10
Cold Acclimation Proteome Analysis Reveals Close Link between the Up-Regulation of Low-Temperature Associated Proteins and Vernalization Fulfillment Elham Sarhadi, †,‡,§ Siroos Mahfoozi, §,| Seyed Abdollah Hosseini, and Ghasem Hosseini Salekdeh* ,†,Department of Systems Biology, Agricultural Biotechnology Research Institute, Karaj, Iran, Science and Research Branch, Islamic Azad University, Tehran, Iran, Physiology-Agronomy unit of Department of Cereals Research, Seed and Plant Improvement Institute, P.O. Box 31585-4119, Karaj, Iran, and Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, ACCER, Tehran, Iran Received May 15, 2010 Low-temperature (LT) stress is one of the major limiting factors in cereal production in cold high- altitude mountainous areas of Iran where cereals are exposed to variable periods of temperatures in the vernalization range during the autumn season. Cereals regulate their development through adaptive mechanisms that are responsive to low but nonfreezing temperatures. We exploited a proteomic approach to determine the interrelationship between vernalization fulfillment and expression of low- temperature (LT)-induced protein in most hardy Norstar and semi-hardy Azar2 wheat (Triticum aestivum L. em. Thell). These cultivars were subjected to 12 h of cold acclimating temperature (2 °C) over a period of 0-89 days. LT tolerance, as measured by LT50, and vernalization fulfillment, as estimated from final leaf number (FLN), was determined at intervals throughout the acclimation period. A significant decrease in FLN associated with LT treatment indicated that Norstar and Azar2 had vernalization responses. Azar2 achieved its vernalization fulfillment and maximum LT tolerance (-8 °C) by 28 days of acclimation. However, Norstar had a longer vernalization requirement (between 35 and 42 days) and reached vernalization fulfillment and maximum LT tolerance (-18.7 °C) about the same time as vernalization fulfillment. We applied a two-dimensional electrophoresis-based proteomics approach to analyze changes in the leaf proteome of two genotypes, Norstar and Azar2, during cold acclimation. Using MALDI-TOF/TOF mass spectrometry, 66 LT-associated proteins could significantly be identified. These proteins were categorized into cold-regulated proteins, antifreezing proteins, oxidative stress defense, photosynthesis, chloroplast post-transcriptional regulation, metabolisms, and protein synthesis. A close association between the vernalization fulfillment and the start of a decline in the protein accumulation of hardy Norstar with a long vernalization requirement and semi-hardy Azar2 with a short vernalization requirement was observed. This finding supported the hypothesis that developmental trait which was regulated by vernalization had a regulatory influence over LT proteome response and highlight a close link between the up-regulation of LT-associated proteins and vernalization fulfillment at the molecular level in wheat. Keywords: low-temperature proteomic vernalization wheat Triticum aestivum Introduction Low-temperature (LT) stress is a major factor limiting cereal production in cold high-altitude mountainous areas of Iran where cereals are exposed to variable periods of temperatures in the vernalization range during the autumn season. 1 Because cereals regulate their development through adaptive mecha- nisms that are responsive to low but nonfreezing temperatures, the unpredictable nature of the over wintering period makes the selection of highly adapted genotypes for cold regions a difficult challenge. On LT exposure, vernalization-requiring plants continue to reduce their final leaf number (FLN) up to the point of vernalization fulfillment. 2 Vernalization require- ment is critical to winter plants as it prevents transition to the reproductive phase in regions with cold winters. Fulfillment of vernalization requirement has been suggested for the loss of LT tolerance of over wintering cereals. 1 In regions with cold winters, vernalization requirement is an important adaptive feature that delays heading by postponing the transition from * Corresponding author: Ghasem Hosseini Salekdeh, Agricultural Bio- technology Research Institute of Iran, P.O. Box 31535-1897, Karaj, Iran. E-mail: [email protected]. Fax: +98-261-2704539. Agricultural Biotechnology Research Institute. Islamic Azad University. § These authors contributed equally. | Seed and Plant Improvement Institute. Royan Institute for Stem Cell Biology and Technology. 5658 Journal of Proteome Research 2010, 9, 5658–5667 10.1021/pr100475r 2010 American Chemical Society Published on Web 08/31/2010

Cold Acclimation Proteome Analysis Reveals Close Link between the Up-Regulation of Low-Temperature Associated Proteins and Vernalization Fulfillment

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Cold Acclimation Proteome Analysis Reveals Close Link between the

Up-Regulation of Low-Temperature Associated Proteins and

Vernalization Fulfillment

Elham Sarhadi,†,‡,§ Siroos Mahfoozi,§,| Seyed Abdollah Hosseini,† andGhasem Hosseini Salekdeh*,†,⊥

Department of Systems Biology, Agricultural Biotechnology Research Institute, Karaj, Iran, Science andResearch Branch, Islamic Azad University, Tehran, Iran, Physiology-Agronomy unit of Department of Cereals

Research, Seed and Plant Improvement Institute, P.O. Box 31585-4119, Karaj, Iran, and Department ofMolecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, ACCER, Tehran, Iran

Received May 15, 2010

Low-temperature (LT) stress is one of the major limiting factors in cereal production in cold high-altitude mountainous areas of Iran where cereals are exposed to variable periods of temperatures inthe vernalization range during the autumn season. Cereals regulate their development through adaptivemechanisms that are responsive to low but nonfreezing temperatures. We exploited a proteomicapproach to determine the interrelationship between vernalization fulfillment and expression of low-temperature (LT)-induced protein in most hardy Norstar and semi-hardy Azar2 wheat (Triticum aestivumL. em. Thell). These cultivars were subjected to 12 h of cold acclimating temperature (2 °C) over aperiod of 0-89 days. LT tolerance, as measured by LT50, and vernalization fulfillment, as estimatedfrom final leaf number (FLN), was determined at intervals throughout the acclimation period. Asignificant decrease in FLN associated with LT treatment indicated that Norstar and Azar2 hadvernalization responses. Azar2 achieved its vernalization fulfillment and maximum LT tolerance (∼ -8°C) by 28 days of acclimation. However, Norstar had a longer vernalization requirement (between 35and 42 days) and reached vernalization fulfillment and maximum LT tolerance (∼ -18.7 °C) about thesame time as vernalization fulfillment. We applied a two-dimensional electrophoresis-based proteomicsapproach to analyze changes in the leaf proteome of two genotypes, Norstar and Azar2, during coldacclimation. Using MALDI-TOF/TOF mass spectrometry, 66 LT-associated proteins could significantlybe identified. These proteins were categorized into cold-regulated proteins, antifreezing proteins,oxidative stress defense, photosynthesis, chloroplast post-transcriptional regulation, metabolisms, andprotein synthesis. A close association between the vernalization fulfillment and the start of a decline inthe protein accumulation of hardy Norstar with a long vernalization requirement and semi-hardy Azar2with a short vernalization requirement was observed. This finding supported the hypothesis thatdevelopmental trait which was regulated by vernalization had a regulatory influence over LT proteomeresponse and highlight a close link between the up-regulation of LT-associated proteins and vernalizationfulfillment at the molecular level in wheat.

Keywords: low-temperature • proteomic • vernalization • wheat • Triticum aestivum

Introduction

Low-temperature (LT) stress is a major factor limiting cerealproduction in cold high-altitude mountainous areas of Iranwhere cereals are exposed to variable periods of temperaturesin the vernalization range during the autumn season.1 Because

cereals regulate their development through adaptive mecha-nisms that are responsive to low but nonfreezing temperatures,the unpredictable nature of the over wintering period makesthe selection of highly adapted genotypes for cold regions adifficult challenge. On LT exposure, vernalization-requiringplants continue to reduce their final leaf number (FLN) up tothe point of vernalization fulfillment.2 Vernalization require-ment is critical to winter plants as it prevents transition to thereproductive phase in regions with cold winters. Fulfillmentof vernalization requirement has been suggested for the lossof LT tolerance of over wintering cereals.1 In regions with coldwinters, vernalization requirement is an important adaptivefeature that delays heading by postponing the transition from

* Corresponding author: Ghasem Hosseini Salekdeh, Agricultural Bio-technology Research Institute of Iran, P.O. Box 31535-1897, Karaj, Iran.E-mail: [email protected]. Fax: +98-261-2704539.

† Agricultural Biotechnology Research Institute.‡ Islamic Azad University.§ These authors contributed equally.| Seed and Plant Improvement Institute.⊥ Royan Institute for Stem Cell Biology and Technology.

5658 Journal of Proteome Research 2010, 9, 5658–5667 10.1021/pr100475r 2010 American Chemical SocietyPublished on Web 08/31/2010

the vegetative to the reproductive phase. In contrast, springcereals, which do not have a vernalization requirement,normally develop rapidly into their reproductive phase whengrown under long days.1,3

The ability of wheat plants to survive freezing increases uponexposure to low nonfreezing temperatures, a phenomenonknown as cold acclimation.4 This is a relatively slow, adaptiveresponse of plants to relatively mild cold stress (e.g., +2 °C)that results in various changes in morphological, biochemical,and physiological characters of plants that greatly enhancetolerance to more severe conditions.

Plants apply several stress adaptive strategies, one of whichis regulation of gene expression. The hypothesis that cold-induced proteins are likely to be involved in cold tolerance hasled to great efforts to discover these proteins for the improve-ment of crops in temperate and cold countries. Biochemicaland molecular analysis have demonstrated differential geneexpression and the accumulation of specific proteins duringthe induction of freezing tolerance.5-7

A proteomics approach proved to be a powerful approachto identify plant response to stresses.8 This approach has beenapplied to investigate the temporal changes of total proteinsin rice leaves after chilling treatment.9,10 The effects of LT stresson proteome of rice anther,11 rice young microspore12 andArabidopsis nuclei13 were also studied.

Although these studies provide initial insight into plantresponse to LT stress, proteome analysis of LT tolerance andthe interrelationship between vernalization fulfillment and theexpression pattern of LT-associated proteins has not beenstudied in wheat cultivars with different vernalization require-ment at various acclimation periods. To fill this gap, we haveanalyzed the leaf proteomes of two winter bread wheat (Triti-cum aestivum L. em. Thell) cultivars, Norstar and Azar2, withdifferent vernalization requirements. The results indicated thatseveral LT-associated proteins were up-regulated in cold-hardygenotype compared to semi-hardy genotype. This study verifiesthe evidence of a close link between the up-regulation of LT-associated proteins and vernalization fulfillment at the molec-ular level in wheat.

Materials and Methods

Sample Preparation. Two wheat (Triticum aestivum L. em.Thell) cultivars, most hardy Norstar and semi-hardy Azar2, wereplanted in 2006 at the green house and cold room of Seed andPlant Improvement Institute (SPII) in Karaj, Iran for vernaliza-tion fulfillment and LT tolerance determinations. Seeds of twocultivars were placed on moist filter paper in Petri dishes andimbibed in the dark at 4 °C for 48 h and then germinated at aconstant temperature of 20 °C in the dark for 24 h. Activelygerminating seeds were grown at 20 °C for 12 h at a lightintensity (mixture of fluorescent and incandescent lamps) of350 µmol m-2 s-1. After 14 days, plants were subjected to 2 °Cacclimation and samples collected for final leaf number (FLN)and LT50 (temperature at which 50% of the plants are killed byLT stress) determination. Light intensity during LT acclimationat 2 °C was 200 µmol m-2 s-1 with a 12 h day/12 h night.

Determination of LT Tolerance. The procedure outlined byLimin and Fowler14 was used to determine the LT50 of eachcultivar at the end of each LT acclimation period. At the endof each acclimation period (d0, d14, d28, d42, d56, and d89) at2 °C, plant crowns were covered in moist sand in aluminumweighing cans and placed in a programmable freezer that washeld at -3 °C for 12 h. After 12 h, they were cooled at a rate of

2 °C/h down to -17 °C, then cooled faster with a rate of 8 °C/h. Five crowns were removed at 2 °C intervals for each of fivetest temperatures selected for each cultivar in each treatment.Samples were then thawed overnight at 4 °C. Thawed crownswere planted into flats containing soil, sand, and soft moldleaves (2:1:1) that were kept moist. The flats were placed in agrowth room maintained at 20 °C with a 16 h day/8 h night.Plant recovery was rated (alive vs dead) after 3 weeks and aLT50 was calculated for each treatment.

The experimental design for LT tolerance (LT50) was a 2cultivar × 6 acclimation period factorial in a three-replicaterandomized complete block design (RCBD). Analysis of vari-ance was conducted to determine the level of significance ofdifferences due to main effects and their interactions in eachexperiment. Least significant difference (LSD) was used todetermine significant differences between means of treatments.

Determination of Vernalization Fulfillment. At the end ofeach vernalization periods (0, 14, 21, 28, 35, 42, 49, 56, and 70days), pots containing two plants (for each vernalizationtreatment in each replication) were moved to 20 °C until flagleaf emergence and leaf numbered on the main shoot. Thecontrol nonvernalized seedlings were maintained continuouslyat 20 °C with the same light intensity and length. Vernalizationfulfillment was considered when the FLN became constant.15,16

The experimental design for vernalization determination asmeasured by FLN was a 2 (cultivar) × eight (vernalizationperiod) factorial in a RCBD with two replicates. Analysis ofvariance was performed as described for the determination ofLT tolerance. Plants for FLN measurements were grown in 15cm pots filled with mixture of field soil, loamy sand and softmold leaves (2:1:1) (2 plants/pot). Plants were uniformlywatered and fertilized with sustained-release fertilizer and anutrient-complete water-soluble solution.

Protein Extraction. Proteomics analysis was performed onleaf samples of three independent replications collected fromtwo genotypes during LT acclimation periods at the time ofcold treatment initiation (d0) and 14 (d14), 28 (d28), 42 (d42),and 56 (d56) days after treatment initiation. Leaf samples (1 g)were pulverized to a fine powder with liquid nitrogen and amortar and pestle. Protein extraction was performed as previ-ously described.17 The samples were ground in liquid nitrogenand suspended in 10% (w/v) trichloroacetic acid in acetonewith 0.07% (w/v) dithiothreitol (DTT) at -20 °C for 1 h, followedby centrifugation for 15 min at 35 000× g. The pellets werewashed using ice-cold acetone containing 0.07% DTT, incu-bated at -20 °C for 1 h and centrifuged at 4 °C. Washing andsedimentation of the pellets repeated three times and thenpellets were freeze-dried. The sample powders were thensolubilized in lysis buffer [9.5 M urea, 2% (w/v) CHAPS, 0.8%(w/v) Pharmalyte pH 3-10, 1% (w/v) DTT] and the proteinconcentration was determined by the Bradford assay (Bio-Rad)with BSA as the standard.

Two-Dimensional Electrophoresis. The isoelectric focusingand the second dimension were performed as previouslydescribed.18 For analytical and preparative gels, the 24 cm IPGstrips (pH 4-7) (Bio-Rad) were rehydrated overnight with 350µL of rehydration buffer [8 M urea, 0.5% CHAPS, 20 mM DTT,0.5% (v/v) IPG buffers] in a reswelling tray (GE Healthcare) atroom temperature. For analytical and preparative gels, 120 µgand 1.2 mg of protein were loaded, respectively. Isoelectricfocusing (IEF) was conducted at 20 °C with Mutiphor II and aDryStrip kit (GE Healthcare). The running condition was asfollows: 500 V for 1 h, followed by 1000 V for 1 h, and finally

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3000 V for 16 h. The focused strips were equilibrated twice for15 min in 10 mL equilibration solution. The focused strips wereequilibrated twice for 15 min in 10 mL equilibration solution.The first equilibration was performed in a solution containing6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT,and 50 mM Tris-HCl buffer, pH 8.8. The second equilibrationwas performed in a solution modified by the replacement ofDTT by 2.5% (w/v) iodoacetamide. Separation in the seconddimension was performed by SDS-PAGE in a vertical slab ofacrylamide (12% total monomer, with 2.6% cross-linker) usinga PROTEAN II Multi Cell (BioRad). The protein spots inanalytical and preparative gels were visualized by silver nitrate19

and colloidal CBB G-250,20 respectively.Staining and Image Analysis. GS-800 densitometer (Bio-Rad)

at a resolution of 600 dots per square inch (dpi) was used forscanning of silver stain gels. The scanned gels saved as TIFimages for subsequent analysis. Gels were analyzed using theMelanie 4 software (GeneBio, Geneva, Switzerland). Spotdetection, protein quantification, and spot pairing were carriedout based on software settings. The molecular masses of proteinon gels were determined by coelectrophoresis of standardprotein markers (GE Heathcare) and pI of the proteins weredetermined by migration of the protein spots on 18 cm IPG(pH 4-7, linear) strips. The percent volume of each spot wasestimated and analyzed to protein abundance determination.One two-dimensional gel per plant was run for three biologi-cally independent replicates and percent volume of each spotwas estimated and analyzed. Considering the two genotypes(Norstar and Azar2) and two treatments (control and treated),genotype X treatment combinations were analyzed by one-wayanalyses of variance (ANOVA) for each stage (d14, d28, d42,and d56) separately. ANOVA was performed by MSTAT-Csoftware (East Lansing, MI), and mean comparisons wereperformed by Duncan’s multiple range test at P < 0.05, whenappropriate. Spots were determined to be significantly up- ordown-regulated when P e 0.05. The induction factor (IF) wascalculated by dividing the percent volume of spots in gelscorresponding to treated to the percent volume of spots in d0samples.

Protein Identification and Database Search. Protein spotsof interest were cut from the 2DE gels and destained for 1 h atroom temperature using a freshly prepared wash solutionconsisting of 100% acetonitrile/50 mM ammonium biocarbon-ate (NH4CHO3) (50:50 v/v). Wash solution was removed andspots were left to dry for 30 min at 37 °C. Proteins were digestedusing a trypsin solution containing 12 ng/µL (10 µL) trypsin in50 mM ammonium bicarbonate solution. This reaction was leftto proceed for 45 min at 4 °C. Excess of trypsin solution wasremoved and 20 µL of 50 mM ammonium bicarbonate wasadded before gel pieces were placed in a 37 °C incubatorovernight.

A 1 µL aliquot of each fraction was applied directly to theground steel MALDI target plate, followed immediately by anequal volume of a freshly prepared 5 mg/mL solution of4-hydroxy-R-cyano-cinnamic acid (Sigma) in 50% aqueous(v:v) acetonitrile containing 0.1%, trifluoroacetic acid (v:v).

Positive-ion MALDI mass spectra were obtained using aBruker ultraflex III in reflectron mode, equipped with aNd:YAG smart beam laser. MS spectra were acquired over amass range of m/z 800-4000. Final mass spectra were exter-nally calibrated against an adjacent spot containing 6 peptides(des-Arg1-Bradykinin, 904.681; Angiotensin I, 1296.685; Glu1-Fibrinopeptide B, 1750.677; ACTH (1-17 clip), 2093.086; ACTH

(18-39 clip), 2465.198; ACTH (7-38 clip), 3657.929). Monoiso-topic masses were obtained using a SNAP averaging algorithm(C 4.9384, N 1.3577, O 1.4773, S 0.0417, H 7.7583) and a S/Nthreshold of 2.

For each spot, the 10 strongest peaks of interest, with a S/Ngreater than 10, were selected for MS/MS fragmentation.Fragmentation was performed without the introduction of acollision gas. The default calibration was used for MS/MSspectra, which were baseline-subtracted and smoothed (Sav-itsky-Golay, width 0.15 m/z, cycles 4); monoisotopic peakdetection used a SNAP averagine algorithm (C 4.9384, N 1.3577,O 1.4773, S 0.0417, H 7.7583) with a minimum S/N of 3. BrukerflexAnalysis software was used to perform the spectral process-ing and peak list generation for both the MS and MS/MSspectra.

Combined mass spectral and tandem mass spectral datawere submitted to database searching using a locally runningcopy of the Mascot program (Matrix Science Ltd., version 2.1),through the Bruker BioTools interface (version 3.1). Searchcriteria included: Enzyme, Trypsin; Variable modifications,Oxidation (M); Peptide tolerance, 200 ppm; MS/MS tolerance,0.8 Da; Instrument, MALDI-TOF-TOF. The search criteria alsoincluded Carbamidomethyl (C) as a fixed modification for allalkylated samples. The database search was run against NCBInonredundant protein database Viridiplantae (20080218; 5519594sequences; 1911975371 residues).

Results and Discussion

Vernalization Fulfillment. Two cultivars that representdifferent reproductive strategies were selected for detailedevaluation in these studies. Norstar is a Canadian winter-habitcultivar with a long vernalization requirement and excellentLT tolerance that has been widely used as a parent in breedingprograms targeting regions with high stress winters. It has alsobeen used extensively in the investigation of low-temperatureresponse mechanisms in wheat.21 “Azar2” is a recently releasedwinter habit cultivar with a relatively short vernalizationrequirement that has been gaining wide acceptance as areplacement for Sardari local cultivar in the dry and coldnorthwest areas of Iran. Azar2 was derived from a single crossbetween a CIMMYT winter habit cultivar and Sardari.

Analysis of variance for the FLN showed that differences dueto cultivar, vernalization periods, and the cultivar × vernaliza-tion periods interactions were all highly significant (P < 0.001;Supplementary Table 1, Supporting Information). These resultsindicated that there were important differences in the magni-tude and pattern of FLN in different vernalization requirementcultivars (Figure 1). In either Azar2 or Norstar winter wheatcultivars, the marked differences of FLN due to the days ofvernalization were highly significant (P < 0.001). When grownat continuous 20 °C compared to a 28 and 35 d pretreatmentat 2 °C, Norstar and Azar2 wheat showed a reduced FLN from23 to 10.0 and 18.5 to 10.0, respectively (Figure 1), indicatingthat these are winter habit genotypes with a vernalizationrequirement.22 In Norstar, FLN became constant between 35and 42 days of vernalization (vernalization fulfillment), indicat-ing that a long vernalization requirement period in this wintercultivar causes a delay in its transition from the vegetative tothe reproductive phase. Azar2 winter cultivar with a shortvernalization requirement, achieved minimum FLN or vernal-ization fulfillment between 21 and 28 days of vernalization.

research articles Sarhadi et al.

5660 Journal of Proteome Research • Vol. 9, No. 11, 2010

These observations agree with earlier reports that Norstarhas a long vernalization requirement when grown in controlledand field conditions.1,2,16

On LT exposure, vernalization-requiring cultivars reducetheir final leaf number (FLN) until they reach a minimum leafnumber, which is the point of vernalization fulfillment.15,16,22,23

Consequently, transition from the vegetative to the reproduc-tive phase was considered complete when the FLN for con-secutive sampling dates became near constant.16,24

LT Tolerance. Analysis of variance indicated that differencesin LT50 due to cultivars, acclimation period, and the acclimationperiod × cultivar interactions were highly significant (P < 0.001)(Supplementary Table 1, Supporting Information). Plants grownat 2 °C started to acclimate at a rapid rate. The rate of changein LT tolerance then gradually slowed until LT tolerance beganto be lost. Norstar wheat reached its maximum LT tolerance(LT50 ) -18.7 °C) between 34 and 42 d, which is about thesame time as vernalization saturation occurred (Figure 2). Thisis in agreement with previous reports2,16 and indicates that thesignaled end of the vegetative phase, as indicated by FLNmeasurements (Figure 1), corresponds to the loss of LTtolerance. Azar2 cultivar with a relatively short vernalizationrequirement (about 28 d) achieved maximum LT tolerance (LT50

) -8 °C) about the same time as vernalization fulfillmentoccurred (Figure 1). Greater LT tolerance was maintained forNorstar for the period from 28 to 89 d (Figure 2). This appearsto be the result of an extended vegetative period (compared to

Azar2) that delayed the transition to the reproductive phaseas illustrated in Figure 1. It has been recently reported thatAzar2 winter cultivar obtains its maximum LT tolerance (LT50

) ∼ -14 °C) about the first week of December which is alsoabout the same time as vernalization saturation occurs underfield conditions of northwest Iran.1 Satisfaction of the vernal-ization requirement has been associated with a decline in LTtolerance of overwintering cereals.1,2,16,25

Proteome Response. Leaf proteins from d0, d14, d28, d42,and d56 samples were analyzed using 2-DE gels in threeindependent replicates. Out of 1094 reproducibly detectedproteins, 102 proteins showed significant changes in abundancein at least one stage compared with d0 (Supplementary Table2 and Supplementary Figure 1, Supporting Information). Theirestimated molecular masses and pIs are listed in Supplemen-tary Table 2 (Supporting Information), along with their abun-dance ratios (abundance in treated plants as a fraction of theirabundance in d0 plants). As shown in Supplementary Table 2(Supporting Information) and Figure 3, both the number andthe accumulation of some LT-associated proteins were in-creased during vegetative stage of Norstar winter wheat, whilea gradual decline was observed after vernalization fulfillmentindicating the regulatory role of vernalization response on theexpression of some LT-associated genes in wheat. In general,the number of proteins responding to treatments increased asthe treatment progressed. However, the numbers of differen-tially expressed proteins were much higher in Norstar com-pared to Azar2. Forty-nine proteins (32 up- and 17 down-regulated, respectively) showed significant changes during coldacclimation in Azar2, whereas 91 proteins (71 up- and 21 down-regulated, respectively) changed significantly in Norstar (Figure3).

Protein Identification by Mass Spectrometry. One hundred-two proteins listed in Supplementary Table 2 (SupportingInformation) were analyzed by MALDI-TOF/TOF mass spec-trometry leading to significant identification of 66 LT-associatedproteins (Table 1 and Supplementary Table 3, SupportingInformation). Figure 4 shows the positions of the 66 identifiedprotein using mass spectrometry on a 2-DE gel of leaf fromNorstar plant harvested at d56.

Figure 1. Final leaf number (FLN) of Norstar and Azar2 winterwheat cultivars acclimated at 2 °C for 0-89 d and then moved to20 °C conditions (SE ) 0.74).

Figure 2. Low-temperature tolerance of Norstar and Azar2 winterwheat acclimated at 2 °C for 0-98 days. SE ) 0.64.

Figure 3. Number of leaf proteins differing significantly inabundance during cold acclimation in wheat genotypes Norstaran Azar2 compared to d0. Based on data in Supplementary Table1 (Supporting Information). Solid bars, proteins more abundantduring cold acclimation. Open bars, proteins less abundantduring cold acclimation.

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Table 1. Abundance Ratio of Proteins at d14, d28, d442, and d56 Compared with d0a

induction factor

Norstar Azar2

spot ID protein name GI number d14/d0 d28/d0 d42/d0 d56/d0 d14/d0 d28/d0 d42/d0 d56/d0

Cold responsive proteins111 Cold acclimation protein (WCS19) 28273091 4.14 5.85 6.57 9.73 P P P P112 Cold acclimation protein (WCS19) 28273091 4.11 6.13 8.26b 10.82b P P P P120 Actin-binding protein (WCOR719) 1381154 1.82 4.05c 2.54c 3.90c 5.51 5.66 5.71b 6.45c

121 Actin-binding protein (WCOR719) 11066101 6.19c 8.92c 6.64c 9.30c 7.01b 6.23 7.46b 8.88c

125 Cold-responsive LEA/RAB-relatedCOR protein (Wrab17)

7716956 2.36 1.69 3.40c 4.00c 0.99 0.86 1.64b 1.28

126 Cold regulated protein (Wcor18) 26017213 3.43 6.23c 6.85c 6.91b 3.54b 3.33 4.90c 4.59128 Cold regulated protein (Wcor18) 26017213 1.20 1.50b 1.92b 1.30 0.93 0.68 1.30 1.19130 Cold regulated protein (Wcor18) 26017213 3.25 5.11c 3.92c 5.50c 0.93 0.59 0.42 1.24131 Group3 LEA protein 157073742 2.48 1.66 4.90 4.44c 0.94 0.62 1.39 1.15136 Cold acclimation protein (Wcor615) 1657857 0.47b 0.52b 0.77 0.63 0.76 0.69 1.31 1.08139 Cold-responsive LEA/RAB-related

COR protein (Wrab17)7716956 2.38 2.57 3.748b 3.69c 1.21 1.21 2.16c 1.47

141 Cold-responsive LEA/RAB-relatedCOR protein (Wrab17)

7716956 2.21b 3.86b 1.89c 2.02 1.29 1.43 0.90 1.53

848 Cold-responsive protein (Wcor14) 20067227 P P P P A A A A849 Cold-responsive protein (Wcor14a) 6561859 P P P P A A A A850 Cold-responsive protein (Wcor14 c) 20067227 P P P P A A A A

Antifreezing53 PR-4 6048567 AA P P P 0.64 0.88 0.24 0.42383 Chitinase 563489 1.72 4.09b 9.81b 12.63c 0.21 1.41 3.06 2.83838 Thaumatin-like protein 115454465 AA AA P P 1.63 0.65 2.10 1.10933 beta-1,3-glucanase 109150358 P P P P P P P P1011 beta-1,3-glucanase 34787356 3.63 6.58c 12.01b 14.48c 1.38 0.48 2.68 2.10

Oxidative stress/defense58 USP family protein 60100214 1.41 1.82b 1.62 2.4b 1.59 1.44 1.41 1.8269 USP family protein TA50110_4565 3.16 3.63c 4.01c 3.19c 1.60 5.03 6.63 2.1087 Nucleoside diphosphate kinase 2 42733490 2.75c 2.27 3.00b 2.13b 1.28 0.80 1.38 0.6690 Cu/Zn superoxide dismutase 1568639 1.96c 2.25c 2.03 1.86 0.47 0.32c 0.41b 0.33c

177 2-Cys peroxiredoxin 2829687 1.12 1.23 1.71 1.07 1.28 1.97b 2.89b 2.48b

309 Manganese superoxide dismutase 1654387 1.11 1.36 1.65b 2.08c 1.29 1.23 1.22 1.49482 S-like RNase 41387691 1.89b 1.95b 2.96c 1.90b 0.96 0.97 2.06 0.79486 2-Cys peroxiredoxin 2829687 AA P P P 0.54 0.67 1.48 0.82500 Cu/Zn superoxide dismutase 1568639 1.66b 2.56 4.31c 4.76c 0.83 1.15 1.17 2.05b

508 Glutathione transferase 20067423 2.06 3.16b 3.64b 4.40b 0.70 1.46 1.75 0.00562 Cyclophilin 115472829 P P P P 3.49 7.10b 7.33b 7.38c

Photosynthesis41 RuBisCO small subunit 62176930 0.85 0.79 0.62c 0.44c 0.68 0.64b 0.50b 0.4952 RuBisCO small subunit 132107 3.11b 4.06b 4.35c 4.40 P P P P62 RuBisCO small subunit 11990897 1.36 1.89b 1.02 2.02b 0.76 1.08 1.04 1.6864 RuBisCO small subunit 11990897 1.63b 1.67c 1.55b 1.42b 1.73 1.50 1.26 1.39247 RuBisCO small subunit 132107 0.46b 0.51b 0.60b 0.65b 1.14 1.42 1.19 1.71248 RuBisCO small subunit 132107 0.54b 0.51b 0.51b 0.77 0.75 0.51 0.59 0.54400 Oxygen-evolving enhancer protein 2 131394 1.40 1.53b 1.75c 1.12 1.02 1.10 1.22 0.89532 Oxygen-evolving enhancer protein 1 147945622 4.80 5.80c 10.54b 1.76 3.29 3.92 5.46c 2.65536 Oxygen-evolving enhancer protein 1 739292 1.62 1.99b 2.48c 1.87b 1.25 1.12 1.23 1.19705 RuBiCo activase isoform 1 167096 0.49c 0.51c 0.46c 0.77 0.78 0.93 0.56 0.701036 Oxygen-evolving enhancer protein 2,

chloroplast precursor21836 3.10b 4.10c 5.52c 4.27 0.28 0.55 0.57 1.24

1087 Plastocyanin 115465862 2.53 2.80 4.59 3.14 1.56b 1.66b 1.01 1.49

Metabolism106 Thioredoxin M-type 11135474 1.53b 1.47 1.94c 1.42 2.91 2.58 3.16b 3.02b

246 Enoyl-ACP reductase 115475922 0.45c 0.50c 0.40c 0.65b 0.66 0.88 0.67 0.73315 Thioredoxin M-type, chloroplast

precursor (TRX-M)11135474 1.62b 1.93c 2.26 1.71b 2.35 2.73 3.62 1.73

371 3-beta hydroxysteroid dehydrogenase/isomerase

115461679 0.59b 0.59c 0.47c 0.65b 0.71 0.69 0.55 0.64

415 Oxalate oxidase 22138786 0.72 1.54 2.58c 3.25b 1.20 0.65 1.56 1.84640 Plastidic cysteine synthase 1 125529334 0.55c 0.51c 0.54c 0.67b 0.62 0.64 0.60 0.65647 Fructose-bisphosphate aldolase,

chloroplast precursor108864053 3.63b 2.75c 4.02c 1.87 1.62 1.35 1.54 1.23

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Of 66 differentially expressed proteins, the expression levelof 41 proteins showed significant changes during cold acclima-tion in only one genotype. The expression of four proteinschanged only in Azar2, while 37 protein spots showed changesin expression only in Norstar. The asterisks in Table 1 identifythe abundance ratios that are significantly different from 1.00based on three biologically independent replicates. Of 66proteins, 25 proteins changed significantly in both genotypesduring cold acclimation of them 16 proteins increased while 4

proteins decreased significantly in both. Five protein spotschanged significantly in opposite directions in the two genotypes.

The expression pattern of cold-induced proteins in pro-teomic analyses revealed significant changes in abundance ofcold-induced proteins during the vegetative/reproductive tran-sition between cultivars. Even though there were large differ-ences in cold tolerance, similar proteins were expressed bythese winter-habit cultivars. In Norstar, the maximum ac-cumulation of proteins was detected on day 42 of vernalization

Table 1. Continued

induction factor

Norstar Azar2

spot ID protein name GI number d14/d0 d28/d0 d42/d0 d56/d0 d14/d0 d28/d0 d42/d0 d56/d0

649 Thiamine biosynthetic enzyme 115472485 4.27c 3.56b 3.12c 4.23c 3.34 3.05 2.838 2.70815 Glycosyl hydrolases 125542150 P P P P 1.37 1.96 3.12 2.26935 fructose-bisphosphate aldolase 15227981 1.54 1.61 2.39 1.43 1.64 2.354b 2.546b 1.291092 Oxalate oxidase 22138786 1.60 2.70c 3.94c 4.27c 2.40 1.63c 2.34 2.30

Protein synthesis80 Ribosomal protein S12 115470601 1.59 1.82b 1.99b 1.49 1.58 1.59 1.18 0.66134 Chloroplast 50S ribosomal protein L12 115439005 1.29 2.56b 6.98c 8.21c 0.72 0.66b 1.51 1.89135 Chloroplast 50S ribosomal protein L12 115439005 0.72 0.60b 0.31c 0.51c 0.61b 0.52c 0.47c 0.63166 Translational elongation factor P 125535556 0.75 0.65b 0.71 0.81 0.61c 0.50c 0.53c 0.71

Chloroplast post-transcriptional regulation514 Harpin binding protein 1 38679333 2.33c 2.67c 3.03b 2.88c 1.12 0.91 0.96 1.10462 29 kDa ribonucleoprotein (cp29) 149392545 2.19 2.09c 1.67 3.03c 1.91 2.15 3.29b 3.35b

529 RNA-binding protein (cp31) 3550467 0.51b 0.21c 0.43c 0.45c 0.50b 0.40b 0.53b 0.45c

586 RNA-binding protein (cp33) 3550485 3.22b 2.51 4.44c 4.27c 1.98 2.24 3.57b 1.87599 RNA-binding protein (cp33) 3550485 AA AA P P 1.96 1.40 2.78 3.011073 RNA-binding protein (cp31) 3550483 1.25 3.79 5.92c 6.28c 1.77 1.90 2.67 1.95

Others866 DAG protein 115460334 0.84 0.62b 0.37b 0.40b 1.28 2.75 0.53 1.161094 TGB12K interacting protein 2 29826242 2.75 2.04 2.64 2.12 1.58 1.96c 2.51b 1.97b

a The abundance ratio is expressed as average %volume in treated plants/average %volume in control plants (d0)). The numbering corresponds to the2-DE gel in Figure 4. P, not detected in d0; A, detected only in d0; AA, not detected in d0 and in cold-acclimation stage. b Treatment effect is significant, P< 0.05. c Treatment effect is significant, P < 0.01.

Figure 4. 2-DE gel analysis of proteins extracted from leaves of genotype Norstar harvested at d56. In the first dimension, 120 µg ofprotein was loaded on an 24 cm IPG strip with a linear gradient of pH 4-7. In the second dimension, 12% SDS-PAGE gels were used,with a well for Mr standards. Proteins were visualized by silver staining. Numbered spots correspond to the proteins identified bymass spectrometry.

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at the same time that vernalization fulfillment occurred. Thisprobably is due to the influence of vernalization fulfillment,which affected the transition from the vegetative to thereproductive phase and caused down-regulation of cold-induced genes and loss of LT tolerance. The maximum ac-cumulation of proteins in Azar2 was also observed on day 42of vernalization but 14 days after vernalization fulfillment. Thismay suggest the influence of other mechanisms in modulatingdown-regulation of cold-induced proteins.26

The regulatory role of vernalization on phenological devel-opment and their effects on the expression of LT tolerance incereals has been demonstrated in different studies.27 Resultsof the current experiments explain the role that developmentalgenes, such as the vrn alleles, play in the regulation of LTtolerance gene expression and clearly demonstrate that thepoint of vernalization fulfillment is pivotal in the expressionof LT tolerance genes. On the basis of the developmentalhypothesis of LT tolerance, cereal plants have different waysof increasing the length of vegetative phase, all of which extendthe time that low temperature tolerance genes are more highlyexpressed. Recent researches have shown that transition to thereproductive phase, which is determined by clusters of co-adapted flowering genes such as vrn, is the critical switch thatinitiates down-regulation of the LT tolerance genes.3,6,27,28

The identified proteins were classified into several functionalcategories including cold-regulated proteins, antifreeze pro-teins, oxidative stress defense, photosynthesis, chloroplast post-transcriptional regulation, metabolisms, and protein synthesis(Figure 5). The largest functional category was Cor/Lea proteins(23%), which was greatly affected during cold acclimation(Figure 5).

Several Up-Regulated LT-Associated Proteins in NorstarWere Identified as Cor/Lea Proteins. In wheat and its relatedcereal species, which grow under widely different climaticconditions, freezing-tolerant cultivars show greater ability forcold acclimation than susceptible cultivars.29 A growing num-ber of genes have been shown to be induced during coldacclimation, many of their encoded proteins could potentiallycontribute to freezing tolerance.4,30 The cold-responsive geneshave classified as Cor (cold-responsive or -regulated), Lea (lateembryogenesis abundant), Dhn (dehydrin), Rab [responsive toabscisic acid (ABA)], Lt (LT responsive), ERD (early dehydra-tion-inducible), and RD (responsive to desiccation) or others.4

Many of polypeptides encoded by these cold-responsive geneshave repeated amino acid sequence motifs and share the

property of being extremely hydrophilic. These genes arecollectively called Cor/Lea gene family according to the des-ignation by Thomashow.4 There is strong evidence that Cor/Lea gene can contribute to freezing tolerance.31-33

We identified 15 Cor/Lea proteins including cold acclimationprotein WCS19 (spots 111 and 112), WCOR719 (spots 120 and121), cold-responsive LEA/RAB-related COR protein (spot 125,139, and 141), cold regulated protein (spots 126, 128, and 130),group3 late embryogenesis abundant protein (spot 131), coldacclimation protein WCOR615 (spot 136), Cold-responsiveprotein WCOR14c (spots 848 and 850), and cold-responsiveprotein WCOR14a (spot 849).

We questioned whether the level of LT tolerance after theexposure of wheat seedlings to the LT condition can beexplained by the expression profiles of the LT-responsive Cor/Lea proteins in hardy Norstar and semi-hardy Azar2 wheat. Weobserved that Cor/Lea protein expression was highest for theLT-tolerant, Norstar, and lowest for the tender genotypes,Azar2. WCS19 (spots 111 and 112) were highly up-regulated inNorstar and detected in Azar2 under stress condition. Howeverthe expression level of these spots were lower in Azar2 asmeasured by percent volume of spots. WCS19 is a stromalprotein that belongs to a new class of organelle-targeted group3 LEA proteins. It was shown that the overexpression of anotherchloroplast-targeted wheat COR/LEA protein, WCS19, improvedfreezing tolerance of cold-acclimated leaves.34 We also identi-fied three other proteins belong group 3 LEA proteins. Theseinclude WCOR14a (spots 848 and 850) and WCOR14a (spot 849)which were detected only in Norstar. Wcor14 is chloroplast-targeted protein and are induced by LT and influenced bylight.35,36 Despite the observed cryoprotective activity of theseproteins, their exact roles remain to be elucidated.

The signal for up-regulation of Cor proteins have beeninvestigated in Arabidopsis. It has been demonstrated that coldstress induces the expression of C-repeat binding factors(CBFs), also known as dehydration-responsive element-bindingprotein 1s or DREB1s, which can bind to cis-elements in thepromoters of COR genes and activate their expression andconfer freezing tolerance.37 Similar CBF genes may also func-tions as a transcription factor for the Cor/Lea genes to developfreezing tolerance in common wheat.38

Genotypes with a winter growth habit (vrn-1 allele) showvery low VRN-1 transcript levels and certain CBF genes athigher levels until plants are vernalized. Once the wintergenotypes carrying the vrn-1 allele are vernalized, CBF tran-

Figure 5. Functional category distribution of the 66 identified proteins.

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script levels are dampened relative to levels detected innonvernalized plants.39 We observed the highest number of up-regulated Cor/Lea genes in freezing tolerant genotype, Norstar,at the same time that vernalization fulfillment occurred whichis likely due to the influence of vernalization fulfillment. It isexpected that several CBF and COR genes are down-regulatedin response to high VRN-1 transcript levels after vernalizationfulfillment. However, no significant decrease in the accumula-tion of Cor/Lea protein at d56 was observed. This might bedue to a time lag between vernalization fulfillment, the increasein VRN-1 protein level and the full effect of the resultingincrease in down-regulation of Cor/Lea proteins. In addition,it has been suggested that VRN-1-mediated mechanism mayplay a role in the regulation of a specific subset of cold-responsive genes and VRN-1 transcription is necessary but notsufficient to down-regulate the COR genes.26

Further studies are needed to determine the mode of actionand regulation of Cor/Lea genes in wheat during cold acclima-tion and vernalization.

Up-Regulation of Antifreeze Proteins May Offer Protec-tion against Freezing. When plants are exposed to freezingtemperatures, ice crystals form in the intercellular spaces.Antifreeze proteins bind irreversibly to the ice crystals andinhibit their growth (for review see ref 40). Antifreeze proteinsfrom plant are homologous to pathogenesis-related (PR) pro-teins and include �-1,3-glucanases, chitinases, thaumatin-likeproteins, and polygalacturonase inhibitor protein (Hon et al.1995; Worrall et al., 1998). Five of Lt-associated proteins wereidentified as antifreezing proteins including PR-4 protein (spot53), chitinase (spot 383), thaumatin-like protein (spot 838),�-1,3-glucanase (spots 933 and 1011). PR-4 protein was de-tected only in Norstar and one beta-1,3-glucanase (spot 933)was observed in two genotypes only during cold acclimation.Thaumatin-like protein (spot 838) was present in Norstarduring cold acclimation but did not show change in Azar2. One�-1,3-glucanase (spot 1011) and chitinase (spot 383) were up-regulated only in Norstar. Plant �-1,3-glucanases are docu-mented to contribute to several physiological processes in-cluding in response to abiotic stresses.41,42 It has been suggestedthat glucanases in winter rye inhibit the formation of fatal icecrystals, in addition to its potential role in resisting infectionby psychrophilic pathogens.41

Post-translational modifications of chitinase antifreeze pro-teins were studied in winter rye using mass spectrometry. Itwas shown that neither the pathogen-induced chitinases northe cold induced chitinase-AFPs were post-translationallymodified beyond cleavage of the sequence targeting them forsecretion.43 Further studies are needed to elucidate whatfeatures of distinguish PR proteins with and without antifreezeactivity. It is possible that the activities of PR and antifreezeproteins are regulated through interactions with other mol-ecule.40

Stress/Defense Proteins Were Up-Regulated to a GreaterExtent in Norstar. We identified 11 LT-associated proteinsinvolved in oxidative stress tolerance. These proteins includeUSP family proteins (spots 58 and 69), nucleoside diphosphatekinase 2 (spot 87), Cu/Zn superoxide dismutase (SOD) (spots90, 309, and 500), 2-Cys peroxiredoxin (spot 177 and 486), S-likeRNase (spot 482), and glutathione transferase (spot 508), andcyclophilin (spot 562).

ROS appear to have a strong influence on cold regulation ofgene expression.57 ROS can induce Ca2+ signatures that impactcold signaling. ROS signals can also exert their effects directly

through the activation of redox-responsive proteins, such astranscription factors and protein kinases. It has been demon-strated that CBFs also regulate the expression of genes involvedin ROS detoxification and many others with known or pre-sumed cellular protective functions.37

All three identified SOD were up-regulated in Norstar.Whereas in Azar2, spot 90 was down-regulated, spot 309 didnot show any significant change, and spot 500 was up-regulatedonly at d56. SOD converts superoxide to the less toxic hydrogenperoxide (H2O2) molecule and its differential expression hasbeen reported in response to various abiotic stresses.42,44-46

The detoxification of H2O2 is the accomplished with enzymessuch as ascorbate peroxidase and 2-cys peroxiredoxin. Interest-ingly, spots 177 and 486 were identified as 2-Cysteine perox-iredoxins, a member of a ubiquitous group of peroxidases thatreduce H2O2 and alkyl hydroperoxide.47 Spot 177 was up-regulated during cold acclimation only in Azar2 whereas spot486 was present only during cold acclimation in Norstar anddid not show any significant changes in Azar2. It has beensuggested that 2-Cys peroxiredoxin-dependent water/watercycle may be an important alternative to detoxify H2O2 underoxidative stress conditions.18,48-50

We also identified a nucleoside diphosphate kinase (NDPK)2 (spot 87) up-regulated only in Norstar. This protein is believedto be a housekeeping enzyme that uses ATP to maintainintracellular levels of CTP, UTP, and GTP. In addition, NDPKmay play significant roles in stress responses49,51 and mitogen-activated protein kinase (MAPK)-mediated H2O2 signaling.52

It has been shown that The overexpression of AtNDPK2 led todecreased constitutive ROS levels and enhanced tolerance tomultiple environmental stresses.53

A glutathione transferase (GST) (spot 508) and a s-like RNase(spot 482) were up-regulated only in Norstar. GST limitsoxidative damage by removing reactive oxygen species (ROS).54

The S-like RNases are closely related to S-RNases but they haveimportant differences in structure, expression, and function.55

Their genes are induced in response to developmental andenvironmental cues.51 It suggests that this S-RNase like proteinsmay be involved in a responsive or signaling pathway understresses. However, the exact function of these proteins understress remains to be elucidated.

Two up-regulated proteins (spots 58 and 69) in Norstar wereidentified as Two USP family proteins (spots 58 and 69), a planthomologue of the bacterial universal stress protein family, wereup-regulated only in Norstar. It was shown that the expressionof a member of USP family, OsUsp1, was induced by submer-gence and ethylene treatments in rice.56 The precise functionand the mode of action of these novel proteins remained tobe established.

Overall, the predominance of proteins related to oxidativestress defense in Norstar underscores the importance ofmanaging ROS and oxidative damage during cold acclimationto protect plant cells.

Stabilizing Factors for Nonribosome-Bound mRNAs inthe Stroma Showed Higher Expression during Cold Acclima-tion. We identified six chloroplast ribonucleoproteins (cpRNPs)involved in stabilization of nonribosomal-bond mRNAs instromal. These proteins include Hairpin binding protein 1 (spot514), 29 kDa ribonucleoprotein (cp29) (spot 462), RNA-bindingprotein (cp31) (spots 529 and 1073), and RNA-binding protein(cp33) (spots 586 and 599). Five out of six proteins showedhigher expression during cold acclimation (Table 1). It has beensuggested that cpRNPs associate with nascent RNAs or pre-

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RNAs immediately after transcription and confer stability andrsibonuclease resistance to the RNAs. These cpRNP-RNAcomplexes also act as a scaffold for the specific catalyticmachinery involved in RNA maturation and RNA editing.58

Further studies of cpRNPs are necessary to clarify the mode oftheir regulation by external stimuli in plant Cells and theirinvolvement in plant adaptation to stresses.

Abundance of Proteins Associated with PhotosynthesisChanged during Cold Acclimation. Totally 12 LT-associatedproteins were classified as photosynthesis related proteins.These proteins were classified in three subcategories: (1) lightharvesting reaction including two oxygen-evolving enhancerprotein 1 (spots 532, 536), an oxygen-evolving enhancer protein2 (spots 400 and 1036), and a plastocyanin (spot 1092), (2)Calvin cycle including six Rubisco small subunits (spots 41, 52,62, 64, 247, 248), and (3) regulation of photosynthesis includinga RuBiCo activase isoform 1 (spot 705) (Table 1).

All light harvesting reaction proteins were significantly up-regulated only in Norstar whereas six differentially expressedRubisco small subunits fragments did not show any specifictrend in two genotypes. Partial degradation of photosynthetichave been reported in abiotic stressed such as chilling9 anddrought51 stresses.

Rubisco activase isoform 1 was down-regulated in bothgenotypes. These results pointed that changes in expressionsof photosynthetic proteins happened during cold acclimationbut further studies are required to find out possible linkbetween these changes and cold-acclimation of wheat.

Concluding Remarks. Our results suggest that proteomicsapproach can be a powerful method to broaden our knowledgeabout plant-environment interaction at proteome level. Theseresults include the following:

1. Both the total number of cold associated proteins and thenumber of up-regulated proteins (P < 0.05) were higher in themost hardy genotype, Norstar, than in the semi-hardy geno-type, Azar2. The results provide evidence that cold causes aredirection in protein synthesis, to a much greater extent infreezing tolerant genotype.

2. The main contrasting results observed here was clearinduction of Cor/Lea and antifreezing proteins during coldacclimation in freezing tolerant genotype, Norstar, whereas lessinduction was observed in Azar2. Furthermore, the predomi-nance of proteins related to ROS handling in Norstar under-scores the importance of managing ROS and oxidative damageduring cold acclimation to protect plant cells. It has beenshown that CBFs regulate the expression of Cor/Lea and ROSdetoxification genes and many others with known or presumedcellular protective functions.37 As these genes are modulatedby known classes of transcription factors and other regulatorymechanisms, these underlying regulatory genes become can-didate genes for molecular breeding.

3. A close association between the vernalization fulfillmentand the start of a decline in the protein accumulation of hardyNorstar with long vernalization requirement and semi-hardyAzar2 with a short vernalization requirement supports thehypothesis that developmental traits which is regulated byvernalization16,23 has a regulatory influence over low-temper-ature gene expression.

4. In addition to differential expression of Cor/Lea andoxidative stress defense proteins in two genotypes, the identi-fied proteins are known to be involved in several importantputative mechanisms of plant adaptation to freezing conditions

including photosynthesis, chloroplast post-transcriptional regu-lation, metabolisms, and protein synthesis.

While this work provides experimental evidence on thecontribution of several candidate proteins and mechanisms infreezing tolerance in wheat, further investigation is requiredto precisely clarify the function of these proteins in coldtolerance in planta. Proteome analysis of organelles such asthe plasma membrane may be applied to broaden our knowl-edge about cold tolerance.59

More work is also needed to determine the relationship oftiming of proteomic changes and cold acclimation/vernaliza-tion fulfillment. Deacclimation treatment of acclimated plantsmight be an effective approach to understand the role of cold-rinduced proteins in cold acclimation/vernalization fulfillment.Proteome analysis of additional cultivars with different require-ment of cold acclimation and/or vernalization may help tobetter understand wheat behavior under low temperatureconditions. The difference in expression pattern can be exam-ined genetically by further studies on mapping populationderived from the cross between contrasting lines.

Acknowledgment. This study was supported by grantsfrom Agricultural Biotechnology Research Institute of Iran(to G.H.S.) and from Seed and Plant Improvement Institute(to M.R.).

Supporting Information Available: Supplementarytables and figure. This material is available free of charge viathe Internet at http://pubs.acs.org.

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Cold Acclimation Proteome Analysis research articles

Journal of Proteome Research • Vol. 9, No. 11, 2010 5667