Proteome Analysis of Cold Acclimation in Sunflower - Journal of

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Proteome Analysis of Cold Acclimation in Sunflower Tiago S. Balbuena,*,† Joaquín J. Salas,‡ Enrique Martínez-Force,‡ Rafael Garces,‡ 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 ABSTRACT: Cold acclimation is the phenomenon in which plants are exposed to low, but nonfreezing, temperatures before exposure to drastic temperatures. To investigate how sunflower plants adjust their metabolism during cold treatment, a comparative proteomic approach, based on spectral counting data, was adopted to identify differentially expressed proteins in leaves of freezing susceptible (Hopi) and tolerant (PI 543006 and BSD-2-691) lines after cold acclimation. In total 718, 675, and 769 proteins were confidently identified by tandem mass spectrometry in Hopi, PI 543006, and BSD-2-691 sunflower lines. Tolerant lines PI 543006 and BSD-2-691 showed the highest number of differentially expressed proteins, as 43, 72, and 168 proteins changed their expression in Hopi, PI 543006, and BSD-2-691 sunflower lines, respectively, at 95% confidence. Cold-responsive proteins were mostly involved in metabolism, protein synthesis, energy, and defense processes in all sunflower lines studied. Hierarchical clustering of all differentially expressed proteins resulted in the characterization of 14 different patterns of expression across Hopi, PI 543006, and BSD-2-691 and indicated that tolerant lines showed different proteome responses to cold acclimation. KEYWORDS: cold acclimation, GeLCMS, Helianthus annuus, mass spectrometry, spectral counting

’ INTRODUCTION Plants are sessile organisms that frequently face different environmental variations and have to adapt their metabolism to overcome adverse conditions. Low temperature has a great impact on plant productivity, mostly because it significantly alters plant metabolism and physiology.1 The development of tolerance mechanisms in response to this environmental stress is essential to ensure correct growth and development.2 A particular characteristic developed by plants long exposed to temperature stress is the phenomenon of acquired tolerance.2 Cold acclimation is the term assigned for the metabolic changes that occur in the organism if exposed to low, but nonfreezing, temperatures conferring tolerance to subsequent subzero temperatures.3 Cold acclimation has been shown to involve reprogramming of metabolism, changes in gene expression, changes in membrane fluidity, alterations in carbohydrate metabolism, increase in ability to withstand oxidative stress and synthesis of cold acclimation induced proteins.4 Although the analysis of gene transcripts may provide comprehensive information about the physiological state of an organism in a particular condition, analysis of the protein complement is also critical, as studies have demonstrated that the levels of transcripts are not strictly correlated to the levels of the translated proteins.57 In addition, many crucial post-translational modifications may occur that can not be screened by transcript analysis. Proteome analyses of cold responses have been carried out in different plant organisms. For woody species, r 2011 American Chemical Society

Prunus persica and Picea obovata cold responses were studied through two-dimensional gel electrophoresis (2-DE) and detection of differential expression by DIGE technology.4,8 Differentially expressed proteins during cold acclimation in these woody species were mostly involved in carbohydrate metabolism, defensive and protective mechanisms, energy production and cytoskeleton organization. As for model plant species, the same proteomic strategy was performed for Arabidopsis thaliana plants under two different cold acclimation conditions, resulting in the identification of 22 proteins involved in cold response.9 In depth comparative analysis of cold response in the nuclear proteome of Arabidopsis revealed differential expression of 54 proteins out of the 184 proteins identified in the nuclear map.10 Thellungiella halophila is a newly emerging model species for the molecular elucidation of abiotic stress tolerance.11 Two dimensional electrophoresis comparison in leaves of this species during short and long-term exposure to cold stress, revealed significant change in 66 protein spots out of 1500 detected protein spots.12 Subsequent mass spectrometry driven identification revealed that identified proteins mainly participate in photosynthesis, RNA metabolism, defense response, energy pathway, protein synthesis, folding, cell wall, cytoskeleton and signal transduction. In Physcomitrella patens, a highly tolerant species to a variety of abiotic stresses, 2-DE-based proteome analysis also revealed Received: November 12, 2010 Published: February 23, 2011 2330

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

Journal of Proteome Research changes in the expression of proteins involved in signaling, cytoskeleton and defense after cold acclimation.13 Late development consequences after cold treatment at the young microspore stage were detected in rice anthers, where 70 protein spots were up- or down-expressed.14 The seedling leaves of Oryza sativa were also screened for proteome responses, using the 2-DE approach, after progressive low temperature treatment.15 From the 60 differentially changed protein spots, 41 were successfully identified including proteins involved in protein synthesis, folding and degradation, antioxidative/detoxifying enzymes, energy pathway and signal transduction. Although some of the mechanisms involved in cold acclimation have been unveiled using the 2-DE approach, this strategy has the limitation of under-representing “extreme” proteins such as hydrophobic membrane proteins and proteins with high/low isoelectric point. In order to overcome these limitations, we performed in gel digestion of SDS-PAGE separated proteins, followed by liquid chromatography-tandem mass spectrometry (GeLCMS). Helianthus annuus is one of the most widely cultivated oil crops in the world and part of the “big four” oil crops, which collectively make up to over 87% of global oil production.16 In this species, oil yield per plant is the result of the number of seeds per capitulum, weight per seed and oil concentration. These three components are determined by genetic factors, but they can be highly modified by the environment and growth conditions.17 Large scale production of this oil crop involves sowing in early spring and harvesting at the end of summer.18 Sowing sunflower seeds in autumn could overcome early plant desiccation problems associated with high temperatures during summer, especially in warm and dry climates; however, late sowing implies copying with low temperatures, including freezing conditions during winter season. Plants grown in temperate climates face wide fluctuations of diurnal and seasonal temperatures.19 Acclimation to ambient temperature is important not only to protect the plant from chilling damage, but also for adaptation to optimal metabolic conditions.19 Although sunflower plants tolerate chilling, they may be highly affected by freezing temperatures. In order to select and characterize sunflower lines tolerant to freezing conditions and possibly use them in early seed sowing, we have previously screened 100 lines from the sunflower collection of the Instituto de la Grasa (Sevilla, Spain) for freezing tolerance. Sunflower susceptible line Hopi and tolerant lines PI 543006 and BSD-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 that are responsive to cold acclimation in freezing susceptible and tolerant sunflower lines. We adopted the procedure of SDSPAGE prefractionation prior to reverse-phase liquid chromatography-tandem mass spectrometry of in-gel digested peptides, a technique also referred to as GeLCMS. As a result, 243 differentially expressed, nonredundant proteins were identified across the studied sunflower lines using this approach (Figure 1). The implications of these proteins are discussed in the context of the complex events occurring during cold acclimation.

’ EXPERIMENTAL PROCEDURES Plant Material and Cold Treatment

Helianthus annuus freezing susceptible line Hopi and tolerant sunflower lines PI 543006 (both from USDA, ARS, National Genetic Resources Program) and BSD-2-69120 were germinated in soil under fluorescent light (200 μm m2 s1, 16 h

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photoperiod) at 25/15 °C day/night temperatures. After 6 weeks of germination, leaves of non- acclimated plants (control) were sampled and the chamber temperature was decreased progressively during a week to reach 15/5 °C day/night temperatures with a 12 h photoperiod at 200 μm m2 s1. Then, plants were kept in the chamber for an additional 7 days period before sampling. All samples were frozen in liquid nitrogen and immediately submitted to protein extraction. The experiment was repeated to give four independent biological replicates. Protein Extraction and Gel Electrophoresis

Leaf tissues from each sunflower line in control and coldacclimated conditions were frozen in liquid nitrogen and ground in a mortar to produce a fine powder. Aliquots of 1 g of the powder were resuspended in 10 mL of 10% (w/v) trichloroacetic acid and 0.07% 2-mercaptoethanol in acetone and incubated at 20 °C for 45 min. Protein precipitates were collected by centrifugation at 10 000 g for 10 min at 4 °C, washed twice with cold acetone containing 0.07% 2-mercaptoethanol and vacuum-dried at room temperature. Aliquots of 200 mg of leaf acetone powder were transferred into sterile microtubes containing 1 mL resuspension buffer [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 5 min at 4 °C. Finally, supernatants were transferred to fresh tubes and 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 each sunflower line studied. Prior to gel electrophoresis, sample aliquots containing 150 μg of proteins were mixed with an equal volume of loading buffer containing 0.5 M Tris (pH 6.8), 20% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol and traces of bromophenol blue and incubated for 5 min at 99 °C. Gel electrophoresis was performed under denaturing conditions in 12% polyacrylamide gels for 3 h using 10 mA per gel. After protein migration, gels were stained with colloidal Coomassie under standard conditions.21 In Gel Trypsin Digestion

Prior to protein digestion, the gel lane for each biological replicate was sliced into 10 equal size segments, diced into approximately 1 mm cubes with a clean scalpel and transferred into 1.5 mL sterile polypropylene tubes for in gel digestion. In gel trypsin digestion was carried out according to Shevchenko et al.22 Protein digestion was performed by the addition of 200 μL of sequencing grade porcine trypsin (Promega, Madison, WI) at 5 ng/μL. After 120 min of cold incubation at 4 °C, samples were digested overnight at 37 °C. Upon in gel digestion, gel pieces were 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 centrifuge and kept at 80 °C until LCMS/MS analyses. LCMS/MS Analyses

For each round of LCMS/MS analysis, extracted peptides were reconstituted in 0.1% (v/v) FA and separated at the flow rate of 150 nL/min into a 10 cm 150 μm ID nanocolumn (C18, 100 Å, 5 μm) (Michrom Bioresources) using the following mobile phase gradient: from 5 to 35% of solvent B in 25 min, 35 to 70% in 25 min, back to 5% in 10 min. Solvent A was water containing 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 spectrometry data were acquired in a ProteomeX LTQ Workstation 2331

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Journal of Proteome Research

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Figure 1. Downstream analysis for the identification of differentially expressed proteins in freezing susceptible and tolerant sunflower lines after cold acclimation. 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. The number of assigned MS/MS spectra for each identified protein was determined across the whole gel lane and spectral index (SpI) calculated. For cutoff estimation, 1,000 randomized spectral indexes were obtained for each protein and the SpI threshold determined for 95% confidence level. Proteins with SpI 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.

(Thermo, San Jose, CA) in data-dependent acquisition (DDA) mode controlled by XCalibur 2.0 software (Thermo Fisher Scientific). Typical DDA cycle consisted of a survey scan within m/z 200 to 2000 followed by MS/MS fragmentation of the seven most abundant precursor ions under normalized collision energy of 35%. Fragmented precursor ions were dynamically excluded according to the following: repeat counts: 3, repeat duration: 30 s, exclusion duration: 30 s. Database Search and Protein Identification

The 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 were translated and the open reading frames (ORFs) scanned using the Virtual Ribosome version 1.1 software.23 For each H. annuus TIGR nucleotide sequence, one amino acid sequence was reported, corresponding to the longest ORF reported. To estimate the peptide false discovery rate (FDR), randomized (decoy) sequences were combined with the forward database, resulting in a concatenated database of 89 324 entries. Peak list was generated 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 2332


Journal of Proteome Research count: 10. Protein identification was performed using SEQUEST algorithm within the Bioworks 3.3.1 software package (Thermo). Search parameters were set as follows: oxidation of methionine was allowed as a variable modification and carbamidomethylation of cysteine as a static modification; enzyme: trypsin; number of allowed 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, SEQUEST output files were uploaded and analyzed by Scaffold 2.2.1 software (Proteome Software, Portland, OR). Each ten SEQUEST search results files resulting from the searches of the ten gel lane segments from each biological replicate were combined and the number of assigned peptides and spectra in each biological replicates used for confident protein identification and quantification. To obtain high-confidence and unambiguous protein assignments, ProteinProphet and PeptideProphet Probabilities24 equal to 99 and 95%, respectively, and all protein identifications required detection of at least two unique peptides in at least one biological replicate. Peptides that were common to more than one protein sequence were not excluded from data analyses as they comprised less than 0.4% of total spectra in each sample (Supplementary Table 1, Supporting Information). Estimated FDR, for the filter settings specified above, showed an average value of 0.7%. Relative Protein Quantification

Spectral counts were used to estimate the protein amount in each sample. To assess differences in the relative protein abundance between control and cold treatments for each sunflower line, we used a metric named spectral index, which is based on the spectral counts associated to each identified protein.25 The spectral index for a protein is defined as ! Sacclimated N D acclimated SpI ¼ Sacclimated þ Scontrol N T acclimated ! Scontrol N D control ð1Þ Sacclimated þ Scontrol N T control where Scontrol and Sacclimated correspond to the mean spectral count values for the control and cold-acclimated conditions, respectively, and ND and NT correspond to the number of biological replicates in which a protein was detected in the given treatment and the total number of biological replicates evaluated for 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 can range from 1 to þ1 where values close to zero indicate that relative protein abundance is about equal in cold and control treatments. Positive values suggest enrichment of the proteins of interest in cold acclimatized plants, whereas negative values suggest enrichment in control treatment. To determine statistically significant cutoff values for the SpIs, we performed a random permutation analysis, in which the proteome profiles (spectral counts) of the control and cold replicates were permuted 1000 times and the SpI for each protein was recalculated in each permutation event.25 From the created null distribution of randomized SpIs (Supplementary Figure 1, Supporting Information), we selected the upper and lower SpI limits that encompassed 95% of the randomly generated indices. This approach indicated that absolute SpI cutoff values g0.558, 0.55, 0.558 and e0.525, 0.542, 0.552 for Hopi, PI 543006 and BSD-2-691, respectively, were significant at a 95% confidence

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level; meaning that a protein with an SpI higher or lower than the cutoff values had less than 5% probability of being part of the null distribution and therefore of being mistakenly identified as enriched in one of the two groups. Functional Classification

Each TIGR assembly or singleton was aligned to the UniProtUniref database and the annotation for the protein with the best alignment to each assembly was used as the annotation for that sequence.26 Functional classifications of differentially expressed proteins were performed according to the catalog established for Arabidopsis.27 Two subclasses, protein/polypeptide transporters and protein repair were added to the functional catalogue. A redox homeostasis class was also added to the original catalogue. These changes were performed in order to increase the accuracy on the description of the functions of the proteins identified in the present work and to better represent changes that may occur during the studied abiotic stress. Protein Expression Profiling

To access information on similarities of the expression profile of the differentially expressed proteins across the studied sunflower lines, we performed hierarchical clustering analysis, using the freely available software Permutmatrix.28 For this, spectral count data from control and cold treatments were normalized using the spectral index formula (eq 1). The indices for each protein were used to analyze the expression pattern across the sunflower lines. From the normalized data, the Euclidean metric was performed to calculate dissimilarities and clustering (aggregation) was performed according to the unweighted paired group average linkage (UPGMA) procedure.29

’ RESULTS Detection of Differentially Expressed Proteins Induced by Cold Acclimation

To maximize the number of identifications and improve stastical confidence, four biological replicates were used in this study. Proteins in each replicate were prefractionated by 12% SDS-PAGE and segmented into 10 gel slices prior to trypsin digestion to reduce sample complexity prior to LCMS/MS. This approach resulted in high number of identifications for all sunflower lines in both control and cold conditions (Figure 2). In the present work, a total number of 3519 redundant proteins were confidently detected and analyzed. The Hopi susceptible sunflower line showed an increase of 22.5% in the number of detected proteins after cold treatment, as 533 and 653 proteins were confidently assigned in control and cold acclimation treatments, respectively. Tolerant sunflower lines showed a distinct pattern in the number of proteins detected before and after cold treatment. Total number of assigned proteins increased only 3.4% in PI 543006 sunflower line in response to cold stress. In contrast, a reduction of 14% was detected in BSD-2-691 as detected proteins changed from 642 to 552 after cold acclimation. Detection of the differential expression of a protein (the term differential expression is used here in the sense of differential protein abundance, which can be the result of several processes, including changes in de novo protein biosynthesis, protein modification and protein degradation) is challenging for largescale proteomic experiments due to the large number of peptide and protein identifications. One strategy to analyze differential expression between two samples is by comparison of the presence or absence of a particular protein in different samples 2333



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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 of identified proteins containing 718, 675 and 769 identifications for Hopi, PI 543006 and BSD-2-691 sunflower lines, respectively; lower panel, number of common and differentially expressed proteins at 95% confidence level.

(e.g., de novo synthesis or degradative turnover). However, a comprehensive and sensitive analysis should also take into account quantitative differences in the expression of proteins identified. Although spectral counting has been successfully used in label-free plant proteomic analyses,30 the very nature of the spectral counting makes statistical analysis difficult as the underlying distribution is not normal due to the overwhelming number of zero entries, particularly when differential expression is stark.31 For the detection of differentially expressed proteins after cold acclimation in Hopi, PI 543006 and BSD-2-691 sunflower lines, the spectral index25 for each protein was calculated, as described in eq 1. This strategy is based on relative abundance as measured by the number of spectral counts across replicates; and reproducibility is determined by the number of replicates for each treatment in which the protein was detected. This was particularly important to assess the significance of proteins detected in only one biological replicate, especially those with a low number of associated spectral counts. In addition, random permutation analysis of spectral counts across biological replicates, followed by recalculation of spectral indexes, establishes confidence intervals for assessing authentic differential protein expression, and not experimental variation, without assumptions about data

distribution. This approach indicated that from the 718 nonredundant proteins detected in the Hopi sunflower line, the expression of 34 and 9 proteins were induced in cold-acclimated and control treatments, respectively, with 95% confidence. Also, with the same confidence level, the expression of 56 and 16 proteins were induced in cold and control treatments, respectively, in PI 543006 line. BSD-2-691 line showed the highest number of differentially expressed proteins, a total of 168, and a higher number of induced proteins in the control than in coldacclimation treatment, indicating that the expression of a substantial number of proteins is suppressed in response to cold stress in this tolerant sunflower line. Functional Classification of Cold-Responsive Proteins

One of the first steps in interpreting protein expression data is to group differentially expressed proteins into functional categories. Since no functional classification is available for sunflower, the categorization performed in the present study was based on the criteria established for the EU Arabidopsis genome project27 (Figure 3). In accordance with the detection of the lowest number of cold-responsive proteins across all lines, the number of proteins in Hopi sunflower line was never higher than those 2334



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

found in PI 543006 and BSD-2-691 in any category. Also, BSD-2691 tolerant sunflower line showed the highest number of associated proteins in all functional categories. Proteins involved in metabolism, protein synthesis and energy were the most responsive to cold-stress. These three categories comprised 56, 53 and 48% of total cold-responsive proteins in Hopi, PI 543006 and BSD-2-691 sunflower lines, respectively. The number of proteins involved in energy metabolism accounted for 23, 17 and 13% of total differentially expressed proteins in Hopi, PI 543006 and BSD-2-691, respectively, being the most represented class in Hopi susceptible sunflower line. Among the three most abundant categories, metabolism class comprised the highest number of associated proteins within the studied lines (50 in total). Metabolism category showed the highest number of cold-responsive proteins in PI 543006 and BSD-2-691 sunflower lines (15 and 29, respectively); while this class accounted only for six proteins in the susceptible line. Protein synthesis class was also highly represented in BSD-2-691 line after cold stress (29 proteins), while this group accounted only for eight and eleven proteins in Hopi and PI 543006 sunflower lines, respectively. Although less abundant than the other categories, transporters comprised ten responsive proteins in BSD-2-691 line, while this class was represented only by two proteins in PI 543006 and Hopi lines. Similar profile was detected for cell structure related proteins, as eight proteins were differentially expressed in BSD-2-691 line; while only one protein identification was assigned in PI 543006 tolerant line. Proteins involved in cell structure and transcription were absent in Hopi after cold treatment, being detected only in the tolerant lines. Coldresponsive proteins belonging to secondary metabolism was represented only by geranylgeranyl pyrophosphate synthase-related protein (TA11185_4232), only found in BSD-2-691 sunflower line. Expression Profile of Cold-Responsive Proteins

To facilitate the biological interpretation of proteins that displayed differential expression, proteins were sorted on the basis of the expression pattern across the susceptible and tolerant sunflower lines (Figure 4). Cold-responsive proteins varied widely in their patterns of expression, resulting in a total of fourteen protein clusters. In terms of global protein expression, two-way hierarchical clustering indicated that cold-associated proteome responses in the Hopi susceptible line were closer to PI 543006 than those shown in BSD-2-691 sunflower line. Clustering of the

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differentially expressed proteins yielded five minor clusters, formed by less than five proteins, and nine major clusters with five or more proteins in each group. Major cluster A accounted for six proteins, all of them with low expression profile in Hopi susceptible line. Cluster B proteins, formed by nine identifications, showed high expression in Hopi and low expression in BSD-2-691 sunflower lines. Cluster D was the largest cluster found in the present study. This group was formed by 80 proteins in which expression was reduced only in the BSD-2-691 tolerant sunflower line. Functional classification of clustered proteins indicated that this group was mainly formed by accessions involved in protein synthesis, including initiation factors, ribosomal proteins and translation inhibitors (Table 1). Protein cluster E accounted for five identifications that showed induced expression in PI 543006 and reduced expression in BSD-2-691 sunflower lines after cold treatment. Cluster G was the second largest cluster found in the present study, comprising 57 cold-responsive proteins. This cluster consisted of proteins only detected in BSD-2-691 sunflower line and had an opposite expression profile in relation to the largest cluster D, as all identifications were induced after cold acclimation. However, functional classification indicated that proteins in this cluster were mostly involved in metabolism and energy processes, contrary to vast suppression in the expression of proteins involved in translation as found for cluster D. Cluster J consisted of accessions only detected in Hopi sunflower line; while cluster L accounted for 38 PI 543006 induced identifications, most of 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-binding protein (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 tolerant sunflower lines. Minor cluster C showed low protein expression in PI 543006 and BSD-2-691 tolerant sunflower lines. This group was formed only by the SAM Mg-protoporphyrin IX methyltranserase enzyme (TA16226_4232) and the RuBisCO large subunitbinding protein (TA7124_4232). Cluster F also constituted two proteins, NADP-G3P dehydrogenase (CX943741) and the cell division protein ftsH homologue (DY911512); however, the expression profile showed differential response between PI 543006 (suppression) and BSD-2-691 (induction) tolerant sunflower lines, an opposite expression profile in relation to cluster E. Cluster H proteins showed induced expression in Hopi and BSD-2-691 lines and the group was formed by the rubber synthesis protein (TA6858_4232) and the plasma membrane polypeptide (TA7585_4232). In the same manner as cluster H, cluster K consisted of induced proteins in Hopi, but instead of induction of expression in BSD-2-691, high expression was observed in the PI 543006 tolerant sunflower line. This cluster was formed by two vacuolar ATP synthase catalytic subunit accessions (TA8692_4232, DY924246), G protein beta-subunit-like protein (TA8126_4232) and the mitochondrial phosphate translocator (TA11282_4232). The final minor cluster detected in the present work is cluster N. This group consisted of proteins that had overexpression after cold acclimation in all sunflower lines. These proteins were the myo-inositol1-phosphate synthase (TA11211_4232), temperature-induced lipocalin (TA12632_4232), H. annuus homologous dehydrin (TA7293_4232) and the TIGR assembly TA6741_4232 (Table 1). 2335



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

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Table 1. List of Differentially Expressed Proteins, Classified According to the Expression Profile across Hopi, PI 543006 and BSD2-691 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) Glyceraldehyde-3-phosphate dehydrogenase (A. majus)

TA10610_4232 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) ATP synthase gamma chain, mitochondrial precursor (I. batatas)

TA9801_4232 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) RuBisCO large subunit-binding protein (A. thaliana)

TA16226_4232 TA7124_4232

Cluster D (suppressed in tolerant BSD-2-691) 33 kd chloroplast ribonucleoprotein precursor (N. sylvestris)

BQ916500

60S ribosomal protein L42 (A. thaliana)

BQ976230

TGB12K interacting protein 3 (N. tabacum)

TA10299_4232

Geranylgeranyl pyrophosphate synthase-related (A. thaliana) Nucleoid DNA-binding-like protein (A. thaliana)

TA11185_4232 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) Initiation factor eIF4A-15 (H. annuus)

TA15214_4232 TA6872_4232

Polygalacturonase inhibitor protein precursor (L. esculentum)

TA7256_4232

60S ribosomal protein L10A (O. sativa) PREDICTED: similar to tubulin, beta 2 (R. norvegicus)

TA7653_4232 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) Probable ATP synthase 24 kDa subunit (A. thaliana)

TA11460_4232 TA8456_4232

Hypothetical protein (R. communis)

TA10628_4232


TA9021_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) Putative nuclear RNA binding protein A (O. sativa)

TA11875_4232 TA6549_4232


TA9282_4232 2337



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Table 1. Continued protein namea

accessionb

Adenylate kinase B (O. sativa) CD846470_rframe1_ORF

BQ972050 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) 3-oxoacyl-[acyl-carrier-protein] reductase (C. lanceolata)

DY929446 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) Putative ribosomal protein S12 (O. sativa)

TA7421_4232 TA7467_4232

Chromosome 9 SCAF14729 (T. nigroviridis)

TA7510_4232

40S ribosomal protein S3a (H. annuus)

TA7643_4232


TA7852_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) 60S ribosomal protein L34 (P. sativum)

TA8276_4232 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) Phosphoglucomutase (S. tuberosum)

TA8290_4232 DY910004

Emb|CAB62460.1 (A. thaliana)

TA16225_4232 TA16410_4232

Proteasome subunit alpha type 3 (O. sativa) 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) Cysteine synthase (V. pseudoreticulata)

TA7804_4232 DY925536

31 kDa ribonucleoprotein (N. plumbaginifolia)

DY917463

Oxygen-evolving enhancer protein 3 (P. sativum)

TA8258_4232

40S ribosomal protein S112 (A. thaliana)

TA7499_4232

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

TA7019_4232


TA7095_4232


TA8219_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) Hypothetical protein (S. tuberosum)

TA10138_4232 TA10793_4232

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

TA7576_4232

Serine hydroxymethyltransferase (A. thaliana)

TA7117_4232 2338



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accessionb

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) Serine-glyoxylate aminotransferase (S. polyrrhiza)

TA7287_4232 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) Chloroplast 30S ribosomal protein S3 (P. ginseng)

TA7645_4232 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) GDP-mannose 3,5-epimerase 1 (O. sativa)

TA10968_4232 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) Peptide methionine sulfoxide reductase (L. sativa) Phosphoenolpyruvate carboxylase (F. trinervia)

DY921192 TA10603_4232 TA14451_4232


TA7775_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) Beta-1,3-glucanase (C. intybus)

TA11778_4232 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) Proteasome subunit beta type 2-A (A. thaliana)

TA7827_4232 AJ828950

Protease Do-like 1 (A. thaliana)

DY914432

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

DY921054

HADIP_rframe1_ORF

HADIP 2339



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accessionb

ATP synthase A chain (P. ginseng) TA15511_4232_rframe3_ORF

TA15435_4232 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) 50S ribosomal protein L12 (N. sylvestris)

DY903749 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) Light harvesting chlorophyll a/b binding PSII (E. gracilis)

CD847964 TA8295_4232


TA11343_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) Cyclophilin 1 (C. lanceolata)

TA10175_4232 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) Heat shock protein 812 (A. thaliana) Remorin-like protein (A. thaliana)

CD857897 TA9004_4232 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

60S ribosomal protein L19-like protein (S. tuberosum) 60S ribosomal protein L30 (E. esula)

TA6538_4232 TA7641_4232

Cyclophilin (K. candel)

TA8568_4232

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

DY916177

2340



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accessionb

Hypothetical protein T16L4.100 (A. thaliana) TA13261_4232_rframe-2_ORF

TA10152_4232 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) Putative UDP-glucose dehydrogenase (S. bicolor)

TA7000_4232 TA7983_4232

Ribosomal protein L37 (G. max)

TA8029_4232

60s ribosomal protein L21 (T. aestivum)

TA7462_4232

Heat shock protein 813 (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) TA7075_4232_rframe1_ORF

TA11077_4232 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


TA10262_4232

ADP ribosylation factor 002 (D. carota) Aldehyde dehydrogenase 1 precursor (L. corniculatus)

TA7206_4232 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) L-ascorbate peroxidase (P. sativum)

TA7528_4232 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) H.annuus homologous dehydrin (H. annuus)

TA12632_4232 TA7293_4232


TA6741_4232

a

UniProt-UniRef protein annotation with the best alignment to the TIGR transcript assembly. b TIGR transcript assembly identifier or GenBank accession number for the identified protein.

’ DISCUSSION Spectral Counting Approach Resulted in the Identification of a Large Number of Cold-Responsive Proteins

In the present work, we aimed to study changes in protein expression induced by cold acclimation in freezing susceptible and tolerant sunflower lines. Previous studies reported changes

in the proteome profile in cold susceptible species, such as rice14,15 and Arabidopsis,9,10 or freezing tolerant species, such as the woody species P. obovata4 and P. persica.8 Here, GeLCMS was performed to investigate cold responses integrating it with a spectral counting-based, label-free proteomic approach. One of the advantages the GeLCMS approach has over 2-DE is the better representation of “extreme” proteins, 2341


Journal of Proteome Research 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 for quantitative LCMS/MS-based proteomics because of their simplicity, affordability and flexibility.32 Although in theory ion suppression could affect quantification, previous studies have demonstrated that spectral counting and also the spectral ion intensity and peak area correlates well with protein abundance in complex samples.30,3338 Investigation of plant proteome changes related to cold stress are exclusively based on fractionation of complex protein samples using 2-DE followed by image analysis and MS protein identification. In these studies, 2-DE gel maps presented a resolving power ranging from 1000 to 1700 protein spots per gel; however, the detection of cold-responsive protein spots was limited to a maximum of 252, from which mass spectrometry analysis resulted in the identification of 43 proteins.4 Short and long exposure to low temperatures also resulted in the detection of 60 spots presenting significant differences in the intensity and identification of 50 differentially expressed proteins in T. halophila leaves.12 In the present work, 3519 redundant proteins were confidently identified across the three sunflower lines in both control and cold treatments. From the 3519 proteins, 718 were unique and 243 were differentially expressed at 95% confidence interval. The strategy used here, that is, one-dimensional gel electrophoresis prefractionation, associated with a fast mass spectrometer scanning, and robust software and statistical framework to detect the differential expression proteins, resulted in the identification of the largest number of proteins involved in plant cold acclimation. Tolerant Lines Are More Responsive to Low, Non-Freezing Temperatures

Cold acclimation is an adaptative response where plants acquire an increase in freezing tolerance upon a prior low nonfreezing temperature treatment.39 Cold acclimation may involve a wide array of metabolic changes governed by extensive reprogramming at both gene expression, post-transcriptional, and translational levels.40 In Arabidopsis, a comparative analysis of the transcriptional changes induced by cold acclimation across natural ecotypes presenting different tolerances to freezing temperatures indicated an increase of 10% to 25% of the mean expression change for the accessions that acclimate well in comparison to those that acclimate poorly.41 Although increased freezing tolerance during cold acclimation is associated with many metabolic changes,42,43 no clear relationship between global metabolic changes and differences between ecotypes with different freezing tolerances was found in Arabidopsis.41 Although proteome data do not provide direct evidence of the regulation of gene expression, in the present work the proteome responses of H. annuus presented a similar profile in relation to the transcriptional changes observed in different Arabidopsis ecotypes, as sunflower freezing tolerants PI 543006 and BSD-2-691 showed higher number of regulated proteins in relation to susceptible line Hopi. The metabolic shift that occurs in plants exposed to cold temperature varies according to its tolerance to this stress. While most differentially expressed proteins in sunflower tolerant lines BSD-2-691 and PI 543006 are related to primary metabolism, that is, amino acids, nitrogen, nucleotides, phosphate, sugars, lipids and cofactor metabolism, most of the proteome responses in the susceptible line Hopi were related to energy metabolism. Proteome analysis of O. sativa exposed to progressive cold resulted in the induction of mainly metabolic related proteins, comprising 39%

ARTICLE

of all identified proteins.15 Photosynthesis, energy and primary metabolism related proteins were mainly affected after cold treatment in T. halophila and P. patens.12,13 Comprehensive transcriptional analysis of Arabidopsis ecotypes presenting different freezing tolerance spectra indicated that the suppression of photosynthesis and induction of photoprotective flavonoids are associated with improved acclimated freezing tolerance; however, metabolic profiling indicated a central role of primary metabolism, especially carbohydrate metabolism, in the mechanism of tolerance,41 which is in accordance with the high number of proteins involved in primary metabolism that were differentially expressed after cold acclimation in H. annuus tolerant lines. Successful Cold Acclimation Requires Differential Expression of Several Proteins

Cold acclimation involves the remodeling of cell and tissue structures and the reprogramming of metabolism and gene expression.40 Sensitivity to freezing mutants have been identified in Arabidopsis that have lost their ability to fully cold acclimate due to single-gene mutations, indicating that, despite the evident complexity, several genes are essential for freezing tolerance and that the inactivation of any of these genes may severely affect acclimated freezing tolerance.41 In the present work, four proteins showed induced expression after cold treatment in all three sunflower lines. Myo-inositol-phosphate synthase (MIPS) catalyzes the conversion of D-glucose 6-phosphate to myo-inositol-1-phosphate, the first committed and rate-limiting step in inositol and inositol-containing compound biosynthesis.44 Besides its essential role in plant embryo development,45 high expression of MIPS confers tolerance to salt4648 and cold44,49 stress. Cold tolerance induced by MIPS may be related to accumulation of myo-inositol, a carbohydrate. Glucose, fructose and sucrose were identified in high concentrations in coldacclimated Arabidopsis ecotypes presenting freezing tolerance.41 The function in plant stress tolerance of soluble sugars has been highlighted by many investigators; however, this accumulation alone is neither necessary nor sufficient for cold acclimation in Arabidopsis plants.50 In the tropical species Passiflora edulis, expression of MIPS is correlated with this species ecological adaptation to short chilling winters without injury, but does not confer severe or prolonged freezing tolerance.44 Temperature-induced lipocalin identification was also clustered together with MIPS. Lipocalins are an ancient and functionally diverse family of proteins found in bacteria, plants, arthropods and chordates.51 Recently, the identification of a true plant lipocalin was reported in wheat and Arabidopsis.52 Expression analysis of wheat lipocalin genes suggest a differential accumulation in various wheat cultivars showing different levels of freezing tolerance, indicating that their expression is associated, or may be involved, with the plant’s capacity to develop freezing tolerance.51 In P. obovata proteome analysis, lipocalin was found to be progressively induced throughout the winter season.4 In winter rye (Secale cereale), photosynthetic acclimation to low temperature mimics the photosynthetic acclimation to high light because both conditions result in a comparable reduction state of PSII.53 Chloroplastic lipocalins and lipocalinlike proteins properties, their tissue specificity, and their transcript accumulation in response to temperature stress suggest a possible role protecting the photosynthetic apparatus against deleterious effect of temperature stress.51 Another protein that showed induced expression in all three sunflower lines was an H. annuus homologous dehydrin. Induction of dehydrin by cold stress has been reported for rice,54 2342


Journal of Proteome Research wheat,55 in fruits of Citrus species56 and Arabidopsis.9,57,58 Dehydrin proteins were also induced in P. obovata needles across winter season and in T. halophila after short and long exposure to low temperatures.4,12 Studies on boreal species indicate strong relation between the acclimation to lower temperatures during autumn and accumulation of dehydrins.5961 Although the biochemical mode of action is still unclear, most of the studies so far suggest that dehydrins stabilize membranes and macromolecules, thus preventing structural damage and maintaining essential enzyme activity during cellular frost dehydration.62 These proteins also interact with sugars to promote low temperature cytoplasmic vitrification which is proposed as a general mechanism for tolerance of extreme frost dehydration.4 In addition, the EST sequences of the assembly identified as TA6741_4232 match, with 47% identity, to the UNIGENE transcript locus NP_173468.1, a cold-responsive 47 protein that has 100% sequence coverage with the CAA62449.1 dehydrin accession. Sunflower Tolerant Lines PI 543006 and BSD-2-691 Showed Different Proteome Responses to Cold Acclimation

Although showing tolerance to freezing, the sunflower lines PI 543006 and BSD-2-691 showed different proteome responses after cold treatment. Proteins exclusively induced in one or the other sunflower tolerant line comprised the vast majority of all identified proteins, 177 proteins, all of them grouped into the major clusters D, L, G and I. Interestingly, exclusively overexpressed proteins presented a different functional classification pattern between tolerant sunflower lines PI 543006 and BSD-2691. Clusters G and L, comprising exclusive proteins induced in BSD-2-691 and PI 543006 lines, respectively, were mainly constituted by proteins involved in primary metabolism. However, recruitment of proteins involved in energy metabolism, especially glycolytic, mitochondrial electron transport and photosynthesis related proteins, and transport was higher in BSD-2-691 than in PI 543006 line after cold acclimation. On the other hand, exclusive induced proteins in PI 543006 after cold acclimation suggested a major role played by proteins involved in protein synthesis. Besides exclusively expressed proteins, accessions showing opposite expression profile also indicate differential response to cold acclimation. Seven proteins showed opposite regulatory profile between PI 543006 and BSD-2-691 lines (clusters E and F). Cluster E comprised five proteins, phosphoglycerate kinase, a hypothetical protein, S-adenosyl-Lmethionine synthetase, serine hydroxymethyltransferase and a nascent polypeptide-associated protein. Phosphoglycerate kinase is a key glycolytic enzyme, a pathway that is strong influenced by cold stress,63 that catalyzes the reversible conversion of 1,3diphosphoglycerate to 3-phosphoglycerate.64 This enzyme was already found to be induced in Arabidopsis10 after cold treatment and in T. halophila soon after low temperature exposure.12 Sequence alignment of the identified hypothetical protein TA10793_4232 indicated high identity (83%) to a prohibitin 1-like protein from Brassica napus (accession Q9AXM0). Although no clear role during cold acclimation was detected for this protein, it has been reported that plant prohibitins may be involved in the defense response and senescence and that prohibitins are essential for mitochondrial homeostasis in plant cells.65 The enzyme S-adenosyl-L-methionine synthetase, which catalysis the formation of S-adenosylmethionine from methionine and ATP, was already reported to be induced after cold acclimation in rice15 and Arabidopsis leaves.9 In the same manner, gene expression of the enzyme serine hydroxymethyltransferase

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was induced after cold treatment in leaves of Arabidopsis.66 Interestingly, this protein was also shown to be induced in protection from oxidative degradation by drought stress in rice leaves.67 Nascent polypeptide associated complex (NAC) promotes proper folding of proteins and/or prevent aggregation of nascent or damaged proteins.68 It is also suggested that NAC is involved in protein sorting and translocation by preventing mistargeting of nascent polypeptide chains to endoplasmic reticulum.69 Suppression of the expression of this protein is already reported in Arabidopsis roots after exposure to high NaCl concentrations. In addition, suppression of NAC was also found in a comparative proteome analysis of rice seedlings exposed to low temperatures.69 The differential proteome responses, induction in tolerant PI 543006 and suppression of BSD-2-691, of these different proteome components, in which most of them were already reported to be involved in general plant stress responses, suggest that there is a complicated and unique mechanism controlling adaptation in each tolerant sunflower line. Differential expression of proteins between tolerant lines PI 543006 and BSD-2-691 was also detected in the accessions grouped in cluster F. Glyceraldehyde 3-phosphate dehydrogenase 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 woody species P. obovata, this enzyme was found to be suppressed throughout winter season.4 FtsH are membrane bound ATP dependent metalloproteases universally found in living organisms.70,71 The function executed by this ancient class of chaperone-like proteins includes assembly, operation and disassembly of protein complexes.70 Cell division protein ftsH was found to be differentially expressed during cold acclimation in rice seedlings,15 P. patens13 and P. obovata.4 Although the expression of these two proteins was reduced in the tolerant line PUB-50, while induced in BSD-2-691, the accessions were also grouped in cluster A (suppression in Hopi), G (induction in BSD-2-691) and I (suppression in PUB50). The identification of isoforms of these proteins in other clusters corroborates the complex and line-unique regulatory mechanisms that may include post translational modification of already cold responsive proteins.72,73

’ CONCLUDING REMARKS Previous studies aimed at identifying cold-responsive proteins were performed using exclusively 2-DE-based strategies, and frequently working only with susceptible plant varieties. In the present study we performed comparative proteomics of cold stress using a less biased approach for protein separation, SDSPAGE, coupled to spectral counting. This approach was used to identify proteins that significantly change after cold treatment in both susceptible and resistant sunflower lines. The strategy resulted in the detection of the largest number of proteins involved in cold acclimation. Proteome responses reported here resemble changes previously described in other stress related treatments, with exceptions. Comparative analysis of tolerant and susceptible lines showed significant proteome differences. However, due to the sometimes divergent responses observed between the lines studied it is not possible to generalize and specify the cold acclimation responses in the H. annuus species. Further analysis of cold acclimation in near-isogenic sunflower lines may also help to understand the complex physiological changes that 2343


Journal of Proteome Research occur in this event and that probably require the targeting of several genes for successful genetic engineering in this species.

’ ASSOCIATED CONTENT

bS

Supporting Information Supplemental 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 Christopher S. Bond Life Sciences Center, 109 Bond Life Sciences Center, 1201 Rollins Ave., Columbia, MO 65211. E-mail: balbuenat@ missouri.edu. Tel.: 573-884-5979. Fax.: 573-884-9676.

’ ACKNOWLEDGMENT Our thanks are due to A. Gonzalez-Callejas and B. LopezCordero for their skillful technical assistance. This work was supported by Junta de Andalucía, Instituto Andaluz de Biotec NDALUS 08/9/L3.1. nología, project BIOA ’ REFERENCES (1) Timperio, A. M.; Egidi, M. G.; Zolla, L. Proteomics applied on plant abiotic stresses: role of heat shock proteins (HSP). J. Proteomics 2008, 71 (4), 391–411. (2) Mahajan, S. Tuteja. Cold, salinity and drought stresses: an overview. Arch. Biochem. Biophys. 2005, 444 (2), 139–158. (3) John, U. P.; Polotnianka, R. M.; Sivakumaran, K. A.; Chew, O.; Mackin, L.; Kuiper, M. J.; Talbot, J. P.; Nugent, G. D.; Mautord, J.; Schrauf, G . E.; Spangenberg, G. C. Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv. Plant, Cell Environ. 2009, 32 (4), 336–348. (4) Kjellsen, T. D.; Shiryaeva, L.; Schroder, W. P.; Strimbeck, G. R. Proteomics of extreme freezing tolerance in Siberian spruce (Picea obovata). J. Proteomics 2010, 73 (5), 965–975. (5) Gygi, S. P.; Rochon, Y.; Franza, R.; Aebersold, R. Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 1999, 19 (3), 1720–1730. (6) Ideker, T.; Thorsson, V.; Ranish, J. A.; Christmas, R.; Buhler, J.; Eng, J. K.; Bumgarner, R.; Goodlett, D. R.; Aebersold, R.; Hood, L. Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Science 2001, 292 (5518), 929–934. (7) Hajduch, M.; Hearne, L. B.; Miernyk, J. A.; Casteel, J. E.; Joshi, T.; Agrawal, G. K.; Song, Z.; Zhou, M.; Xu, D.; Thelen, J. J. Systems analysis of seed filling in Arabidopsis: using general linear modeling to assess concordance of transcript and protein expression. Plant Physiol. 2010, 152 (4), 2078–2087. (8) Renaut, J.; Hausman, J. F.; Bassett, C.; Artlip, T.; Cauchie, H. M.; Witter, E.; Wisniewski, M. Quantitative proteomic analysis of short photoperiod and low-temperature responses in bark tissues of peach (Prunus persica L. Batsch). Tree Genet. Genomes 2008, 4 (4), 589–600. (9) Amme, S.; Matros, A.; Schlesier, B.; Mock, H. P. Proteome analysis of cold stress response in Arabidopsis thaliana using DIGEtechnology. J. Exp. Bot. 2006, 57 (7), 1537–1546. (10) Bae, M. S.; Cho, E. J.; Choi, E. Y.; Park, O. K. Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J. 2003, 36 (5), 652–663. (11) Bressan, R. A.; Zhang, C.; Zhang, H.; Hasegawa, P. M.; Bohnert, H. J.; Zhu, J. K. Learning from the Arabidopsis experience: the next gene search paradigm. Plant Physiol. 2001, 127 (4), 1354–1360.

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