Proteome Analysis of Cold Acclimation in Sunflower

Published: February 23, 2011

r 2011 American Chemical Society 2330 dx.doi.org/10.1021/pr101137q | J. Proteome Res. 2011, 10, 2330–2346

ARTICLE

pubs.acs.org/jpr

Proteome Analysis of Cold Acclimation in SunflowerTiago S. Balbuena,*,† Joaquín J. Salas,‡ Enrique Martínez-Force,‡ Rafael Garc�es,‡ and Jay J. Thelen†

†Department of Biochemistry and Interdisciplinary Plant Group, Christopher S. Bond Life Sciences Center, University of Missouri,Columbia, Missouri 65211, United States‡Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, E-41012 Sevilla, Spain

bS Supporting Information

’ INTRODUCTION

Plants are sessile organisms that frequently face differentenvironmental variations and have to adapt their metabolismto overcome adverse conditions. Low temperature has a greatimpact on plant productivity, mostly because it significantly altersplant metabolism and physiology.1 The development of toler-ance mechanisms in response to this environmental stress isessential to ensure correct growth and development.2 A parti-cular characteristic developed by plants long exposed to tem-perature stress is the phenomenon of acquired tolerance.2 Coldacclimation is the term assigned for the metabolic changes thatoccur in the organism if exposed to low, but nonfreezing, tempera-tures conferring tolerance to subsequent subzero temperatures.3

Cold acclimation has been shown to involve reprogramming ofmetabolism, changes in gene expression, changes in membranefluidity, alterations in carbohydratemetabolism, increase in ability towithstand oxidative stress and synthesis of cold acclimation inducedproteins.4

Although the analysis of gene transcripts may provide com-prehensive information about the physiological state of anorganism in a particular condition, analysis of the proteincomplement is also critical, as studies have demonstrated thatthe levels of transcripts are not strictly correlated to the levels ofthe translated proteins.5�7 In addition, many crucial post-trans-lational modifications may occur that can not be screened bytranscript analysis. Proteome analyses of cold responses havebeen carried out in different plant organisms. For woody species,

Prunus persica and Picea obovata cold responses were studiedthrough two-dimensional gel electrophoresis (2-DE) and detec-tion of differential expression by DIGE technology.4,8 Differen-tially expressed proteins during cold acclimation in these woodyspecies were mostly involved in carbohydrate metabolism,defensive and protective mechanisms, energy production andcytoskeleton organization. As for model plant species, the sameproteomic strategy was performed for Arabidopsis thaliana plantsunder two different cold acclimation conditions, resulting in theidentification of 22 proteins involved in cold response.9 In depthcomparative analysis of cold response in the nuclear proteome ofArabidopsis revealed differential expression of 54 proteins out ofthe 184 proteins identified in the nuclear map.10 Thellungiellahalophila is a newly emerging model species for the molecularelucidation of abiotic stress tolerance.11 Two dimensional elec-trophoresis comparison in leaves of this species during short andlong-term exposure to cold stress, revealed significant change in66 protein spots out of 1500 detected protein spots.12 Subse-quent mass spectrometry driven identification revealed thatidentified proteins mainly participate in photosynthesis, RNAmetabolism, defense response, energy pathway, protein synthe-sis, folding, cell wall, cytoskeleton and signal transduction. InPhyscomitrella patens, a highly tolerant species to a variety ofabiotic stresses, 2-DE-based proteome analysis also revealed

Received: November 12, 2010

ABSTRACT: Cold acclimation is the phenomenon in which plantsare exposed to low, but nonfreezing, temperatures before exposure todrastic temperatures. To investigate how sunflower plants adjust theirmetabolism during cold treatment, a comparative proteomic ap-proach, based on spectral counting data, was adopted to identifydifferentially expressed proteins in leaves of freezing susceptible(Hopi) and tolerant (PI 543006 and BSD-2-691) lines after coldacclimation. In total 718, 675, and 769 proteins were confidentlyidentified by tandem mass spectrometry in Hopi, PI 543006, andBSD-2-691 sunflower lines. Tolerant lines PI 543006 and BSD-2-691showed the highest number of differentially expressed proteins, as 43,72, and 168 proteins changed their expression inHopi, PI 543006, andBSD-2-691 sunflower lines, respectively, at 95% confidence. Cold-responsive proteins were mostly involved in metabolism, proteinsynthesis, energy, and defense processes in all sunflower lines studied. Hierarchical clustering of all differentially expressed proteinsresulted in the characterization of 14 different patterns of expression across Hopi, PI 543006, and BSD-2-691 and indicated thattolerant lines showed different proteome responses to cold acclimation.

KEYWORDS: cold acclimation, GeLC�MS, Helianthus annuus, mass spectrometry, spectral counting

2331 dx.doi.org/10.1021/pr101137q |J. Proteome Res. 2011, 10, 2330–2346

Journal of Proteome Research ARTICLE

changes in the expression of proteins involved in signaling,cytoskeleton and defense after cold acclimation.13 Late develop-ment consequences after cold treatment at the youngmicrosporestage were detected in rice anthers, where 70 protein spots wereup- or down-expressed.14 The seedling leaves of Oryza sativawere also screened for proteome responses, using the 2-DEapproach, after progressive low temperature treatment.15 Fromthe 60 differentially changed protein spots, 41 were successfullyidentified including proteins involved in protein synthesis, fold-ing and degradation, antioxidative/detoxifying enzymes, energypathway and signal transduction. Although some of the mechan-isms involved in cold acclimation have been unveiled using the2-DE approach, this strategy has the limitation of under-repre-senting “extreme” proteins such as hydrophobic membraneproteins and proteins with high/low isoelectric point. In orderto overcome these limitations, we performed in gel digestion ofSDS-PAGE separated proteins, followed by liquid chromatogra-phy-tandem mass spectrometry (GeLC�MS).

Helianthus annuus is one of themost widely cultivated oil cropsin the world and part of the “big four” oil crops, which collectivelymake up to over 87% of global oil production.16 In this species, oilyield per plant is the result of the number of seeds per capitulum,weight per seed and oil concentration. These three componentsare determined by genetic factors, but they can be highlymodified by the environment and growth conditions.17 Largescale production of this oil crop involves sowing in early springand harvesting at the end of summer.18 Sowing sunflower seedsin autumn could overcome early plant desiccation problemsassociated with high temperatures during summer, especially inwarm and dry climates; however, late sowing implies copyingwith low temperatures, including freezing conditions duringwinter season. Plants grown in temperate climates face widefluctuations of diurnal and seasonal temperatures.19 Acclimationto ambient temperature is important not only to protect the plantfrom chilling damage, but also for adaptation to optimal meta-bolic conditions.19 Although sunflower plants tolerate chilling,they may be highly affected by freezing temperatures. In order toselect and characterize sunflower lines tolerant to freezingconditions and possibly use them in early seed sowing, we havepreviously screened 100 lines from the sunflower collection ofthe Instituto de la Grasa (Sevilla, Spain) for freezing tolerance.Sunflower susceptible line Hopi and tolerant lines PI 543006 andBSD-2-691 showed opposite survival rates after cold acclimation,followed by exposure to freezing condition. In the present work,we carried out a global proteomic investigation of proteins thatare responsive to cold acclimation in freezing susceptible andtolerant sunflower lines. We adopted the procedure of SDS-PAGE prefractionation prior to reverse-phase liquid chromatog-raphy-tandem mass spectrometry of in-gel digested peptides, atechnique also referred to as GeLC�MS. As a result, 243differentially expressed, nonredundant proteins were identifiedacross the studied sunflower lines using this approach (Figure 1).The implications of these proteins are discussed in the context ofthe complex events occurring during cold acclimation.

’EXPERIMENTAL PROCEDURES

Plant Material and Cold TreatmentHelianthus annuus freezing susceptible line Hopi and tolerant

sunflower lines PI 543006 (both from USDA, ARS, NationalGenetic Resources Program) and BSD-2-69120 were germinatedin soil under fluorescent light (200 μm � m�2 � s�1, 16 h

photoperiod) at 25/15 �Cday/night temperatures. After 6 weeksof germination, leaves of non- acclimated plants (control) weresampled and the chamber temperature was decreased progres-sively during a week to reach 15/5 �C day/night temperatureswith a 12 h photoperiod at 200 μm � m�2 � s�1. Then, plantswere kept in the chamber for an additional 7 days period beforesampling. All samples were frozen in liquid nitrogen and im-mediately submitted to protein extraction. The experiment wasrepeated to give four independent biological replicates.

Protein Extraction and Gel ElectrophoresisLeaf tissues from each sunflower line in control and cold-

acclimated conditionswere frozen in liquid nitrogen and ground ina mortar to produce a fine powder. Aliquots of 1 g of the powderwere resuspended in 10 mL of 10% (w/v) trichloroacetic acid and0.07% 2-mercaptoethanol in acetone and incubated at�20 �C for45 min. Protein precipitates were collected by centrifugation at10 000� g for 10 min at 4 �C, washed twice with cold acetonecontaining 0.07% 2-mercaptoethanol and vacuum-dried at roomtemperature. Aliquots of 200 mg of leaf acetone powder weretransferred into sterile microtubes containing 1 mL resuspensionbuffer [50 mM Tris (pH 6.8), 2% (w/v) SDS], briefly vortexed,incubated on ice for 30 min and then centrifuged 11 000� g for 5min at 4 �C. Finally, supernatants were transferred to fresh tubesand protein concentration was estimated by the BCA Protein Kit(Thermo Fisher Scientific, Houston, TX) using BSA as standard.Protein extracts were prepared in four biological replicates for eachsunflower line studied. Prior to gel electrophoresis, sample aliquotscontaining 150 μg of proteins were mixed with an equal volume ofloading buffer containing 0.5MTris (pH 6.8), 20% (v/v) glycerol,2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol and traces ofbromophenol blue and incubated for 5 min at 99 �C. Gelelectrophoresis was performed under denaturing conditions in12% polyacrylamide gels for 3 h using 10mA per gel. After proteinmigration, gels were stained with colloidal Coomassie understandard conditions.21

In Gel Trypsin DigestionPrior to protein digestion, the gel lane for each biological

replicate was sliced into 10 equal size segments, diced intoapproximately 1 mm cubes with a clean scalpel and transferredinto 1.5 mL sterile polypropylene tubes for in gel digestion. In geltrypsin digestion was carried out according to Shevchenko et al.22

Protein digestion was performed by the addition of 200 μL ofsequencing grade porcine trypsin (Promega, Madison, WI) at5 ng/μL. After 120 min of cold incubation at 4 �C, samples weredigested overnight at 37 �C. Upon in gel digestion, gel pieceswere saturated with 700 μL of extraction buffer 5% formic acid(FA)/acetonitrile (1:2, v/v) and incubated for 30 min at 37 �C.Supernatants were collected, dried down in a vacuum centrifugeand kept at �80 �C until LC�MS/MS analyses.

LC�MS/MS AnalysesFor each round of LC�MS/MS analysis, extracted peptides

were reconstituted in 0.1% (v/v) FA and separated at the flowrate of 150 nL/min into a 10 cm � 150 μm ID nanocolumn(C18, 100 Å, 5 μm) (MichromBioresources) using the followingmobile phase gradient: from 5 to 35% of solvent B in 25 min, 35to 70% in 25 min, back to 5% in 10 min. Solvent A was watercontaining 0.1% FA, solvent B was acetonitrile containing 0.1%FA. After LC separation, peptides were positively ionized at 2.1 kV,at 250 �C and injected in the mass spectrometer. Mass spectro-metry data were acquired in a ProteomeX LTQ Workstation



(Thermo, San Jose, CA) in data-dependent acquisition (DDA)mode controlled by XCalibur 2.0 software (Thermo FisherScientific). Typical DDA cycle consisted of a survey scan withinm/z 200 to 2000 followed by MS/MS fragmentation of the sevenmost abundant precursor ions under normalized collision energyof 35%. Fragmented precursor ions were dynamically excludedaccording to the following: repeat counts: 3, repeat duration: 30 s,exclusion duration: 30 s.

Database Search and Protein IdentificationThe TIGR transcript assembly database (http://plantta.jcvi.

org) for Helianthus annuus (release 2, June 2006) was used for

querying all MS/MS acquired data. Nucleotide sequences weretranslated and the open reading frames (ORFs) scanned usingthe Virtual Ribosome version 1.1 software.23 For each H. annuusTIGR nucleotide sequence, one amino acid sequence was re-ported, corresponding to the longest ORF reported. To estimatethe peptide false discovery rate (FDR), randomized (decoy)sequences were combined with the forward database, resultingin a concatenated database of 89 324 entries. Peak list wasgenerated by extract_msn.exe program in Bioworks 3.3.1(Thermo) according to the following parameters: MW range:200 to 2000; absolute threshold: 500; precursor ion tolerance:2000 ppm; group scan: 1;minimum group count: 1; minimum ion

Figure 1. Downstream analysis for the identification of differentially expressed proteins in freezing susceptible and tolerant sunflower lines after coldacclimation. First, susceptible Hopi and tolerant PI 543006 and BSD-2-691 lines were exposed to 25/15 �C (control) and 15/5 �C (acclimated) day/night temperatures. Then, proteins from leaves of four biological replicates were extracted and fractionated in 12% SDS-PAGE. After gel segmentation,in gel trypsin digestion was performed in 96 well plates and the extracted peptides were injected in an LC system coupled with a mass spectrometer. Thenumber of assigned MS/MS spectra for each identified protein was determined across the whole gel lane and spectral index (SpI) calculated. For cutoffestimation, 1,000 randomized spectral indexes were obtained for each protein and the SpI threshold determined for 95% confidence level. Proteins withSpI higher than threshold were induced after cold acclimation, or cold enriched, and those SpI lower than threshold were suppressed after cold treatment,or control enriched.



count: 10. Protein identification was performed using SEQUESTalgorithm within the Bioworks 3.3.1 software package (Thermo).Search parameters were set as follows: oxidation of methioninewas allowed as a variable modification and carbamidomethylationof cysteine as a static modification; enzyme: trypsin; number ofallowed missed cleavages: 2; mass range: 200 to 2000; threshold:500; minimum ion count: 10; peptide tolerance: 2000 ppm;fragment ions tolerance: 1000 Da. After searches, SEQUESToutput files were uploaded and analyzed by Scaffold 2.2.1 software(Proteome Software, Portland, OR). Each ten SEQUEST searchresults files resulting from the searches of the ten gel lane segmentsfrom each biological replicate were combined and the number ofassigned peptides and spectra in each biological replicates used forconfident protein identification and quantification. To obtainhigh-confidence and unambiguous protein assignments, Protein-Prophet and PeptideProphet Probabilities24 equal to 99 and 95%,respectively, and all protein identifications required detection of atleast two unique peptides in at least one biological replicate.Peptides that were common to more than one protein sequencewere not excluded from data analyses as they comprised less than0.4% of total spectra in each sample (Supplementary Table 1,Supporting Information). Estimated FDR, for the filter settingsspecified above, showed an average value of 0.7%.

Relative Protein QuantificationSpectral counts were used to estimate the protein amount in

each sample. To assess differences in the relative proteinabundance between control and cold treatments for each sun-flower line, we used ametric named spectral index, which is basedon the spectral counts associated to each identified protein.25

The spectral index for a protein is defined as

SpI ¼ SacclimatedSacclimated þ Scontrol

� NDacclimated

NTacclimated

!

� ScontrolSacclimated þ Scontrol

� NDcontrol

NTcontrol

!ð1Þ

where Scontrol and Sacclimated correspond to the mean spectralcount values for the control and cold-acclimated conditions,respectively, and ND and NT correspond to the number ofbiological replicates in which a protein was detected in the giventreatment and the total number of biological replicates evaluatedfor this treatment, respectively. As the SpI is based on two factors,the relative protein abundance (assessed by the spectral count)and the number of replicates with detectable peptides, values canrange from �1 to þ1 where values close to zero indicate thatrelative protein abundance is about equal in cold and controltreatments. Positive values suggest enrichment of the proteins ofinterest in cold acclimatized plants, whereas negative valuessuggest enrichment in control treatment. To determine statisti-cally significant cutoff values for the SpIs, we performed arandom permutation analysis, in which the proteome profiles(spectral counts) of the control and cold replicates were per-muted 1000 times and the SpI for each protein was recalculatedin each permutation event.25 From the created null distributionof randomized SpIs (Supplementary Figure 1, Supporting In-formation), we selected the upper and lower SpI limits thatencompassed 95% of the randomly generated indices. Thisapproach indicated that absolute SpI cutoff values g0.558,0.55, 0.558 and e0.525, 0.542, 0.552 for Hopi, PI 543006 andBSD-2-691, respectively, were significant at a 95% confidence

level; meaning that a protein with an SpI higher or lower than thecutoff values had less than 5% probability of being part of the nulldistribution and therefore of being mistakenly identified asenriched in one of the two groups.

Functional ClassificationEach TIGR assembly or singleton was aligned to the UniProt-

Uniref database and the annotation for the protein with the bestalignment to each assembly was used as the annotation for thatsequence.26 Functional classifications of differentially expressedproteins were performed according to the catalog established forArabidopsis.27 Two subclasses, protein/polypeptide transportersand protein repair were added to the functional catalogue.A redox homeostasis class was also added to the originalcatalogue. These changes were performed in order to increasethe accuracy on the description of the functions of the proteinsidentified in the present work and to better represent changesthat may occur during the studied abiotic stress.

Protein Expression ProfilingTo access information on similarities of the expression profile

of the differentially expressed proteins across the studied sun-flower lines, we performed hierarchical clustering analysis, usingthe freely available software Permutmatrix.28 For this, spectralcount data from control and cold treatments were normalizedusing the spectral index formula (eq 1). The indices for each proteinwere used to analyze the expression pattern across the sunflowerlines. From the normalized data, the Euclidean metric was per-formed to calculate dissimilarities and clustering (aggregation) wasperformed according to the unweighted paired group averagelinkage (UPGMA) procedure.29

’RESULTS

Detection of Differentially Expressed Proteins Induced byCold Acclimation

To maximize the number of identifications and improvestastical confidence, four biological replicates were used in thisstudy. Proteins in each replicate were prefractionated by 12%SDS-PAGE and segmented into 10 gel slices prior to trypsindigestion to reduce sample complexity prior to LC�MS/MS.This approach resulted in high number of identifications for allsunflower lines in both control and cold conditions (Figure 2). Inthe present work, a total number of 3519 redundant proteinswere confidently detected and analyzed. The Hopi susceptiblesunflower line showed an increase of 22.5% in the number ofdetected proteins after cold treatment, as 533 and 653 proteinswere confidently assigned in control and cold acclimation treat-ments, respectively. Tolerant sunflower lines showed a distinctpattern in the number of proteins detected before and after coldtreatment. Total number of assigned proteins increased only 3.4%inPI 543006 sunflower line in response to cold stress. In contrast, areduction of 14% was detected in BSD-2-691 as detected proteinschanged from 642 to 552 after cold acclimation.

Detection of the differential expression of a protein (the termdifferential expression is used here in the sense of differentialprotein abundance, which can be the result of several processes,including changes in de novo protein biosynthesis, proteinmodification and protein degradation) is challenging for large-scale proteomic experiments due to the large number of peptideand protein identifications. One strategy to analyze differentialexpression between two samples is by comparison of thepresence or absence of a particular protein in different samples



(e.g., de novo synthesis or degradative turnover). However, acomprehensive and sensitive analysis should also take intoaccount quantitative differences in the expression of proteinsidentified. Although spectral counting has been successfully usedin label-free plant proteomic analyses,30 the very nature of thespectral counting makes statistical analysis difficult as the under-lying distribution is not normal due to the overwhelming numberof zero entries, particularly when differential expression is stark.31

For the detection of differentially expressed proteins after coldacclimation in Hopi, PI 543006 and BSD-2-691 sunflower lines,the spectral index25 for each protein was calculated, as describedin eq 1. This strategy is based on relative abundance as measuredby the number of spectral counts across replicates; and reprodu-cibility is determined by the number of replicates for eachtreatment in which the protein was detected. This was particu-larly important to assess the significance of proteins detected inonly one biological replicate, especially those with a low numberof associated spectral counts. In addition, random permutationanalysis of spectral counts across biological replicates, followedby recalculation of spectral indexes, establishes confidence inter-vals for assessing authentic differential protein expression, andnot experimental variation, without assumptions about data

distribution. This approach indicated that from the 718 non-redundant proteins detected in the Hopi sunflower line, theexpression of 34 and 9 proteins were induced in cold-acclimatedand control treatments, respectively, with 95% confidence. Also,with the same confidence level, the expression of 56 and 16proteins were induced in cold and control treatments, respec-tively, in PI 543006 line. BSD-2-691 line showed the highestnumber of differentially expressed proteins, a total of 168, and ahigher number of induced proteins in the control than in cold-acclimation treatment, indicating that the expression of a sub-stantial number of proteins is suppressed in response to coldstress in this tolerant sunflower line.

Functional Classification of Cold-Responsive ProteinsOne of the first steps in interpreting protein expression data is

to group differentially expressed proteins into functional cate-gories. Since no functional classification is available for sunflower,the categorization performed in the present study was based onthe criteria established for the EU Arabidopsis genome project27

(Figure 3). In accordance with the detection of the lowestnumber of cold-responsive proteins across all lines, the numberof proteins in Hopi sunflower line was never higher than those

Figure 2. Schematic illustration of the detection of control and cold-acclimated enriched proteins in Hopi, PI 543006 and BSD-2-691 sunflower lines.Upper panel, total number of identifications in control and acclimated treatments; middle panel, spectral indexes obtained from non-redundant lists ofidentified proteins containing 718, 675 and 769 identifications for Hopi, PI 543006 and BSD-2-691 sunflower lines, respectively; lower panel, number ofcommon and differentially expressed proteins at 95% confidence level.



found in PI 543006 and BSD-2-691 in any category. Also, BSD-2-691 tolerant sunflower line showed the highest number ofassociated proteins in all functional categories. Proteins involvedin metabolism, protein synthesis and energy were the mostresponsive to cold-stress. These three categories comprised 56,53 and 48% of total cold-responsive proteins in Hopi, PI 543006and BSD-2-691 sunflower lines, respectively. The number ofproteins involved in energy metabolism accounted for 23, 17 and13% of total differentially expressed proteins in Hopi, PI 543006and BSD-2-691, respectively, being the most represented class inHopi susceptible sunflower line. Among the three most abundantcategories, metabolism class comprised the highest number ofassociated proteins within the studied lines (50 in total). Metabo-lism category showed the highest number of cold-responsiveproteins in PI 543006 and BSD-2-691 sunflower lines (15 and29, respectively); while this class accounted only for six proteins inthe susceptible line. Protein synthesis class was also highly repre-sented in BSD-2-691 line after cold stress (29 proteins), while thisgroup accounted only for eight and eleven proteins in Hopi and PI543006 sunflower lines, respectively. Although less abundant thanthe other categories, transporters comprised ten responsive pro-teins in BSD-2-691 line, while this class was represented only bytwo proteins in PI 543006 and Hopi lines. Similar profile wasdetected for cell structure related proteins, as eight proteins weredifferentially expressed in BSD-2-691 line; while only one proteinidentification was assigned in PI 543006 tolerant line. Proteinsinvolved in cell structure and transcription were absent inHopi aftercold treatment, being detected only in the tolerant lines. Cold-responsive proteins belonging to secondary metabolism was repre-sented only by geranylgeranyl pyrophosphate synthase-relatedprotein (TA11185_4232), only found in BSD-2-691 sunflower line.

Expression Profile of Cold-Responsive ProteinsTo facilitate the biological interpretation of proteins that

displayed differential expression, proteins were sorted on the basisof the expression pattern across the susceptible and tolerantsunflower lines (Figure 4). Cold-responsive proteins varied widelyin their patterns of expression, resulting in a total of fourteenprotein clusters. In terms of global protein expression, two-wayhierarchical clustering indicated that cold-associated proteomeresponses in the Hopi susceptible line were closer to PI 543006than those shown in BSD-2-691 sunflower line. Clustering of the

differentially expressed proteins yielded five minor clusters,formed by less than five proteins, and nine major clusters withfive or more proteins in each group. Major cluster A accounted forsix proteins, all of them with low expression profile in Hopisusceptible line. Cluster B proteins, formed by nine identifications,showed high expression inHopi and low expression in BSD-2-691sunflower lines. Cluster D was the largest cluster found in thepresent study. This group was formed by 80 proteins in whichexpression was reduced only in the BSD-2-691 tolerant sunflowerline. Functional classification of clustered proteins indicated thatthis group was mainly formed by accessions involved in proteinsynthesis, including initiation factors, ribosomal proteins andtranslation inhibitors (Table 1). Protein cluster E accounted forfive identifications that showed induced expression in PI 543006and reduced expression in BSD-2-691 sunflower lines after coldtreatment. Cluster G was the second largest cluster found in thepresent study, comprising 57 cold-responsive proteins. Thiscluster consisted of proteins only detected in BSD-2-691 sun-flower line and had an opposite expression profile in relation to thelargest cluster D, as all identifications were induced after coldacclimation. However, functional classification indicated thatproteins in this cluster were mostly involved in metabolism andenergy processes, contrary to vast suppression in the expression ofproteins involved in translation as found for cluster D. Cluster Jconsisted of accessions only detected inHopi sunflower line; whilecluster L accounted for 38 PI 543006 induced identifications, mostof them involved in protein synthesis and metabolic functions.The last major cluster detected in the present work was cluster M.This group was constituted by the rab-type small GTP-bindingprotein (TA7528_4232), L-ascorbate peroxidase (TA7734_4232),monodehydroascorbate reductases (DY919001, TA13809_4232),and photosystem II protein reaction center (CD849098) (Table 1),all induced proteins in both PI 543006 and BSD-2-691 tolerantsunflower lines.

Minor cluster C showed low protein expression in PI 543006and BSD-2-691 tolerant sunflower lines. This group was formedonly by the SAM Mg-protoporphyrin IX methyltranseraseenzyme (TA16226_4232) and the RuBisCO large subunit-binding protein (TA7124_4232). Cluster F also constitutedtwo proteins, NADP-G3P dehydrogenase (CX943741) and thecell division protein ftsH homologue (DY911512); however, theexpression profile showed differential response between PI543006 (suppression) and BSD-2-691 (induction) tolerant sun-flower lines, an opposite expression profile in relation to clusterE. Cluster H proteins showed induced expression in Hopi andBSD-2-691 lines and the group was formed by the rubbersynthesis protein (TA6858_4232) and the plasma membranepolypeptide (TA7585_4232). In the same manner as clusterH, cluster K consisted of induced proteins in Hopi, but insteadof induction of expression in BSD-2-691, high expressionwas observed in the PI 543006 tolerant sunflower line. Thiscluster was formed by two vacuolar ATP synthase catalyticsubunit accessions (TA8692_4232, DY924246), G proteinbeta-subunit-like protein (TA8126_4232) and the mitochon-drial phosphate translocator (TA11282_4232). The final minorcluster detected in the present work is cluster N. This groupconsisted of proteins that had overexpression after cold acclima-tion in all sunflower lines. These proteins were the myo-inositol-1-phosphate synthase (TA11211_4232), temperature-inducedlipocalin (TA12632_4232), H. annuus homologous dehydrin(TA7293_4232) and the TIGR assembly TA6741_4232(Table 1).

Figure 3. Functional categorization of differentially expressed proteinsin Hopi, PI 543006 and BSD-2-691 sunflower lines. Bars represent thetotal number of differentially expressed proteins in each sunflower line.The percentage of proteins grouped in a particular functional class inrelation to the total number of cold-responsive proteins in each sun-flower line is indicated at the top of the corresponding bar.



Figure 4. Protein expression profile of differentially expressed proteins across Hopi, PI 543006 and BSD-2-691 sunflower lines. (Left) Hierarchicalclustering of the 231 cold-responsive proteins. (Right) Protein functional classification of the three most abundant clusters.



Table 1. List of Differentially Expressed Proteins, Classified According to the Expression Profile across Hopi, PI 543006 and BSD-2-691 Sunflower Lines (see Supplementary Table 8, Supporting Information, for Complete Quantitative Data)

protein namea accessionb

Cluster A (suppressed expression in susceptible Hopi)

ATP synthase CF1 alpha chain (L. sativa) AJ541270

NClpP4 (Arabidopsis thaliana) TA10610_4232

Glyceraldehyde-3-phosphate dehydrogenase (A. majus) TA7356_4232

Thioredoxin F-type (M. crystallinum) TA7521_4232

Vacuolar ATP synthase subunit B isoform 2 (H. vulgare) TA15023_4232

30S ribosomal protein S31 (A. thaliana) TA10238_4232

Cluster B (induced in susceptible Hopi and suppressed in BSD-2-691)

30S ribosomal protein S13 (A. thaliana) TA6608_4232

Putative pyruvate dehydrogenase E1 (O. sativa) TA10236_4232

Protein disulfide isomerase (I. batatas) TA7253_4232

Hypothetical protein (S. tuberosum) TA7518_4232

Putative chloroplast cysteine synthase 1 (N. tabacum) TA9801_4232

ATP synthase gamma chain, mitochondrial precursor (I. batatas) TA10071_4232

Elongation factor TS (O. sativa) TA12912_4232

TA8800_4232_rframe3_ORF TA8800_4232

Initiation factor eIF4A-15 (H. annuus) TA6874_4232

Cluster C (suppressed in tolerant PI 543006 and BSD-2-691)

SAM Mg-protoporphyrin IX methyltranserase (N. tabacum) TA16226_4232

RuBisCO large subunit-binding protein (A. thaliana) TA7124_4232

Cluster D (suppressed in tolerant BSD-2-691)

33 kd chloroplast ribonucleoprotein precursor (N. sylvestris) BQ916500

60S ribosomal protein L4�2 (A. thaliana) BQ976230

TGB12K interacting protein 3 (N. tabacum) TA10299_4232

Geranylgeranyl pyrophosphate synthase-related (A. thaliana) TA11185_4232

Nucleoid DNA-binding-like protein (A. thaliana) TA11931_4232

Calcium homeostasis regulator CHoR1 (S. tuberosum) TA11974_4232

PS60 protein precursor (N. tabacum) TA12085_4232

Polygalacturonase inhibitor-like protein (A. thaliana) TA12180_4232

3-isopropylmalate dehydrogenase (B. napus) TA13587_4232

GDSL-motif lipase/hydrolase-like protein (A. thaliana) TA14599_4232

Metacaspase 1 (L. esculentum) TA15080_4232

Pyridoxine biosynthesis protein isoform B (N. tabacum) TA15214_4232

Initiation factor eIF4A-15 (H. annuus) TA6872_4232

Polygalacturonase inhibitor protein precursor (L. esculentum) TA7256_4232

60S ribosomal protein L10A (O. sativa) TA7653_4232

PREDICTED: similar to tubulin, beta 2 (R. norvegicus) TA7851_4232

PREDICTED: similar to tubulin, beta 2 (R. norvegicus) TA7853_4232

PREDICTED: similar to Rps15a protein (G. gallus) TA8086_4232

Ketol-acid reductoisomerase (A. thaliana) TA8730_4232

Chloroplast 30S ribosomal protein S4 (L. sativa) TA11460_4232

Probable ATP synthase 24 kDa subunit (A. thaliana) TA8456_4232

Hypothetical protein (R. communis) TA10628_4232


Rubisco subunit binding-protein alpha subunit (O. sativa) DY907657

Perchloric acid soluble translation inhibitor protein (G. triflora) DY930835

Hypothetical protein F5K20_290 (A. thaliana) TA7694_4232

Rubisco subunit binding-protein alpha subunit (T. pretense) TA7123_4232

RuBisCO large subunit-binding protein (P. sativum) TA11875_4232

Putative nuclear RNA binding protein A (O. sativa) TA6549_4232

60S ribosomal protein L4�2 (A. thaliana) TA9282_4232



Table 1. Continuedprotein namea accessionb

Adenylate kinase B (O. sativa) BQ972050

CD846470_rframe1_ORF CD846470

Hypothetical protein (A. thaliana) CD849116

Putative ribosomal protein L29 (O. sativa) CD849622

CD853480_rframe3_ORF CD853480

Seed maturation-like protein (O. sativa) CD854761

Dicarboxylate/tricarboxylate carrier (C. junos) CD858386

Putative nuclear antigen homologue (T. pretense) DY920339

At1g31020/F17F8_6 (A. thaliana) DY929446

3-oxoacyl-[acyl-carrier-protein] reductase (C. lanceolata) TA10324_4232

AT5g14910/F2G14_30 (A. thaliana) TA10497_4232

TA13325_4232_rframe-2_ORF TA13325_4232

TGB12K interacting protein 2 (N. tabacum) TA15283_4232

Similar to nClpP2 (A. thaliana) TA16335_4232

40S ribosomal protein S17 (C. annuum) TA6619_4232

Putative steroid binding protein 2 (A. thaliana) TA7106_4232

ATP synthase delta’ chain (I. batatas) TA7421_4232

Putative ribosomal protein S12 (O. sativa) TA7467_4232

Chromosome 9 SCAF14729 (T. nigroviridis) TA7510_4232

40S ribosomal protein S3a (H. annuus) TA7643_4232


40S ribosomal protein S7 (A. marina) TA7887_4232

Chromosome 9 SCAF14729 (T. nigroviridis) TA8102_4232

Expressed protein (A. thaliana) TA8231_4232

Ribonucleoside-diphosphate reductase (N. tabacum) TA8276_4232

60S ribosomal protein L34 (P. sativum) TA8374_4232

Hypothetical protein (A. thaliana) TA8737_4232

GSDL-motif lipase (A. americana) TA9496_4232

RAD23 protein (L. esculentum) TA9639_4232

T1N6.24 protein (A. thaliana) TA9700_4232

Similarity to unknown protein (A. thaliana) TA7191_4232

AT4g23890/T32A16_60 (A. thaliana) TA14214_4232

Putative acid phosphatase (H. vulgare) TA8290_4232

Phosphoglucomutase (S. tuberosum) DY910004

Emb|CAB62460.1 (A. thaliana) TA16225_4232

Proteasome subunit alpha type 3 (O. sativa) TA16410_4232

Ketol-acid reductoisomerase (P. sativum) TA8731_4232

GF14 protein (F. agrestis) TA12363_4232

Nascent polypeptide-associated complex NAC (M. truncatula) TA7365_4232

CX943630_rframe2_ORF CX943630

NME2 protein (H. sapiens) TA7804_4232

Cysteine synthase (V. pseudoreticulata) DY925536

31 kDa ribonucleoprotein (N. plumbaginifolia) DY917463

Oxygen-evolving enhancer protein 3 (P. sativum) TA8258_4232

40S ribosomal protein S11�2 (A. thaliana) TA7499_4232

GloEL protein; chaperonin, 60 kDa (A. thaliana) TA7019_4232

60S ribosomal protein L32�1 (A. thaliana) TA7095_4232


40S ribosomal protein S2 homologue (A. thaliana) TA6726_4232

Cluster E (induced expression in tolerant PI 543006 and suppressed in tolerant BSD-2-691)

Phosphoglycerate kinase, cytosolic (N. tabacum) TA10138_4232


S-adenosyl-L-methionine synthetase (N. tabacum) TA7576_4232

Serine hydroxymethyltransferase (A. thaliana) TA7117_4232




Nascent polypeptide associated complex (O. sativa) TA8011_4232

Cluster F (suppressed expression in tolerant PI 543006 and induced in tolerant BSD-2-691)

NADP-G3P dehydrogenase (Chlamydomonas sp.) CX943741

Cell division protein ftsH homologue (N. tabacum) DY911512

Cluster G (induced in tolerant BSD-2-691)

NADP-isocitrate dehydrogenase (C. lanceolata) CD854278

Photosystem I reaction center subunit IV (S. oleracea) TA11265_4232

Aquaporin (V. vinifera) TA6603_4232

Oxygen-evolving enhancer protein 1 (S. tuberosum) TA7382_4232

Glyceraldehyde 3-phosphate dehydrogenase (D. carota) TA7287_4232

Serine-glyoxylate aminotransferase (S. polyrrhiza) TA6735_4232

Photosystem II P680 chlorophyll A apoprotein (L. sativa) CX944092

Hypothetical protein (C. paradise) TA8018_4232

GTP-binding nuclear protein Ran/TC4 (V. faba) TA6837_4232

40S ribosomal protein S8 (O. sativa) TA7385_4232

Transketolase (S. oleracea) BQ969769

Cysteine protease-1 (H. annuus) TA8640_4232

Aminomethyltransferase (F. anomala) TA7645_4232

Chloroplast 30S ribosomal protein S3 (P. ginseng) AJ539696

60S ribosomal protein L6 (M. crystallinum) TA8564_4232

Embryonic protein DC-8 (D. carota) BU025744

Photosystem I P700 chlorophyll A apoprotein A1 (O. sativa) CX944063

Photosystem Q(B) protein (C. reflexa) TA8145_4232

Putative serine-glyoxylate aminotransferase (F. agrestis) TA6615_4232

34 kDa outer mitochondrial membrane porin (S. tuberosum) TA7955_4232

Dihydrolipoamide dehydrogenase precursor (S. tuberosum) TA10968_4232

GDP-mannose 3,5-epimerase 1 (O. sativa) TA16325_4232

Thylakoid lumenal protein-like (O. sativa) CD846365

Glucose-1-phosphate adenylyltransferase (A. thaliana) DY904677

Putative pectin methylesterase precursor (P. tremula) TA8320_4232

Peroxiredoxin (I. batatas) TA9259_4232

Endoplasmin homologue precursor (C. roseus) CD850869

Photosystem II stability/assembly factor HCF136 (O. sativa) DY916514

Thylakoid lumenal 15 kDa protein (A. thaliana) DY921192

Peptide methionine sulfoxide reductase (L. sativa) TA10603_4232

Phosphoenolpyruvate carboxylase (F. trinervia) TA14451_4232


Profilin-2 (A. artemisiifolia) TA8485_4232

Pom30 protein (S. tuberosum) TA8920_4232

Basic peroxidase precursor 1 (Z. elegans) TA9224_4232

Adenine phosphoribosyltransferase-like (S. tuberosum) TA9262_4232

Aminomethyltransferase (F. pringlei) TA11778_4232

Beta-1,3-glucanase (C. intybus) TA7722_4232

SOUL heme-binding protein-like (O. sativa) TA7859_4232

NADP-isocitrate dehydrogenase (C. lanceolata) TA9623_4232

Pom30 protein (S. tuberosum) TA12423_4232

Photosystem I reaction center subunit N (Z. mays) TA7748_4232

PSII 32 kDa protein (Photosystem q(B) protein) (O. sativa) TA11848_4232

DY930813_rframe2_ORF DY930813

Photosystem II stability/assembly factor HCF136 (O. sativa) TA7827_4232

Proteasome subunit beta type 2-A (A. thaliana) AJ828950

Protease Do-like 1 (A. thaliana) DY914432

Cytosolic fructose-1,6-bisphosphate (L. sativa) DY921054

HADIP_rframe1_ORF HADIP




ATP synthase A chain (P. ginseng) TA15435_4232

TA15511_4232_rframe3_ORF TA15511_4232

Probable aquaporin PIP1.4 (A. thaliana) TA6930_4232

Aspartic protease-like (S. tuberosum) TA7545_4232

36 kDa outer mitochondrial membrane porin (S. tuberosum) TA7612_4232

Pore protein of 24 kD (P. sativum) TA8696_4232

Ethylene-responsive GTP-binding protein (L. esculentum) TA9242_4232

Hypothetical protein At5g13410 (A. thaliana) TA9403_4232

Cluster H (induced expression in susceptible Hopi and induced in tolerant BSD-2-691)

Rubber synthesis protein (P. argentatum) TA6858_4232

Plasma membrane polypeptide (N. tabacum) TA7585_4232

Cluster I (suppressed in tolerant PI 543006)

Pectinesterase (A. thaliana) DY903749

50S ribosomal protein L12 (N. sylvestris) TA11022_4232

Catalase 4 (H. annuus) TA6831_4232

Fructose-1,6-bisphosphatase (P. sativum) TA9781_4232

ATP synthase gamma chain (P. sativum) TA8602_4232

Proteasome subunit beta type 6 (A. thaliana) TA9822_4232

Cell division protein ftsH homologue 1 (A. thaliana) TA12156_4232

Histone H2A (H. orientalis) TA7311_4232

31 kDa ribonucleoprotein (A. thaliana) CD847964

Light harvesting chlorophyll a/b binding PSII (E. gracilis) TA8295_4232


Cytochrome c (H. annuus) TA8567_4232

Cluster J (induced in Hopi)

Quinone oxidoreductase-like protein (H. annuus) TA7396_4232

Hypothetical protein OSJNBa0073E05.18 (O. sativa) DY923049

Hypothetical protein C7A10.450 (A. thaliana) TA10175_4232

Cyclophilin 1 (C. lanceolata) TA8509_4232

Thiosulfate sulfurtransferase (D. glomerata) TA12269_4232

Phosphoglucomutase (O. sativa) BQ913504

Remorin-like protein (A. thaliana) TA7328_4232

ATP synthase delta chain (I. batatas) TA9348_4232

OSJNBa0020P07.3 protein (O. sativa) DY911323

DnaK protein (O. sativa) TA6709_4232

Ribosome recycling factor (D. carota) CD857897

Heat shock protein 81�2 (A. thaliana) TA9004_4232

Remorin-like protein (A. thaliana) TA16308_4232

Ferredoxin (H. annuus) TA7560_4232

Ubiquitin-conjugating enzyme 1 (A. hypogaea) TA8200_4232

Cluster K (induced expression in susceptible Hopi and induced in tolerant PI 543006)

Vacuolar ATP synthase catalytic subunit A (D. carota) TA8692_4232

G protein beta-subunit-like protein (N. plumbaginifolia) TA8126_4232

Vacuolar ATP synthase catalytic subunit A (D. carota) DY924246

Mitochondrial phosphate translocator (M. truncatula) TA11282_4232

Cluster L (induced in tolerant PI 543006)

Ascorbate peroxidase (P. tomentosa) TA8256_4232

TA6732_4232_rframe3_ORF TA6732_4232

60S ribosomal protein L19-like protein (S. tuberosum) TA6538_4232

60S ribosomal protein L30 (E. esula) TA7641_4232

Cyclophilin (K. candel) TA8568_4232

UTP--glucose-1-P uridylyltransferase (M. acuminata) DY916177



’DISCUSSION

Spectral Counting Approach Resulted in the Identificationof a Large Number of Cold-Responsive Proteins

In the present work, we aimed to study changes in proteinexpression induced by cold acclimation in freezing susceptibleand tolerant sunflower lines. Previous studies reported changes

in the proteome profile in cold susceptible species, such asrice14,15 and Arabidopsis,9,10 or freezing tolerant species, suchas the woody species P. obovata4 and P. persica.8 Here,GeLC�MS was performed to investigate cold responses inte-grating it with a spectral counting-based, label-free proteomicapproach. One of the advantages the GeLC�MS approach hasover 2-DE is the better representation of “extreme” proteins,


Hypothetical protein T16L4.100 (A. thaliana) TA10152_4232

TA13261_4232_rframe-2_ORF TA13261_4232

Photosystem I reaction center subunit X (N. tabacum) TA8031_4232

Thioredoxin peroxidase (N. tabacum) TA6927_4232

Cucumisin-like serine protease (A. thaliana) TA9328_4232

Putative ribose-5-phosphate isomerase (O. sativa) TA10608_4232

Adenosylhomocysteinase 1 (A. thaliana) TA6744_4232

Auxin-binding protein ABP20 precursor (P. persica) TA12426_4232

Photosystem II 10 kDa polypeptide (L. esculentum) TA7000_4232

Putative UDP-glucose dehydrogenase (S. bicolor) TA7983_4232

Ribosomal protein L37 (G. max) TA8029_4232

60s ribosomal protein L21 (T. aestivum) TA7462_4232

Heat shock protein 81�3 (O. sativa) TA6699_4232

Elongation factor Tu, chloroplast precursor (G. max) TA8623_4232

Aspartate aminotransferase-like (S. tuberosum) CD854632

Chloroplast 50S ribosomal protein L2 (N. debneyi) CD855490

Putative 10kd chaperonin (CPN10) (A. thaliana) TA11077_4232

TA7075_4232_rframe1_ORF TA7075_4232

Heat shock protein 70 (C. sativus) TA7579_4232

Glutathione S-transferase GST1 (C. chinense) TA8350_4232

AT5g14030/MUA22_2 (A. thaliana) TA9959_4232

Putative ribosomal protein (S. demissum) TA7024_4232

Similarity to RNA binding protein (A. thaliana) BU026783


ADP ribosylation factor 002 (D. carota) TA7206_4232

Aldehyde dehydrogenase 1 precursor (L. corniculatus) TA8118_4232

DNA-binding protein-related-like (O. sativa) TA11122_4232

Beta-hydroxyacyl-ACP dehydratase (P. mariana) TA13535_4232

Putative glycine-rich RNA binding protein 3 (C. roseus) TA6606_4232

Glutamate--ammonia ligase (B. napus) TA7345_4232

Cluster L (induced in tolerant PI 543006)

Phosphoethanolamine N-methyltransferase (A. tripolium) TA7702_4232

60S ribosomal protein L7A-like protein (S. tuberosum) TA8464_4232

Cluster M (induced in tolerant PI 543006 and BSD-2-691)

Rab-type small GTP-binding protein (C. arietinum) TA7528_4232

L-ascorbate peroxidase (P. sativum) TA7734_4232

Monodehydroascorbate reductase (L. esculentum) DY919001

Photosystem II protein reaction center W (O. sativa) CD849098

Monodehydroascorbate reductase (M. crystallinum) TA13809_4232

Cluster N (induced in all sunflower lines)

Myo-inositol-1-phosphate synthase (G. max) TA11211_4232

Temperature-induced lipocalin (P. armeniaca) TA12632_4232

H.annuus homologous dehydrin (H. annuus) TA7293_4232

TA6741_4232_rframe1_ORF TA6741_4232aUniProt-UniRef protein annotation with the best alignment to the TIGR transcript assembly. bTIGR transcript assembly identifier or GenBankaccession number for the identified protein.



including hydrophobic membrane proteins as well as extreme(high or low) molecular weight and isoelectric point proteins.32

Label-free quantitative strategies are attractive alternatives forquantitative LC�MS/MS-based proteomics because of theirsimplicity, affordability and flexibility.32 Although in theory ionsuppression could affect quantification, previous studies havedemonstrated that spectral counting and also the spectral ionintensity and peak area correlates well with protein abundance incomplex samples.30,33�38

Investigation of plant proteome changes related to cold stressare exclusively based on fractionation of complex protein samplesusing 2-DE followed by image analysis and MS protein identifica-tion. In these studies, 2-DE gel maps presented a resolving powerranging from 1000 to 1700 protein spots per gel; however, thedetection of cold-responsive protein spots was limited to amaximum of 252, from which mass spectrometry analysis resultedin the identification of 43 proteins.4 Short and long exposure to lowtemperatures also resulted in the detection of 60 spots presentingsignificant differences in the intensity and identification of 50differentially expressed proteins in T. halophila leaves.12 In thepresent work, 3519 redundant proteins were confidently identifiedacross the three sunflower lines in both control and cold treat-ments. From the 3519 proteins, 718 were unique and 243 weredifferentially expressed at 95% confidence interval. The strategyused here, that is, one-dimensional gel electrophoresis prefractio-nation, associated with a fast mass spectrometer scanning, androbust software and statistical framework to detect the differentialexpression proteins, resulted in the identification of the largestnumber of proteins involved in plant cold acclimation.

Tolerant Lines Are More Responsive to Low, Non-FreezingTemperatures

Cold acclimation is an adaptative response where plantsacquire an increase in freezing tolerance upon a prior lownonfreezing temperature treatment.39 Cold acclimation mayinvolve a wide array of metabolic changes governed by extensivereprogramming at both gene expression, post-transcriptional,and translational levels.40 In Arabidopsis, a comparative analysisof the transcriptional changes induced by cold acclimation acrossnatural ecotypes presenting different tolerances to freezingtemperatures indicated an increase of 10% to 25% of the meanexpression change for the accessions that acclimate well incomparison to those that acclimate poorly.41 Although increasedfreezing tolerance during cold acclimation is associated withmany metabolic changes,42,43 no clear relationship betweenglobal metabolic changes and differences between ecotypes withdifferent freezing tolerances was found in Arabidopsis.41 Althoughproteome data do not provide direct evidence of the regulation ofgene expression, in the present work the proteome responses ofH. annuus presented a similar profile in relation to the transcrip-tional changes observed in different Arabidopsis ecotypes, assunflower freezing tolerants PI 543006 and BSD-2-691 showedhigher number of regulated proteins in relation to susceptible lineHopi. The metabolic shift that occurs in plants exposed to coldtemperature varies according to its tolerance to this stress. Whilemost differentially expressed proteins in sunflower tolerant linesBSD-2-691 and PI 543006 are related to primary metabolism, thatis, amino acids, nitrogen, nucleotides, phosphate, sugars, lipids andcofactor metabolism, most of the proteome responses in thesusceptible line Hopi were related to energy metabolism. Pro-teome analysis of O. sativa exposed to progressive cold resulted inthe induction of mainly metabolic related proteins, comprising 39%

of all identified proteins.15 Photosynthesis, energy and primarymetabolism related proteins were mainly affected after cold treat-ment in T. halophila and P. patens.12,13 Comprehensive transcrip-tional analysis of Arabidopsis ecotypes presenting different freezingtolerance spectra indicated that the suppression of photosynthesisand induction of photoprotective flavonoids are associated withimproved acclimated freezing tolerance; however, metabolic profil-ing indicated a central role of primary metabolism, especiallycarbohydrate metabolism, in the mechanism of tolerance,41 whichis in accordance with the high number of proteins involved inprimary metabolism that were differentially expressed after coldacclimation in H. annuus tolerant lines.

Successful Cold Acclimation Requires Differential Expres-sion of Several Proteins

Cold acclimation involves the remodeling of cell and tissuestructures and the reprogramming of metabolism and geneexpression.40 Sensitivity to freezing mutants have been identified inArabidopsis that have lost their ability to fully cold acclimate due tosingle-genemutations, indicating that, despite the evident complexity,several genes are essential for freezing tolerance and that theinactivation of any of these genes may severely affect acclimatedfreezing tolerance.41 In the present work, four proteins showedinduced expression after cold treatment in all three sunflower lines.Myo-inositol-phosphate synthase (MIPS) catalyzes the conversion ofD-glucose 6-phosphate to myo-inositol-1-phosphate, the first com-mitted and rate-limiting step in inositol and inositol-containingcompound biosynthesis.44 Besides its essential role in plant embryodevelopment,45 high expression of MIPS confers tolerance tosalt46�48 and cold44,49 stress. Cold tolerance induced by MIPS maybe related to accumulation of myo-inositol, a carbohydrate. Glucose,fructose and sucrose were identified in high concentrations in cold-acclimated Arabidopsis ecotypes presenting freezing tolerance.41 Thefunction in plant stress tolerance of soluble sugars has been high-lighted by many investigators; however, this accumulation alone isneither necessary nor sufficient for cold acclimation in Arabidopsisplants.50 In the tropical species Passiflora edulis, expression ofMIPS iscorrelated with this species ecological adaptation to short chillingwinters without injury, but does not confer severe or prolongedfreezing tolerance.44

Temperature-induced lipocalin identification was also clus-tered together with MIPS. Lipocalins are an ancient and func-tionally diverse family of proteins found in bacteria, plants,arthropods and chordates.51 Recently, the identification of a trueplant lipocalin was reported in wheat and Arabidopsis.52 Expres-sion analysis of wheat lipocalin genes suggest a differentialaccumulation in various wheat cultivars showing different levelsof freezing tolerance, indicating that their expression is asso-ciated, or may be involved, with the plant’s capacity to developfreezing tolerance.51 In P. obovata proteome analysis, lipocalinwas found to be progressively induced throughout the winterseason.4 In winter rye (Secale cereale), photosynthetic acclima-tion to low temperature mimics the photosynthetic acclimationto high light because both conditions result in a comparablereduction state of PSII.53 Chloroplastic lipocalins and lipocalin-like proteins properties, their tissue specificity, and their tran-script accumulation in response to temperature stress suggest apossible role protecting the photosynthetic apparatus againstdeleterious effect of temperature stress.51

Another protein that showed induced expression in all threesunflower lines was an H. annuus homologous dehydrin. Induc-tion of dehydrin by cold stress has been reported for rice,54



wheat,55 in fruits of Citrus species56 and Arabidopsis.9,57,58 Dehy-drin proteins were also induced in P. obovata needles across winterseason and in T. halophila after short and long exposure to lowtemperatures.4,12 Studies on boreal species indicate strong relationbetween the acclimation to lower temperatures during autumnandaccumulation of dehydrins.59�61 Although the biochemical modeof action is still unclear, most of the studies so far suggest thatdehydrins stabilize membranes and macromolecules, thus pre-venting structural damage and maintaining essential enzymeactivity during cellular frost dehydration.62 These proteins alsointeract with sugars to promote low temperature cytoplasmicvitrification which is proposed as a general mechanism fortolerance of extreme frost dehydration.4 In addition, the ESTsequences of the assembly identified as TA6741_4232match, with47% identity, to the UNIGENE transcript locus NP_173468.1, acold-responsive 47 protein that has 100% sequence coverage withthe CAA62449.1 dehydrin accession.

Sunflower Tolerant Lines PI 543006 and BSD-2-691 ShowedDifferent Proteome Responses to Cold Acclimation

Although showing tolerance to freezing, the sunflower lines PI543006 and BSD-2-691 showed different proteome responsesafter cold treatment. Proteins exclusively induced in one or theother sunflower tolerant line comprised the vast majority of allidentified proteins, 177 proteins, all of them grouped into themajor clusters D, L, G and I. Interestingly, exclusively over-expressed proteins presented a different functional classificationpattern between tolerant sunflower lines PI 543006 and BSD-2-691. Clusters G and L, comprising exclusive proteins induced inBSD-2-691 and PI 543006 lines, respectively, were mainlyconstituted by proteins involved in primary metabolism. How-ever, recruitment of proteins involved in energy metabolism,especially glycolytic, mitochondrial electron transport andphotosynthesis related proteins, and transport was higher inBSD-2-691 than in PI 543006 line after cold acclimation. On theother hand, exclusive induced proteins in PI 543006 after coldacclimation suggested a major role played by proteins involved inprotein synthesis. Besides exclusively expressed proteins, acces-sions showing opposite expression profile also indicate differen-tial response to cold acclimation. Seven proteins showedopposite regulatory profile between PI 543006 and BSD-2-691lines (clusters E and F). Cluster E comprised five proteins,phosphoglycerate kinase, a hypothetical protein, S-adenosyl-L-methionine synthetase, serine hydroxymethyltransferase and anascent polypeptide-associated protein. Phosphoglycerate kinaseis a key glycolytic enzyme, a pathway that is strong influenced bycold stress,63 that catalyzes the reversible conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate.64 This enzyme wasalready found to be induced in Arabidopsis10 after cold treatmentand in T. halophila soon after low temperature exposure.12

Sequence alignment of the identified hypothetical proteinTA10793_4232 indicated high identity (83%) to a prohibitin1-like protein from Brassica napus (accession Q9AXM0).Although no clear role during cold acclimation was detectedfor this protein, it has been reported that plant prohibitins may beinvolved in the defense response and senescence and thatprohibitins are essential for mitochondrial homeostasis in plantcells.65 The enzyme S-adenosyl-L-methionine synthetase, whichcatalysis the formation of S-adenosylmethionine from methio-nine and ATP, was already reported to be induced after coldacclimation in rice15 andArabidopsis leaves.9 In the samemanner,gene expression of the enzyme serine hydroxymethyltransferase

was induced after cold treatment in leaves of Arabidopsis.66

Interestingly, this protein was also shown to be induced inprotection from oxidative degradation by drought stress in riceleaves.67 Nascent polypeptide associated complex (NAC) pro-motes proper folding of proteins and/or prevent aggregation ofnascent or damaged proteins.68 It is also suggested that NAC isinvolved in protein sorting and translocation by preventingmistargeting of nascent polypeptide chains to endoplasmicreticulum.69 Suppression of the expression of this protein is alreadyreported in Arabidopsis roots after exposure to high NaCl con-centrations. In addition, suppression of NAC was also found in acomparative proteome analysis of rice seedlings exposed to lowtemperatures.69 The differential proteome responses, induction intolerant PI 543006 and suppression of BSD-2-691, of thesedifferent proteome components, in which most of them werealready reported to be involved in general plant stress responses,suggest that there is a complicated and unique mechanismcontrolling adaptation in each tolerant sunflower line.

Differential expression of proteins between tolerant lines PI543006 and BSD-2-691 was also detected in the accessionsgrouped in cluster F. Glyceraldehyde 3-phosphate dehydrogen-ase is a well-known protein to be involved in cold acclimation.Induction of this protein was already described in leaves of T.halophila,12 Arabidopsis10 and P. patens;13 however, in the woodyspecies P. obovata, this enzyme was found to be suppressedthroughout winter season.4 FtsH are membrane bound ATPdependent metalloproteases universally found in livingorganisms.70,71 The function executed by this ancient class ofchaperone-like proteins includes assembly, operation and disas-sembly of protein complexes.70 Cell division protein ftsH wasfound to be differentially expressed during cold acclimation inrice seedlings,15 P. patens13 and P. obovata.4 Although theexpression of these two proteins was reduced in the tolerant linePUB-50, while induced in BSD-2-691, the accessions were alsogrouped in cluster A (suppression in Hopi), G (induction inBSD-2-691) and I (suppression in PUB50). The identification ofisoforms of these proteins in other clusters corroborates thecomplex and line-unique regulatory mechanisms that may in-clude post translational modification of already cold responsiveproteins.72,73

’CONCLUDING REMARKS

Previous studies aimed at identifying cold-responsive proteinswere performed using exclusively 2-DE-based strategies, andfrequently working only with susceptible plant varieties. In thepresent study we performed comparative proteomics of coldstress using a less biased approach for protein separation, SDS-PAGE, coupled to spectral counting. This approach was used toidentify proteins that significantly change after cold treatment inboth susceptible and resistant sunflower lines. The strategyresulted in the detection of the largest number of proteinsinvolved in cold acclimation. Proteome responses reported hereresemble changes previously described in other stress relatedtreatments, with exceptions. Comparative analysis of tolerant andsusceptible lines showed significant proteome differences. How-ever, due to the sometimes divergent responses observed be-tween the lines studied it is not possible to generalize and specifythe cold acclimation responses in the H. annuus species. Furtheranalysis of cold acclimation in near-isogenic sunflower lines mayalso help to understand the complex physiological changes that



occur in this event and that probably require the targeting ofseveral genes for successful genetic engineering in this species.

’ASSOCIATED CONTENT

bS Supporting InformationSupplemental figure and tables. This material is available free

of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Tiago S. Balbuena, University of Missouri-Columbia ChristopherS. Bond Life Sciences Center, 109 Bond Life Sciences Center,1201 Rollins Ave., Columbia, MO 65211. E-mail: [email protected]. Tel.: 573-884-5979. Fax.: 573-884-9676.

’ACKNOWLEDGMENT

Our thanks are due to A. Gonz�alez-Callejas and B. L�opez-Cordero for their skillful technical assistance. This work wassupported by Junta de Andalucía, Instituto Andaluz de Biotec-nología, project BIO�ANDALUS 08/9/L3.1.

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Proteome Analysis of Cold Acclimation in Sunflower