the accumulation of specific compounds

n response to DP at 20 and 2 ◦C, including nonstructural carbohy-rates, proline, soluble protein, and antioxidant enzyme activities.

n general, we found that cold acclimation at 2 ◦C resulted in anncrease of MDS (predominately sucrose) and SC (predominatelyructans) in leaf and crown tissues. Leaves generally accumulatedigher MDS content, while crowns accumulated higher SC content.rought preconditioning at 20 ◦C resulted in lower leaf sucroseontent and higher crown fructan content for Buccaneer. In addi-ion, exposing plants to DP at 2 ◦C resulted in higher crown fructanontent for both cultivars. Although Sunkissed did not exhibit anncrease in freezing tolerance in response to DP, the DP-inducedccumulation of fructans in crowns of both cultivars may stillontribute to improved overwintering capacity that may not beetected based on direct estimation of LT50. Fructans are derivedrom sucrose and have been reported to improve membrane stabil-ty in response to dehydration-related stresses (Hincha et al., 2002;alluru and Van den Ende, 2008), delay freezing by direct inhibitionf ice crystal growth in the apoplast (Livingston et al., 2009), and areroposed to have roles in the oxidative stress response (Parvanovat al., 2004). As a result, tolerance to both prolonged low temper-ture and drought has been associated with the accumulation ofructans (Pilon-Smits et al., 1995; Hisano et al., 2004; Dionne et al.,010).

In addition to carbohydrates, proline has been associated withrought resistance (Yamada et al., 2005; Wang et al., 2006; DaCostand Huang, 2007) and cold tolerance (Dörffling et al., 1990; Zhangt al., 2006, 2011). Since proline is hydrophilic by nature, it can formydration barriers around proteins, nucleic acids, and cell mem-ranes to help maintain cell stability in response to desiccationonditions (Hare et al., 1999; Hoekstra et al., 2001). In the cur-ent study, cold acclimation at 2 ◦C resulted in the largest increasesn proline content in both leaf and crown tissues. In addition, DPt 2 ◦C induced a significant increase in crown proline content foroth cultivars, whereas no differences were detected at 20 ◦C. Fur-hermore, proline content was higher in Sunkissed following coldcclimation, suggesting that differential proline accumulation inrown tissues could be associated with differences in freezing tol-rance between the two cultivars. These findings are in agreementith results from other investigations, and support the importance

f proline as a protective component in response to low tempera-ure stress (Koster and Lynch, 1992; Cai et al., 2004; Patton et al.,007a).

Along with increased concentrations of compatible solutes,rought and low temperature can lead to the up-regulation of dif-erent proteins, including dehydrins (Jiang and Huang, 2002; Pattont al., 2007b; Kosová et al., 2011), antifreeze proteins (Antikainent al., 1996; Sidebottom et al., 2000; Zhang et al., 2010), and detox-fication enzymes (Kendall and McKersie, 1989; Fu and Huang,001). In addition, down-regulation of specific proteins has also

een observed (Wei et al., 2006; Kosmala et al., 2009; Xu anduang, 2010). In general, total soluble protein content was high-st following exposure to 2 ◦C, which is in agreement with otherold acclimation studies (Vítámvás et al., 2007; Zhang et al., 2006,

perimental Botany 79 (2012) 11– 20

2011). Additionally, soluble protein content was higher in leavescompared to crown tissues for both cultivars. This likely reflectsthe significant contributions of proteins associated with the pho-tosynthetic machinery in leaf tissues, including light harvestingcomponents, electron transport components, and carbon reductionenzymes (Lambers et al., 2008). Drought preconditioning caused asignificant increase in leaf soluble protein content for Buccaneer,whereas no differences were detected for Sunkissed. Furthermore,DP resulted in significantly higher crown soluble protein accumu-lation for both cultivars regardless of temperature regime. Basedon these results, we are conducting additional research to iden-tify specific proteins that are differentially accumulated in leavesand crowns of perennial ryegrass in response to DP at differenttemperature regimes. This will help to provide further insight onimportant proteins and metabolic pathways involved in cross-talkbetween drought and low temperature stress.

Among the different protein classes, we evaluated changes indifferent antioxidant enzymes due to previous research showingthat DP could alleviate oxidative stress by improving reactive oxy-gen species (ROS) scavenging capacity (Selote et al., 2004; Seloteand Khanna-Chopra, 2010). At low temperatures, the uncouplingof metabolic pathways can cause the generation of ROS and resultin oxidative damage to lipids, nucleic acids, and proteins (Huneret al., 1998; Mittler et al., 2004). In turn, enzymatic and non-enzymatic antioxidants are produced to help regulate the levelsof ROS and mitigate oxidative stress in plant tissues. Generally,increased antioxidant enzyme levels have been associated withincreased capacity to survive at low temperatures (Scebba et al.,1999; Janda et al., 2003). In the current study, however, cold accli-mation at 2 ◦C resulted in generally lower antioxidant enzymeactivities in both leaves and crowns compared to plants maintainedat 20 ◦C. It is important to note that we examined enzyme activi-ties following prolonged cold acclimation (2 ◦C for 30 d), which maybe different from other investigations that examined responses ofantioxidant enzymes upon short-term shift to low temperature.Baek and Skinner (2003) investigated changes in gene expressionlevels of several antioxidant enzymes in wheat over a four-weekcold acclimation period. They reported initial up-regulation of glu-tathione reductase and glutathione peroxidase in the first weekof cold acclimation, but then a decline in gene expression levelsby the fourth week of cold acclimation. Additionally, Zhang et al.(2006) reported that leaf SOD initially increased during cold accli-mation in bermudagrass (Cynodon dactylon (L.) Pers. var. dactylon),but then decreased along with CAT and APX activities during 21 d ofcold acclimation. There were no consistent changes in antioxidantenzyme activities in response to DP; although, there were someinstances where DP plants exhibited higher leaf APX, CAT, SOD, orPOD activities compared to WW plants. Consequently, DP did notseem to impart any consistent benefit to improve ROS scavengingcapacity based on the antioxidant enzymes evaluated in this study.

In conclusion, DP resulted in an improvement in freezing toler-ance (lower LT50) of Buccaneer perennial ryegrass at both 20 ◦C and2 ◦C; however, no significant effects were observed on freezing tol-erance of Sunkissed. Although cold acclimation at 2 ◦C induced thegreatest accumulation of carbohydrate, proline, and soluble proteincontents, we also observed changes in these different metabolitesin response to DP that were dependent upon cultivar, tissue, andtemperature regime. Among the different parameters evaluated,DP most consistently induced increases in crown fructans, proline,and total soluble protein content for both cultivars during coldacclimation, which suggested a synergistic effect between droughtexposure and low temperature. Although DP induced accumulation

of these compounds did not necessarily account for the differencesin LT50 between the two cultivars, higher accumulation of thesecompounds in crown tissues of perennial grasses may contributeto better plant overwintering capacity. Additional research is

nd Ex

nsImbr

A

FGa

R

A

A

B

B

B

B

B

BC

C

C

C

D

D

D

D

E

F

F

G

G

G

H

H

H

L. Hoffman et al. / Environmental a

ecessary to evaluate the effects of DP on freezing tolerance ofpecies and cultivars varying in their sensitivities to drought stress.n addition, future research should explore the effects of repeated

ild drought events on freezing tolerance under field conditions,y using strategies such as wilt-based irrigation scheduling, partialootzone drying, or deficit irrigation.

cknowledgments

This research was supported by the University of Massachusettsaculty Research Grant. We also wish to thank Eve Callahan, Lisaolden, Tao Jiang, Peter O’Brien, and Zac Purinton for their technicalssistance.

eferences

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