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Journal of Plant Physiology 168 (2011) 1271–1279

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l homepage: www.e lsev ier .de / jp lph

ifferences in leaf proteome response to cold acclimation between Loliumerenne plants with distinct levels of frost tolerance

leksandra Bociana, Arkadiusz Kosmalaa,∗, Marcin Rapaczb, Barbara Jurczykb,ukasz Marczakc, Zbigniew Zwierzykowskia

Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska 34, 60-479 Poznan, PolandUniversity of Agriculture in Kraków, Department of Plant Physiology, Podłuzna 3, 30-239 Cracow, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland

r t i c l e i n f o

rticle history:eceived 10 November 2010eceived in revised form 5 January 2011ccepted 7 January 2011

eywords:old acclimationrost tolerancerassesolium perennerotein accumulation profiles

a b s t r a c t

Perennial ryegrass (Lolium perenne) is a high quality forage and turf grass mainly due to its excellentnutritive values and rapid establishment rate. However, this species has limited ability to perform in harshwinter climates. Though winter hardiness is a complex trait, it is commonly agreed that frost tolerance(FT) is its main component. Species growing in temperate regions can acquire FT through exposure tolow, non-lethal temperatures, a phenomenon known as cold acclimation (CA). The research on molecularbasis of FT has been performed on the model plants, but they are not well adapted to extreme winterclimates. Thus, the mechanisms of cell response to low temperature in winter crops and agronomicallyimportant perennial grasses have yet to be revealed. Here, two L. perenne plants with contrasting levels ofFT, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomicresearch. The work focused on analyses of leaf protein accumulation before and after 2, 8, 26 h, and 3, 5, 7,14 and 21 days of CA, using a high-throughput two-dimensional electrophoresis, and on the identificationof proteins which were accumulated differentially between the selected plants by the application of massspectrometry (MS). Analyses of 580 protein profiles revealed a total of 42 (7.2%) spots that showed at a

minimum of 1.5-fold differences in protein abundance, at a minimum of at one time point of CA betweenHFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulationbetween the analyzed plants appeared most often on the 5th (18 proteins) and the 7th (19 proteins)day of CA. The proteins derived from 35 (83.3%) spots were successfully identified by the use of MS andchloroplast proteins were shown to be the major group selected as differentially accumulated during CA.The functions of the identified proteins and their probable influence on the level of FT in L. perenne are discussed.

ntroduction

Perennial ryegrass (Lolium perenne L., Lp) is used extensivelys a forage and turf grass due to its high nutritive values (e.g.

Abbreviations: APX, ascorbate peroxidases; ATP, adenosine triphosphate; CA,old acclimation; CS, cysteine synthase; 2-DE, two-dimensional electrophore-is; ESI, electrospray ionization; Fp, Festuca pratensis; FT, frost tolerance; GS,lutamine synthetase; HFT, high frost tolerant; Hsp, heat shock protein; IEF, iso-lectrofocusing; LFT, low frost tolerant; Lp, Lolium perenne; LSR, a large subunitf ribulose-1,5-bisphosphate carboxylase/oxygenase; MALDI-ToF, Matrix Assistedaser Desorption/Ionization Time of Flight; MS, mass spectrometry; MSDB, Modelystem Database; MW, molecular weight; PPFD, photosynthetic photon fluxensity; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; RA, RuBisCOctivase; RBP, RuBisCO binding protein; ROS, reactive oxygen species; ToF – Q, Time-f-Flight – Quadrupole; % Vol, the normalized volumes; WH, winter-hardiness.∗ Corresponding author. Tel.: +48 61 6550285; fax: +48 61 6550301.

E-mail address: akos@igr.poznan.pl (A. Kosmala).

176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.jplph.2011.01.029

© 2011 Elsevier GmbH. All rights reserved.

high digestibility and water-soluble carbohydrates content), rapidestablishment rate and persistence. However, its ability to per-form in harsh winter climates is limited. Meadow fescue (Festucapratensis Huds., Fp), a species closely related to Lp, has lower nutri-tive value but is one of the most winter-hardy species within theLolium–Festuca complex (Humphreys, 1989). The transfer of thegenes governing winter-hardiness (WH) from Fp to Lp is achievable,as both species hybridize readily and their homologous chro-mosomes pair and recombine at high frequency during meiosis(Zwierzykowski et al., 2008). However, not all the physiologicalbases of WH, expressed differently in Fp and Lp, have been iden-tified and comprehensive research is still required to dissect this

complex trait. It has been demonstrated that WH depends on theability of plants to survive low temperatures, extreme temperaturefluctuation, wind desiccation, ice nucleation, and snow mold infec-tion (Humphreys, 1989). The intensity of each factor depends on theyear and the location, and it is commonly agreed that frost tolerance

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272 A. Bocian et al. / Journal of Plan

FT) is the main component of WH and one of the best indicatorsf plant ability to survive harsh winter conditions (Humphreys,989; Hulke et al., 2008). The plant species growing in temperateegions can acquire FT through exposure to low, non-lethal temper-tures, a phenomenon known as cold acclimation (CA). This processs associated with many structural, physiological and biochemicalhanges within plant cells, as well as with altered expression pat-erns of a specific set of genes (Thomashow, 1999; Stefanowskat al., 2002; Hisano et al., 2008; Sandve et al., 2011). To dissectechanisms of CA in plants, the activities of some genes during cold

reatment at the transcriptome (e.g. Vogel et al., 2005) and the pro-eome (e.g. Amme et al., 2006) levels have been analyzed. However,his analysis has been performed mainly for Arabidopsis (Arabidop-is thaliana) and rice (Oryza sativa), and similar research on foragerasses is still rare and incomplete. Moreover, as the model plantsre not adapted to extreme winter climates, their cell metabolismuring CA may not fully reflect that of agricultural plants of tem-erature climate. Thus, further molecular research to recognize theechanisms of cell response to low temperature performed on

gronomically important perennial grass species, such as Lp, is stilleeded.

Transcriptomics and proteomics have both become major fieldsf functional genomics for large-scale analysis of gene expression.omprehensive research on the transcriptome (Rudi et al., 2011)nd the proteome (Kosmala et al., 2009) during cold treatmentas been recently shown for Fp, the most frost tolerant speciesithin the Lolium–Festuca complex. Identification of genes asso-

iated with CA in Lp based on transcriptome screening has recentlyeen performed by Zhang et al. (2009). Here, we present the firstomplex research on Lp proteome during CA to test the hypothesishat the proteomic response to cold treatment varies between Lpenotypes representing different levels of FT. Thus, the presentedork involved: (1) the selection of Lp plants differing in FT, (2)

nalysis of leaf protein accumulation profiles during CA using two-imensional electrophoresis (2-DE), and (3) mass spectrometryMS) identification of proteins that were differentially accumulatedetween the selected plants.

aterials and methods

lant material, growth conditions and selection for frost tolerance

Sixty-four plants of Lolium perenne cv. Arka (Lp 1–64), eacherminated from a single seed, were randomly chosen from aarger population to be selected for FT in two-step experiment asescribed in detail by Kosmala et al. (2006, 2009). Pre-hardeningas performed over 7 days at 12 ◦C, 8 h/16 h photoperiod, and

00 �mol m−2 s−1 photosynthetic photon flux density (PPFD) andold acclimation (CA) for 21 days at 4/2 ◦C, 10 h/14 h photoperiod,nd 200 �mol m−2 s−1 PPFD.

. The 1st step of plant selection – estimation of the regrowth afterfreezing

After spraying with water to ensure ice-nucleation, eightclones of each cold acclimated Lp genotype were then frozenat a cooling rate of approximately 1 ◦C/h to each of the desiredfreezing temperatures: −8 ◦C, −11 ◦C and −14 ◦C. Following an8 h exposure to the target temperature, the plants were trans-ferred to 2 ◦C, 10 h/14 h photoperiod and 200 �mol m−2 s−1 PPFDto defrost, and 24 h later, to 12 ◦C, 12 h/12 h photoperiod and

300 �mol m−2 s−1 PPFD, to recover over 23 days, which is suffi-cient time for the regrowth of tillers in Lolium–Festuca species(Kosmala et al., 2006, 2009). The regrowth of eight clones ofeach genotype/per particular freezing temperature was esti-mated using Larsen’s (1978) visual score (0–9 scale), described

siology 168 (2011) 1271–1279

in detail by Kosmala et al. (2006, 2009), where “0” means “deadplant” and “9” means “no visible sign of injury.” Mean scores foreight clones of each genotype following recovery were deter-mined at individual temperatures. The two most frost-tolerantand two most susceptible genotypes were selected after freezingin three different temperatures by means of the Kruskal–Wallisrank sum test (P = 0.05).

2. The 2nd step of plant selection – profiling of FT changes duringCA in contrasting genotypes

For the four selected genotypes, the TEL50 values (tempera-ture causing a 50% electrolyte leakage) were estimated 1 daybefore CA and after 2, 8, 26 h, and 3, 5, 7, 14 and 21 days of CA,as described in detail by Kosmala et al. (2009). The cell mem-brane damage after freezing the leaves collected at particulartime points of CA was determined by the measuring of electri-cal conductivity of the tissue extracts, as described by Rapacz(1999). The TEL50 values were estimated from a linear regres-sion fitted to the central (linear) part of the sigmoid relationshipbetween the freezing temperature and the electrolyte leakage.Two Lp genotypes with the extreme values of TEL50 after 21 daysof CA, a low frost tolerant and a high frost tolerant plant, werechosen for further steps in the analysis.

Protein extraction and 2-D electrophoresis

The leaf samples (the youngest but fully expanded and undam-aged leaves) were harvested from three clones of each selectedgenotype of Lp (three biological replicates) 1 day before CA andafter 2, 8, 26 h, and 3, 5, 7, 14 and 21 days of CA (every day atthe 2nd hour of the day), frozen in liquid nitrogen and kept at−80 ◦C until further protein extraction for two-dimensional elec-trophoresis (2-DE). The protocol for proteomic research performedhere was the same as that described by Kosmala et al. (2009). Pro-tein extraction was performed according to the method describedby Hurkman and Tanaka (1986) and protein concentration wasdetermined by the use of a 2-D Quant Kit (GE Healthcare) andbovine serum albumin as a standard. In the first dimension, iso-electrofocusing, 24 cm Immobiline DryStrip gels with linear pHrange 4–7 were used to focus 560 �g of protein extracted from100 mg of plant tissue. In the second dimension (dodecyl sulphate-polyacrylamide gel electrophoresis) the proteins were separatedusing 13% polyacrylamide gels (1.5 × 255 × 196 mm). RainbowTM

Molecular Weight Marker (GE Healthcare) was used as a stan-dard to determine molecular weights of proteins for particularspots. Following electrophoresis the gels were stained with col-loidal Coomassie Brilliant Blue G-250, using the modified methodof Neuhoff et al. (1988). Total separated protein spots on the gelswere scanned by ImageScanner III (GE Healthcare) and subjectedto LabScan 6.0 program (GE Healthcare) processing. Spot detectionand image analyses (normalization, spot matching, protein accu-mulation analyses, statistics) were performed with Image Master2-D Platinum software (GE Healthcare). To compensate for subtledifferences in sample loading, gel staining and destaining, the abun-dance of each protein spot was normalized as a relative volume (%Vol). The % Vol of each spot was automatically calculated by ImageMaster software as a ratio of the volume of a particular spot to thetotal volume of all the spots present on the gel. The extraction pro-cedure and electrophoretic separation were performed three times,each time using a different clone of the selected genotype, and the% Vol for the spots from the three replicated gels were then usedto calculate means and standard deviations, which were used to

create the detailed protein accumulation profiles for each of theselected Lp genotypes during CA. The protein abundance for eachspot at a corresponding time point before and during CA betweentwo genotypes was compared, and the significance of the differ-ences was assessed using the Student’s t-test. The spots with at

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by ESI-microQuadrupole Time-of-Flight MS. Finally, the pro-teins derived from 83.3% (35 out of 42) spots were successfullyidentified (Table 1). However, most of them were the homo-

A. Bocian et al. / Journal of Plan

east 1.5-fold differences (P ≤ 0.05) in protein abundance (quanti-ative differences between gels) or spots present only in one Lpenotype and absent in the other Lp genotype (qualitative differ-nces between gels), at a minimum of at one time point of CA, wereubjected to MS analyses and protein identification.

ass spectrometry and protein identification

Peptide samples for MS were prepared using the modifiedethod adapted from Shevchenko et al. (1996). Peptide massesere measured using a MALDI-ToF (Matrix Assisted Laser Des-

rption/Ionization Time of Flight) MS (Autoflex, Bruker Daltonics,ermany) in the Laboratory of Proteomics and Metabolomics, Insti-

ute of Bioorganic Chemistry, Polish Academy of Sciences. Theample ionization was performed by the use of laser beam at37 nm. The analyzer worked in the linear mode and positive ionsere recorded in the mass range between 700 and 4000 Da. Mass

alibration was performed after every four samples using standardsn the range of analytes (Peptide Calibration Standards I, Bruker Dal-onics). The obtained peptide mass fingerprint data were exportedo the Mascot software for MSDB (Model System Database) orwissProt database search (www.matrixscience.com). The follow-ng search parameters were applied: mass tolerance was set to.2 Da, one incomplete cleavage was allowed, alkylation of cysteiney carbamidomethylation as fixed, and oxidation of methionine asariable modification were set.

The proteins that could not be identified based on MALDI-ToFata were further analyzed using ESI (electrospray ionization)-S/MS tandem mass spectrometry to obtain the peptide

ragmentation spectra and partial amino acid sequences. The mix-ure of digested proteins was concentrated and desalted on aP-C18 precolumn (Proxeon Biosystems trap-column, 5 �m i.d.),nd further peptide separation was achieved on a RP-C18 columnProxeon Biosystems, 75 �m i.d., 100 mm long) using 30 min linearradient with acetonitrile and water (both with 0.1% trifluoroaceticcid). Column outlet was directly coupled to the ESI ion sourcef microToF – Q (Quadrupole Time-of-Flight) MS (Bruker Dalton-cs), working in the regime of data dependent MS to MS/MS. Thebtained data were collected by microToF – Q Control softwareBruker Daltonics), analyzed using DataAnalysis software (Brukeraltonics) and matched to the MSDB or SwissProt database using

he Mascot software (www.matrixscience.com). The search param-ters were the same as those applied for MALDI-ToF analyses withne exception: mass tolerance was set to 0.4 Da for MS mode and.2 Da for MS/MS mode.

esults

election for FT

Four genotypes (Lp 13, 21, 36 and 39) were not rooted and devel-ped enough after the initial steps of plant establishment and theyere excluded from the further experiments. The selection of the

emaining 60 Lp genotypes, performed on the basis of their abilityo regrow after freezing, revealed variation in the level of FT withinhe analyzed population (Fig. 1A). However, the freezing at −14 ◦Cas shown to be the worst in discriminating Lp plants according

o FT, as all the plants were strongly damaged. Thus, Lp genotypesere in fact selected towards freezing tolerance on the basis of their

tatistically significant different regrowth after freezing at −11 ◦C.

his temperature, similarly to our previous experiments conductedn Fp under the same conditions (Kosmala et al., 2009), was showno be the best in discriminating Lp plants according to FT. Wherelants showed the same levels of FT after freezing at −11 ◦C, thealues of FT estimated after freezing at −8 ◦C were also taken into

siology 168 (2011) 1271–1279 1273

account. Finally, according to the described manner of plant selec-tion, two genotypes (Lp 40 and Lp 9) with the lowest and two (Lp 15and Lp 44) with the highest levels of FT were chosen for further stepsof the experiment. The results of the TEL50 analysis performed onthe four selected plants revealed that CA significantly increased FTof all the analyzed Lp genotypes (Fig. 1B). For proteomic research,two genotypes with extreme values of TEL50 after 21 days of CAwere selected: Lp 40 (TEL50 = −11.42 ◦C) as a low frost tolerant(LFT) and Lp 44 (TEL50 = −15.13 ◦C) as a high frost tolerant (HFT)plant.

2-D electrophoresis and protein accumulation profiles

To investigate the changes in the protein accumulation profilesin two selected Lp genotypes under cold treatment, 2-DE analy-ses of leaf proteins from three biological replicates at each timepoint of CA for Lp 44 – HFT and Lp 40 – LFT plant were carriedout (Fig. 2). Fifty-four 2-DE gels were obtained here and all the 2-D patterns within pH 4–7 range were shown to be well resolvedprotein maps. Only the spots that were detected within all threereplicate gels were included into the analyses. Based on this crite-rion, 580 protein spots were reproducibly selected by Image Master2-D Platinum software within each gel. The comparative analy-ses of all the protein profiles revealed a total of 42 (7.2%) spotsthat showed qualitative and/or quantitative differences (P ≤ 0.05)in protein abundance, other than those observed under the controlconditions, between HFT and LFT plants, at least at one time point ofCA (Fig. 2; Table 1; Supplementary Fig. S1). The molecular weightsof the selected protein spots ranged from 11 to 63 kDa (Fig. 2). Inthe case of 18 protein spots (no. 9, 11, 13, 15, 18–21, 25, 29–36and 38) the differences were observed only at one time point of CA.Fourteen spots (no. 1–6, 8, 12, 17, 23, 26, 28, 39 and 41) showed dif-ferences in protein abundance between HFT and LFT plants at twodifferent time points of CA, four spots (no. 10, 16, 22 and 37) at three,two (no. 24 and 40) at four, one spot (no. 42) at five, another one (no.27) at six and two spots (no. 7 and 14) at seven out of eight differenttime points of CA. Among them, the time points at which the dif-ferences in protein accumulation were most often observed werethe 5th (18 protein spots) and the 7th (19 protein spots) day of CA.Additionally, eight spots (no. 2, 3, 5, 6, 16, 20–22) showed significantdifferences in protein accumulation between HFT and LFT plantsalso before CA. A total of eleven spots showed differences in proteinaccumulation between the selected plants after the entire periodof acclimation, including seven (no. 3, 7, 15, 23, 35, 40 and 42) withsignificantly higher protein abundance in HFT plants and four (no.18, 19, 27 and 37) with significantly higher abundance in LFT plants.All the protein spots with different levels of protein accumulationbetween HFT and LFT plants were subjected to MS analyses andidentification.

MS and protein identification

Only two proteins were identified based on Matrix AssistedLaser Desorption/Ionization Time of Flight data (spot no. 8and 12) and the other selected spots were further analyzed

logues of the proteins from different species, indicating thegaps in Lp sequence database. Moreover, the peptide mapsof the remaining seven proteins (spot no. 18, 25, 27–29, 37,38) did not match any sequence present in the availabledatabases.

1274 A. Bocian et al. / Journal of Plant Physiology 168 (2011) 1271–1279

Table 1The results of MS analysis performed on proteins that showed at least 1.5-fold differences in abundance at least at one time point of cold acclimation between high frosttolerant (Lp44) and low frost tolerant (Lp40) Lolium perenne plants.

Spot no.a Accessionb Identified proteinc Differences in accumulation patternsd Scoree Coverage (%)f

1 Q2QV45 Heat shock protein 70 kDa, chloroplastic,Triticum aestivum

2 h, 14 days 217 7

2 6B7Q9 RuBisCO large subunit-binding protein subunitbeta, chloroplastic, Zea mays

Before CA#, 5 days#, 7 days 62 2

3 HHWTBA Chaperonin groEL alpha chain, chloroplastic, T.aestivum

Before CA#, 26 h#, 21 days# 119 8

4 Q9LWT6 Chaperonin 60 beta, chloroplastic, Oryza sativa 5 days, 7 days 76 65 Q7X9A7 RuBisCO large subunit-binding protein subunit

alpha, chloroplastic, T. aestivumBefore CA#, 3 days, 5 days# 199 5

6 ATPB AEGCO ATP synthase subunit beta, chloroplastic,Aegilops columnaris

Before CA, 8 h, 5 days 35 2

7 ATPB AEGCO ATP synthase subunit beta, chloroplastic, A.columnaris

2 h#, 8 h#, 26 h#, 3 days#, 5 days#, 7 days#, 21 days# 243 24

8* GLNA2 HORVU Glutamine synthetase leaf isozyme,chloroplastic, Hordeum vulgare

26 h#, 7 days 81 –

9 GLNA2 HORVU Glutamine synthetase leaf isozyme,chloroplastic, H. vulgare

7 days 58 26

10 Q95FQ1 ATP synthase subunit beta, chloroplastic,Lythrum salicaria

2 h, 8 h, 26 h 71 6

11 Q688X2 Hypothetical protein OJ1781 H11.4, O. sativa 3 days# 24 212* CK215364 RuBisCO small chain, chloroplastic, T. aestivum 7 days, 14 days 82 –13 Q8LLS5 Phosphoglycerate kinase, chloroplastic, H.

vulgare7 days# 216 33

14 Q7X9A1 RuBisCO activase beta, chloroplastic,Deschampsia antarctica

2 h#, 8 h#, 26 h#, 3 days#, 5 days#, 7 days#, 14 days# 90 22

15 RCAB HORVU RuBisCO activase beta, chloroplastic, H. vulgare 21 days# 50 816 Q7X9A2 RuBisCO activase alpha, chloroplastic, D.

antarcticaBefore CA, 8 h, 26 h, 5 days 96 6

17 TVWTGY Phosphoglycerate kinase, cytosolic, T. aestivum 3 days#, 5 days# 89 718 – – 21 days – –19 Q7EYM8 Oxidoreductase, zinc-binding, O. sativa 21 days 44 520 Q7X9A2 RuBisCO activase alpha, chloroplastic, D.

antarcticaBefore CA, 8 h 135 6

21 Q1S0L5 Malate dehydrogenase, cytosolic, Medicagotruncatula

Before CA, 5 days# 78 8

22 TA65463 4565 Aldo/keto reductase family-like protein, O.sativa

Before CA#, 2 h#, 3 days#, 5 days# 89 8

23 gi 51859661 RuBisCO large subunit, chloroplastic,Psathyrostachys fragilis

2 h, 21 days# 79 4

24 Q2QLX5 Cysteine synthase, O. sativa 2 h, 5 days, 7 days, 14 days 81 625 – – 8 h – –26 Q949H8 Glu 1 protein, Festuca pratensis 8 h, 5 days# 68 427 – – 26 h#, 3 days, 5 days#, 7 days#, 14 days#, 21 days – –28 – – 5 days, 7 days# – –29 – – 5 days# – –30 Q7XZP6 Ascorbate peroxidase, chloroplastic,

Arabidopsis thaliana2 h 44 3

31 TPIS ARATH Triose phosphate isomerase, cytosolic, A.thaliana

7 days# 41 3

32 ISZMT Triose phosphate isomerase, cytosolic, Z. mays 7 days# 61 533 TA95233 4565 Hypothetical protein T5P19 300, A. thaliana 5 days 63 534 PS17 PINST Cytochrome c oxidase subunit II PS17,

mitochondria, Pinus strobus14 days 32 100

35 RKRZS9 RuBisCO small chain, chloroplastic, O. sativa 21 days# 54 1736 PS17 PINST Cytochrome c oxidase subunit II PS17,

mitochondrial, P. strobus7 days# 32 100

37 – – 3 days, 7 days, 21 days – –38 – – 8 h – –39 Q9ZWF5 50S ribosomal protein L12, chloroplastic, O.

sativa26 h#, 7 days# 76 12

40 Q9SDY9 RuBisCO small chain, chloroplastic, Avenaclauda

8 h#, 26 h#, 7 days#, 21 days# 73 20

41 Q66LL4 RuBisCO small chain, chloroplastic, Loliumperenne

5 days#, 7 days# 73 18

42 Q66LL4 RuBisCO small chain, chloroplastic, L. perenne 2 h#, 3 days#, 5 days#, 7 days, 21 days# 73 18

a Spot numbering was the same as in Fig. 2.b Database accession of a homologous protein.c Homologous protein and organism from which it originates.d The time points where significant differences in protein accumulation patterns during cold acclimation between HFT (Lp44) and LFT (Lp40) plants were found.e Protein identification was performed using the Mascot search probability based Mows score. Ions score was −10 × log(P), where P was the probability that the observed

match was a random event. Mascot defined thresholds which indicated identity or extensive homology (P < 0.05) was 40.f Amino acid sequence coverage for the identified proteins.* Indicates the protein identified using Matrix Assisted Laser Desorption/Ionization Time of Flight mass spectrometry; Abbreviations: ATP, adenosine triphosphate; CA,

cold acclimation.# Indicates the higher protein abundance in HFT plant; the detailed accumulation profiles were shown in Supplementary Fig. S1.

A. Bocian et al. / Journal of Plant Physiology 168 (2011) 1271–1279 1275

regrowth after freezing

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1 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 30 31 32 33 34 35 37 38 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

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Fig. 1. Frost tolerance (FT) of Lolium perenne (Lp) cv. Arka. (A) FT of sixty Lp plants estimated as the capacity to re-grow (scale 0–9) 23 days after freezing at −8, −11, and−14 ◦C following 21 days of cold acclimation (CA). Eight equal-sized clones of each plant were used and the mean scores of FT at a particular temperature are shown. Them −14 ◦CL ◦C = 3T leaf tb −9, −

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ean scores of FT for the underlined genotypes were as follows: Lp 9 (−8 ◦C = 2.0,p40 (−8 ◦C = 1.375, −11 ◦C = 0.875, and −14 ◦C = 0.75), and Lp44 (−8 ◦C = 6.375, −11EL50 means the temperature at which 50% of total electrolytes were released fromasis of freezing tests made in two replicates at five freezing temperatures: −3, −6,

iscussion

A and FT of Lp

Cell membranes were shown to be the primary sites of frostnjury in plants, and it has been shown that CA significantlyncreases membrane stability and integrity (Thomashow, 1999).herefore, estimation of the level of electrolyte leakage and plasmaembrane damage by measuring the electrical conductivity of the

issue extract derived from frozen leaves is thought to be one of theost informative methods to determine the level of FT. Here, the

alues of TEL50 obtained for 3-week cold-acclimated plants (Fig. 1B)ere the base for plant selection for further proteomic research. The

-week period of CA was shown in our earlier work to be necessaryo reach high levels of FT in Fp (Kosmala et al., 2006, 2009). How-ver, the analysis presented here revealed that CA between the 7thnd 21st day only slightly (statistically insignificantly) increasedT of all the studied Lp genotypes. In fact, the differences in FTetween the analyzed plants appeared on the 3rd day of CA andT reached its final levels in all the studied plants on the 7th day ofA. Interestingly, as noted above, the differences in protein accu-ulation were most often observed on the 5th and the 7th day

f CA. The HFT (TEL50 = −15.13 ◦C) and LFT (TEL50 = −11.42 ◦C) Lplants differed by as much as −3.7 ◦C in their FT after 21 days of CA.

xactly the same value of difference in FT was estimated betweenFT (TEL50 = −14 ◦C) and LFT (TEL50 = −10.3 ◦C) Lp plants used in theork of Hoffman et al. (2010), who monitored the level of accumu-

ation of some important metabolites (e.g. proline, water solublearbohydrates, lipids) in response to low temperature.

= 0.875, and −14 ◦C = 0.625), Lp15 (−8 ◦C = 5.375, −11 ◦C = 2.75, and −14 ◦C = 1.25),.375, and −14 ◦C = 1.25). (B) Changes in the level of FT of four Lp plants during CA.issues. Values of TEL50 and coefficient intervals for P = 0.05 were calculated on the12 and −15 ◦C.

Protein accumulation profiles during CA of Lp

In the current study, we demonstrated, for the first time, theprotein accumulation profiles during CA of Lp genotypes differ-ing in their ability to cope with frost (Supplementary Fig. S1). Inmany cases, the same protein was identified in more than onespot. In fact, it was shown that not only post-translational mod-ifications (phosphorylation, methylation and glycosylation), butalso different signals and targeting sequences, in vivo proteoly-sis or in vitro protein degradation during sample preparation canbe a source of “new extra spots” representing the same protein,with different positions (different molecular masses and isoelec-tric points) on the 2-D maps. Here, such multiple spots wereobserved in the case of RuBisCO (ribulose-1,5-bisphosphate car-boxylase/oxygenase) large subunit-binding protein (spots no. 2 and5), ATP (adenosine triphosphate) synthase subunit (spots no. 6, 7and 10), glutamine synthetase (GS, spots no. 8 and 9), RuBisCOsmall chain (spots no. 12, 35, 40–42), RuBisCO activase (RA) beta(spots no. 14 and 15), RA alpha (spots no. 16 and 20), triose phos-phate isomerase (spots no. 31 and 32), and cytochrome c oxidase(spots no. 34 and 36). A differentially accumulated protein, identi-fied here as RuBisCO large subunit, present in spot no. 23, was infact a product of RuBisCO large subunit degradation. Its intact formis indicated in Fig. 2 as a large subunit of ribulose-1,5-bisphosphate

carboxylase/oxygenase (LSR). The reports on the process of degra-dation during low temperature treatment, including, e.g. RuBisCOlarge subunit, RA and ATP synthase alpha chain, have been pub-lished elsewhere (e.g. Yan et al., 2006). A similar situation wasalso observed in our previous research performed on Fp (Kosmala

1276 A. Bocian et al. / Journal of Plant Physiology 168 (2011) 1271–1279

Fig. 2. Representative 2-D protein map obtained for a high frost tolerant Lolium perenne (Lp) plant (Lp 44) before cold acclimation (CA). During isoelectrofocusing the proteinswere separated at pH range 4–7, and after being separated during sodium dodecyl sulphate-polyacrylamide gel electrophoresis they were stained with colloidal CoomassieBrilliant Blue G-250. Spot detection and image analyses were performed using the Image Master 2-D Platinum software (GE Healthcare). The normalized volumes of matchedspots were used for comparisons made between gels to reveal the accumulation profiles of particular proteins during CA (in the case of each spot the means derived fromt 0.05)o gel. Mb n accuo

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hree replicated gels were applied). The spots with at least 1.5-fold differences (P ≤ther Lp genotype, at a minimum of at one time point of CA, are numbered on theisphosphate carboxylase/oxygenase (LSR) was indicated with an arrow. The proteif the presented gel states approximately 60% of the original one).

t al., 2009). In two cases (ATP synthase and cytochrome c oxidase),he accumulation profiles observed in different spots represent-ng the same protein were so distinct that their interpretation

as impossible and requires further experiments. Additionally, thedentity of three other proteins (oxidoreductase, aldo/keto reduc-ase family-like protein and glu-1 protein) was too ambiguous.owever, the other proteins identified here were shown to be

nvolved in numerous physiological processes, and at least some ofhem discussed below could be implicated in the expression of FTn Lp. The detailed protein accumulation profiles discussed here areresented as supplementary results in Fig. S1. First, they revealedhat CA can directly influence the metabolism of various compo-ents of the protein synthesis machinery, as shown previously forp (Kosmala et al., 2009). In the present study, chloroplastic 50Sibosomal protein L12 (spot no. 39) was shown to have higher accu-ulation levels in the HFT Lp genotype during most of the process

f CA. Similar to the current results, the other chloroplastic riboso-al proteins identified in Fp, 50S L10 and 30S S10, showed higher

evels of accumulation in the HFT genotype (Kosmala et al., 2009).

olecular chaperonesHeat shock proteins (Hsps) are molecular chaperones that sta-

ilize native proteins, prevent aggregation of denatured proteins,efold stress-denatured proteins and participate in the foldingnd assembly of nascent polypeptides into functional forms. The

in protein abundance or spots present only in one Lp genotype and absent in theolecular weight (MW) and pH scales are shown. The large subunit of ribulose-1,5-mulation profiles of the selected proteins are shown in Supplementary Fig. S1 (size

increased accumulation level of Hsp70 during CA has been reportedfor many species, e.g. Arabidopsis (Bae et al., 2003), and it was alsoobserved in Lp. The accumulation level of Hsp70 (spot no. 1) washigher in the LFT genotype both before and during the entire CA.Moreover, in LFT plants the accumulation of Hsp70 increased grad-ually up to the maximum on the 7th day of CA, whereas in HFTplant it oscillated with a dramatic drop on the 14th day of CA. Theseresults agree with the fact that the chaperone response is not thedirect response to low temperature but a secondary response tothe loss of cellular homeostasis caused by the direct effects of lowtemperature, such as the accumulation of non-native or denaturedproteins. The Hsp70 accumulation profiles observed here indicatethat in LFT plants cellular homeostasis could be more affected dur-ing cold treatment compared to the homeostasis in HFT plant. Theother molecular chaperones identified in the current study werechaperonin groEL (spot no. 3) and chaperonin 60 (spot no 4). Thefirst showed higher abundance after 3 weeks of CA in both Lp geno-types, compared the conditions before CA, but its accumulationlevel was almost always higher in the HFT genotype. However, therelevance of groEL to the CA in Lp is not clear. Inversely, chaperonin

60 did not show any significant changes in accumulation level whenwe compared conditions before and after full time of CA in bothanalyzed plants, but a significantly higher abundance of this proteinwas observed in LFT genotype between the 5th and 14th day of CA. Ithas been shown that chaperonin 60 protects RA from denaturation

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uring temperature stress (Salvucci, 2008). Thus, lower accumula-ion level of this chaperonin in HFT genotype may indicate that thetability of RA is less affected by low temperature in genotype withigher FT. In fact, RA beta, which is a more active activase form,howed higher abundance in the HFT genotype during the wholerocess of CA (spot no. 14), and a decrease of its accumulation afterhe whole process was observed in the LFT genotype (spot no. 15).

OS-scavengers and antioxidantsReactive oxygen species (ROS) under optimal growth conditions

re produced at relatively low levels in organelles such as chloro-lasts, mitochondria, and peroxisomes. However, during stress,

ncluding low temperature, their rate of production is dramaticallylevated and they have higher potential to cause oxidative dam-ge to cell components (DNA, proteins, and membrane lipids). Torotect the cells plants activate the ROS defense mechanisms that

nvolve ROS-scavenging enzymes, such as a group of ascorbate per-xidases (APX), of which one member, derived from chloroplasts,as identified in this study (spot no. 30). Significant differences inPX accumulation during CA were detected between HFT and LFTlants. First of all, the analyses of LFT genotype revealed almostqual level of this protein abundance during the whole process ofcclimation. On the other hand, HFT genotype, having significantlyower level of APX accumulation during the first hours of CA (mainlyt the 2nd hour), compared to LFT genotype, showed two bursts ofts higher accumulation with the peaks at 26th hour and on the 7thay of CA. Taking into account the fact that at low temperaturehe activity of ROS-scavenging enzymes is lower and their nor-

al abundance in cell insufficient to overcome ROS accumulation,he results obtained in this study may indicate that more tolerantp plants could have significantly higher capacity of ROS scaveng-ng, at least at some time points during CA. Cysteine synthase (CS,pot no. 24), a sulfur metabolism-related enzyme, is responsible forhe final step in cysteine biosynthesis, which is, in turn, one of the

ost important and often limiting steps in the production of glu-athione, an antioxidant implicated in tolerance to both abiotic andiotic stresses. Disturbances in CS accumulation, observed in HFTenotype, especially on the 7th and 14th day of CA, may indicatehat the Lp genotype with higher level of FT used such molecular

echanisms to establish cell homeostasis during cold treatment,hich did not require an involvement of glutathione, at least at the

dvanced steps of CA process. On the other hand, higher levels of CSccumulation between the 3rd and 14th day of CA in LFT genotypeay also suggest that, in this genotype, cellular homeostasis wasore affected by low temperature and elevated levels of CS and

nally glutathione were required to restore it.

omponents of photosynthetic machineryIt has been demonstrated several times that, due to partially

verlapping mechanisms of CA to frost conditions and to cold-nduced photoinhibition, more frost-tolerant plants tend to also be

ore tolerant to cold-induced photoinhibition of photosynthesisRapacz et al., 2004). An increasing rate of photosynthetic carbon

etabolism could be one of the mechanisms that can protect over-intering plants against photoinhibition. The chloroplast-codedart of the key enzyme involved into the dark phase of photosyn-hesis (the Calvin cycle), the intact form of RuBisCO large subunitindicated in Fig. 2 as LSR) did not reveal any significant differ-nces in its accumulation during CA and moreover, between HFTnd LFT genotypes. However, as mentioned above, this proteinas being partially degraded during CA with different intensity at

articular time points of CA in both Lp genotypes. The identifiedroduct of this degradation was found in spot no. 23. It has beenhown that RuBisCO activity increased during CA, e.g. in winterye (Secale cereale) (Hurry et al., 1994). Moreover, after long-termA the increase of its activity was higher in frost tolerant plants

siology 168 (2011) 1271–1279 1277

(Huner et al., 1993), which was attributed to increasing activityrather than increased accumulation. This can be also true in the caseof Lp genotypes analyzed herein as differences in the level of proteinaccumulation were found for RuBisCO binding protein (RBP, spotsno. 2 and 5) and RA (spots no. 14–16 and 20), which were proved tobe involved in the regulation of RuBisCO holoenzyme assembly inchloroplast stroma and its activity, respectively. The pattern of RBPaccumulation was similar for HFT and LFT genotypes during CA,although some disturbances of the accumulation, which differedbetween genotypes, were also observed, e.g. a drastic decreaseof its abundance on the 5th day of CA in the LFT genotype. Evenmore interesting are the accumulation profiles of RA beta, a moreactive activase form. They showed higher activase abundance inthe HFT genotype during the whole process of CA (spot no. 14), anda decrease of its accumulation after the whole process of CA in LFTgenotype (spot no. 15). In our earlier work on Fp, decreased accu-mulation of RA protein was observed after prolonged CA of the LFTgenotype (Kosmala et al., 2009). These results may indicate thatRuBisCO activity could be higher in the HFT Lp genotype not onlyduring the whole process of CA, but also in the control conditionsbefore CA, as a large difference in RA beta accumulation betweenthe analyzed genotypes was observed also before CA. It may comefrom the fact that photosynthetic acclimation to cold, involvingthe increase of RuBisCO activity takes place during pre-hardeningperiod and may be different in LFT and HFT plants (Rapacz and Hura,2002).

The other enzyme involved in the Calvin cycle identified herein spot no. 13, chloroplastic phosphoglycerate kinase, follows thisidea. Disturbance in its accumulation observed on the 7th day of CAin LFT plant may also suggest that the less frost-tolerant genotypewas also less efficient in photosynthetic carbon metabolism. Thesame suggestion was made previously for Fp after the observationthat HFT genotype revealed increased level of chloroplastic phos-phoglycerate kinase accumulation during the whole period of CA(Kosmala et al., 2009).

Proteins involved in energy metabolismThree proteins selected here as differentially accumulated

between Lp genotypes were involved directly into energymetabolism. Two were the enzymes of the glycolytic pathway. Thefirst, triose phosphate isomerase (spots no. 31 and 32), showed adecrease of accumulation on the 7th day of CA in LFT plants. Thesecond enzyme, cytosolic phosphoglycerate kinase, identified herein spot no. 17, showed a decrease of its accumulation on the 3rdand 5th day of CA also in LFT plant. Furthermore, the third proteininvolved in energy metabolism, malate dehydrogenase (spot no.21), an enzyme of the citric acid cycle (the Krebs cycle), showed adecrease of accumulation on the 5th day of CA in LFT plant. Thesethree enzymes are essential for energy production, and thus anydisturbance in their accumulation as noted in the LFT plants couldbe fatal for cell homeostasis, especially during stress conditions.

Other proteinsGS is an essential enzyme of nitrogen metabolism that catalyzes

the fixation of ammonia to glutamate to form glutamine. Higherplants usually have one plastidic and one or more cytosolic GSs.Generally, the abundance of identified here chloroplastic variantsof GS was equal in both genotypes or higher in HFT plants at earlystages of CA, but definitely higher in LFT plants in more advancedstages of CA. It has been documented that GS could be involvedin proline metabolism during low temperature treatment (Charest

and Phan, 1990). However, Hoffman et al. (2010) on the 14th day ofCA found only slight differences in proline content between the Lpcultivars with contrasting levels of FT and no significant differencesafter the full period of CA (21 days), suggesting no close associationbetween proline accumulation and FT in Lp. Conversely, our unpub-

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278 A. Bocian et al. / Journal of Plan

ished metabolomic results (paper in preparation) performed onFT and LFT Lp plants showed that significant differences in pro-

ine accumulation were observed at the early steps of CA (mostlyt 2nd, 8th and 26th hour), where the abundance of this aminocid was higher in HFT plant. Moreover, higher (but statisticallynsignificant) levels of proline accumulation in HFT plant were alsobserved on the 5th, 14th and 21st day of CA. However, the questionhether the differences identified here in GS abundance during CA

etween the analyzed Lp genotypes could be really associated withroline abundance and its different accumulation between HFT andFT plants remains unresolved and requires further work.

he transcriptome and the proteome response to CA in Lp

The relationship between the transcriptome and the proteomeas a complex nature, and it has been demonstrated that mRNA

s a poor indicator of protein expression levels (e.g. Mooneyt al., 2006). This correlation is disturbed not only by the post-ranscriptional steps of gene expression, including the levels ofrotein stability and proteolysis, but also by the experimentalethods used to measure protein and mRNA quantity. On the

ther hand, for the same reasons, the protein accumulation pro-les cannot be regarded as a direct reflection of the correspondingene activities. Zhang et al. (2009) identified genes differentiallyxpressed during CA in Lp by analyzing the abundance of expressedequence tags randomly sampled from two cDNA libraries, oneonstructed from 14-day cold acclimated (TEL50 = −12.2 ◦C) and thether from non-acclimated (TEL50 = −7.6 ◦C) leaves of a relativelyrost-tolerant cultivar of Lp. The results suggest that cold-regulated,ehydration-responsive, and ice recrystalization inhibition genesere up-regulated, and photosynthesis and respiration-related

enes were down-regulated after 14 days of CA in Lp. Only RAas found to be differentially expressed both at the transcriptome

Zhang et al., 2009) and the proteome (results presented herein)evels. However, direct and more detailed correlation of the resultsrom both studies is not possible, as the experimental designs ofhese two experiments were quite different. Furthermore, moremportant than comparison is the integration of data derived fromoth the transcriptomic and proteomic research to fully understandhe mechanisms underlying expression of FT in forage grass species.

he proteome response to CA in Lp and Fp

The time points of CA at which the observed differences inrotein accumulation between HFT and LFT genotypes were mostrequent, were different for Fp and Lp, and involved the 26th hourf CA in Fp and both the 5th and the 7th day of CA in Lp. In the casef both species the similar numbers of differentially accumulatedroteins were selected – 41 for Fp and 42 for Lp. However, the pro-ortions of those proteins to the total protein numbers detected on-D maps were different for particular species, and were 5.1% and.2% for Fp and Lp, respectively. This variation was due mainly to theifferent numbers of total proteins reproducibly selected by Imageaster 2-D Platinum software within Fp and Lp gels. The same pro-

ein amounts (560 �g) were applied for 2-DE and the same systemsor the proteomic experiments were used for each species, includ-ng the method of protein detection, approximately 800 proteinpots for Fp and only 580 ones for Lp were detected. The source ofhat diversity has not been recognized. With the exception of tworoteins, including chloroplastic phosphoglycerate kinase and RAeta, different proteins were found to be differently accumulated

etween LFT and HFT Lp, compared to LFT and HFT Fp genotypes. Onhe other hand, in both species, the chloroplast proteins, includingroteins directly involved in the process of photosynthesis, werehe major group selected as differentially accumulated during CAetween the genotypes differing in FT levels. However, it has been

siology 168 (2011) 1271–1279

well recognized that the proteins located in chloroplasts are usuallythe main component of all the proteins found in leaves.

Conclusions

Our comprehensive research program supports the hypothesisthat the proteomic response to CA varies between Lp genotypeswith different levels of FT, and puts emphasis on function diversityof the differentially accumulated proteins. In all the identified pro-tein groups, clear differences between analyzed Lp genotypes wereindicated. However, gaps in genomic and proteomic databasesof this species strongly disturbed the obtained results. Furtherresearch on Lolium genome sequencing should improve the qualityof proteomic studies.

Acknowledgement

The research was carried out in the framework of the projectof The National Centre for Research and Development (no. PBZ-MNiSW-2/3/2006/21).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jplph.2011.01.029.

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