A Functional Proteomic Approach to the Identification and Characterization of Protein Composition in Wheat Leaf

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A

Functional

Proteomic

Approach

to

the

Identification

and

Characterization

of

Protein

Composition

in

Wheat

Leaf

Jung-Feng Hsieh

1

and Shui-Tein Chen

2,

*

1

Department of Food Science, Fu Jen Catholic University, Xin Zhuang, Taipei 242, Taiwan and

2

Institute of Biological

Chemistry and the Genomics Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan

Abstract:

Proteomics and bioinformatics approach were applied for the analyzing of wheat leaf proteins’ composition

and function. Wheat proteins were precipitated by ammonium sulfate and analyzed by two-dimensional gel

electrophore-sis and mass spectrometry. A total of 200 wheat proteins were selected to identify based on reproducibility and relative

quantity, and 123 proteins were identified with an identification success rate of 61.5%. The classifications of these

pro-teins by BGSSJ (bioinformatic software) were mainly classified by their molecular, biological and cellular function.

Pro-teins grouped under the molecular function were involved in catalytic, binding and antioxidant activity. The catalytic

ac-tivity of identified wheat proteins included oxidoreductase, transferase, hydrolase, lyase and isomerase. Only 10.6 % of

the wheat protein identifications lacked ascertainable functions. These results provided the information to investigate the

composition and function of proteins found in wheat leaf, and enhanced the feasibility of future research on wheat.

Key Words: Proteomics, 2D-PAGE, wheat leaf, protein composition, mass spectrometry.

INTRODUCTION

Wheat is one of the most important cereal crops in the

world. Consumption has doubled in the past 30 years to

nearly 600 million tons per year. The International Maize

and Wheat Improvement Center has stated that the

world-wide demand will increase by over 40% by 2020 (Donnelly,

2005). Knowledge acquirement of wheat’s biochemical

con-stitution and functional biology are required to improve

wheat in ways that can meet this demand. Recently,

scien-tists have shown a great interest in investigating the function

of wheat leaf proteins (Saxena et al., 2000). Two-dimen-

sional gel electrophoresis (2-DE) is a useful tool to explore

the wheat proteins (Rampitsch et al., 2006). For 2-DE

analy-sis, many neutral salts such as ammonium sulfate (AS),

so-dium chloride and soso-dium sulphate have been used to

pre-cipitate or fractionate proteins (Englard and Seifter, 1990).

AS has been the precipitant used most frequently in the

salt-ing out of proteins by stepwise precipitation, and has been

used to concentrate proteins from microorganism, animal

and plant tissues (Farag and Hassan, 2004; Su and Yang,

2000; Kochkina, 2004).

The 2-DE approach to protein profiling has been

success-ful because it is an accessible, inexpensive and powersuccess-ful tool

for analyzing patterns of protein expression. All protein

spots that have been resolved and detected within the 10

4

to

10

5

dynamic range of gel capacity can be studied

qualita-tively and quantitaqualita-tively in relation to each other, and viewed

as a single image (Bahrman et al., 2004). Two-dimensional

difference gel electrophoresis has strengthened the 2D

plat-form by allowing the detection and quantization of diffe-

*Address correspondence to this author at the Institute of Biological Chem-istry and the Genomics Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan; Tel: +886-2-27886230; Fax: +886-2-27883473;

E-mail: bcchen@gate.sinica.edu.tw

rences between samples resolved on the same gel, or across

multiple gels, when linked by an internal standard (Wu,

2006). This technique is based on the protein samples with

fluorescent cyanine dyes, which have distinct excitation and

emission spectra and are movement (charge and size)

matched. Therefore, the same protein labeled with any of the

dyes (Sypro

®

Ruby dye, silver nitrite, Cy3 or Cy5) will move

to the same position within a 2D gel (Trisiriroj et al., 2004;

Dhingra et al., 2005; Topanurak et al., 2005).

2-DE,

combined with protein identification by mass spec-

trometry (MS), has often been employed to identify indivi-

dual protein of interest. For function classification of these

identified proteins, BGSSJ (bioinformatic software, http://

sourceforge.net/projects/bgssj/) is used. This software was

developed by our laboratory, and is an XML-based Java

ap-plication for BGSS (Bulk Gene Search System) that

orga-nizes selected proteins for biological interpretation (Juan

et al., 2002). BGSS integrates UniGene (http://www.ncbi.

nlm.nih.gov/UniGene/), Locus Link (http://www.ncbi.nlm.

nih.gov/LocusLink/index.html), Proteome (http://www.

proteome.com/databases/HumanPD/reports), SWISS-PROT

(http://www.expasy.ch/sprot/), PubMed (http://www.ncbi.

nlm.nih.gov/ PubMed/) and SubtiList (http://genolist.Pasteur.

fr/Subti List) databases. The classifications of functional

annotations of these proteins were mainly classified in

mo-lecular, biological and cellular function. Therefore, the

ob-jective of this study was to investigate the protein

composi-tion and funccomposi-tion in wheat leaf by the proteomic approach

and combined with MS and bioinformatic software.

MATERIALS AND METHODS

Plant Material and Protein Precipitation

Wheat

(Triticum aestivum L.) seeds were planted

indi-vidually in 4 cm diameter x 20 cm high containers. These

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seeds were grown in chambers with 24:18

o

_{C day:night}

tem-perature cycle and 14 h photoperiod for 10 days. Wheat

leaves were harvested, milled by a laboratory-scale milling

machine and filtered through filter paper. The extract was

then salted out with solid AS and collected at 0-40, 40-60,

60-70, 70-80, 80-100 % (w/v) saturation of AS, respectively.

Each fraction was collected by centrifugation (12,000g for

40 min, 4

o

C) and dialyzed extensively against phosphate

buffer (50 mM, pH 7.5) at 4°C for 24 h. The precipitated

proteins were suspended in a chilled (-20

o

C) solution

con-taining 10% TCA, 90% acetone with 0.07% -Me. The

mix-ture was incubated at -20

o

_{C for 4 h, and then centrifuged at}

12,000 xg for 40 min. The pellet was washed three times

with 5 ml of chilled (-20

o

C) acetone with 0.07% -ME

cen-trifuging at 12,000 xg for 40 min between rinses.

Protein Quantification

Protein concentration was determined by the Bio-Rad

protein assay (Bio-Rad Laboratories, Hercules, CA), and the

ovalbumin (Sigma, St. Louis, MO, USA) was adopted as the

standard (Chao and Nylander-French, 2004). Bio-Rad

pro-tein assay dye was diluted with water 3 times the volume,

and then mixed into the standards or samples. Samples were

left at room temperature for 2 min before absorbance, which

was determined at 595 nm using a UV spectrophotometer

(Beckman DU640; Beckman Instruments, Palo Alto, CA).

Sodium Dodecyl Sulfate-Polyacrylamide Gel

Electropho-resis (SDS-PAGE)

Wheat

samples were first analyzed by SDS-PAGE, which

was performed by using the precast Novex

®

_Tris-glycine

gels (Invitrogen Co.). Samples, reducing agent and sample

buffer were denatured by heating in boiling water for 3 min.

The sample buffer contained 10% glycerol, 70 mM Tris-HCl,

pH 6.8, 2% SDS, and 0.02% bromophenol blue. Protein

lad-der and samples (10 μg) were then loaded into separate

wells. After electrophoresis, gels were added in a solution

containing 10% methanol and 7% acetic acid for 30 min,

then stained in 350 ml of the Sypro

®

_{Ruby protein gel stain}

solution overnight, before soaking in deionized water for 20

min to wash the residual dye out (Berggren et al., 2000). The

developed gels were digitally scanned as 2-D images by

us-ing fluorescence image scannus-ing Typhoon 9200 (Amersham

Pharmacia Biotech), and analyzed by ImageMaster software

(Amersham Pharmacia Biotech).

Two-Dimension Gel Electrophoresis and Image Analysis

Wheat samples were dissolved in lysis solution

contain-ing 7M urea (Boehrcontain-inger Mannheim, Germany), 2M thiourea

(Aldrich, WI, USA), and 4% CHAPS (J. T. Baker, NJ,

U.S.A). For the first dimensional separation, 500 μg of total

protein was immobilized and loaded into pH gradient (IPG)

gel strips (pH 4-7, 18-cm long, Amersham Pharmacia

Bio-tech, Uppsala, Sweden), which were rehydrated for 12 hrs in

a solution containing 7M urea, 2M thiourea, 4 % CHAPS, 40

mM Tris-base, 2 % IPG ampholyte, 65 mM DTE and

0.0002% bromophenol blue prior to use. The strips

under-went isoelectric focusing with the use of the IPGphor system

(Amersham Pharmacia Biotech) at 20

o

C with 6000 V for a

total of 65 kVh, followed by having the strips equilibrated

for 15 minutes in the equilibration solution (50 mM

Tris-HCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 2% DTE),

then added with 0.5% agarose to the top of a vertical 8-18%

linear gradient SDS-polyacrylamide gel. Second dimensional

electrophoresis was carried out with PROTEAN II

MULTI-CELL (Bio-Rad, Hercules, CA, U.S.A) at 45 mA per gel for

5 h until the bromophenol blue reached the bottom of the gel.

The gels were immersed in 10% methanol and 7% acetic

acid for 30 minutes, then left in 350 ml of the Sypro

®

Ruby

protein gel stain solution overnight, before soaking in

deion-ized water for 20 minutes to wash residual dye out. The de-

veloped gels were digitally scanned as 2-D images by using

fluorescence image scanning Typhoon 9200, and analyzed

with ImageMaster software.

Protein Digestion

Selected spots were excised and de-stained by washing in

a solution containing 250 μl of acetonitrile/50 mM

ammo-nium bicarbonate (1:1 v/v) for 15 min twice. The gels were

dried by using a centrifugal vacuum concentrator. Reduction

and alkylation for cysteine residues were performed on

sam-ples by using DTE and iodoacetamide, respectively. For

tryptic digestion, the gel was rehydrated in trypsin solution

(12.5 ng/ml) and incubated at 37

o

_{C for 16 h. Peptides}

frag-ments were then extracted with equal volume 100%

acetoni-trile/2% trifluoroacetic acid (TFA), sonicated in a bath for 10

minutes. The extracted peptides were concentrated by

cen-trifugation in a vacuum centrifuge.

MALDI-MS and MS/MS Analysis

For MALDI-MS and MS/MS analysis, samples were

premixed in a ratio of 1:1 with a matrix solution (5 mg/ml

CHCA in 50% ACN, 0.1% v/v TFA and 2% w/v ammonium

citrate) and spotted onto the 96-wells format MALDI sample

stage (Tantipaiboonwong et al., 2005). Data were obtained

directly

on

the

Q-TOF

Ultima

MALDI

instrument

(MALDI

TM

,

Micromass, UK), which was fully automatic with predefined

probe motion pattern and the peak intensity threshold for

switching over from MS survey scanning to MS/MS, and

from one MS/MS to another.

Protein Identification

Peptide mass fingerprint data from MALDI-Q-TOF were

used to match the protein candidates in NCBInr, MSDB and

Swiss-Prot protein databases using Mascot (http://www.

matrixscience.com) search program (Gygi and Aebersold,

2000; Yates, 2000, Patterson and Aebersold, 1995). Search

parameters allowed for methionine oxidation, cysteine

car-bamidomethylation, one missed cleavage site, and a peptide

mass tolerance of 0.15 Da (Morrissey and Downard, 2006;

Rashidi and Buehler, 2000). The product ion spectra

gener-ated by Q-TOF MS/MS were then compared against the

NCBInr and Swiss-Prot databases and an exact match was

found through the Mascot search program (Wan et al., 2001;

Ahram et al., 2002). In addition, the identified proteins were

searched for their annotation in description in the Swiss-Prot

and NCBI protein databases.

Functional Classification of Identified Proteins

For functional classification of identified wheat proteins,

we used BGSSJ, which is an XML-based Java application

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that organizes selected genes or proteins for biological

inter-pretation in the context of Gene Ontology. It organizes

in-formation according to molecular function, biological

proc-esses and cellular components for a number of different

or-ganisms. The application allows for easy and interactive

search in different protein identifiers (GenBank ID,

Uni-Gene, SwissProt), and generates a summary page that lists

the frequencies of Gene Ontology annotations for each

func-tional category (cluster). The visualization browser allows

users to navigate the cluster hierarchy displayed in a tree

diagram and explores the associated proteins of each cluster

through a user-friendly interface.

RESULTS AND DISCUSSION

Protein Precipitation and Wheat Leaf Proteome

Wheat leaf proteins were salted out at 0-40, 40-60, 60-70,

70-80 and 80-100% respectively (w/v) AS saturation (Table

1). A total of 5 fractions were collected and total yield of

these fractions was 95.8%. Total protein of each fraction was

88.6, 491.3, 163.2, 53.6 and 34.1 mg respectively, while

pro-tein content was 10.2, 56.7, 18.8, 6.2 and 3.9%, respectively.

Among these fractions, the highest and lowest protein

con-tent was present in the fractions precipitated with 40-60%

(56.7%) and 80-100% (3.9%) saturation of AS, respectively.

Proteins obtained from each fraction were separated

electro-phoretically in the precast Novex

®

Tris-glycine gel (Fig. 1).

SDS-PAGE showed significant differences in protein

pat-terns for each fraction (L1-L6). However, L3 with highest

protein content and its protein pattern was similar with L1.

This indicated that most of the proteins could be precipitated

at 40-60% (w/v) AS saturation. Furthermore, these protein

fractions were also separated electrophoretically in the 2D

gels. We first analyzed the protein patterns of each fraction

using 2-DE in the pH range of 3-10 (data not shown). The

visualized wheat proteins crowded, appeared in a pH range

of 4-7. Therefore, we further analyzed the protein patterns of

these fractions by using DE in the same pH range. The

2-DE images of wheat leaf proteins (L2-L6) are shown in Fig.

(2). The wheat leaf proteome was mapped and partially

char-acterized to function as a comparative template for future

wheat research. These protein maps will also enable future

Table 1.

Fractional Precipitation of Wheat Leaf Proteins Using Ammonium Sulfate

Ammonium Sulfate (%) Volume (mL) Total Protein (mg) Protein Content (%) Yield (%) Wheat extracts 410 866.6 100.0 100.0 0 – 40 47 88.6 10.2 10.2 40 – 60 65 491.3 56.7 66.9 60 – 70 58 163.2 18.8 85.7 70 – 80 49 53.6 6.2 91.9 80 – 100 54 34.1 3.9 95.8

Fig. (1). SDS-PAGE analysis of wheat leaf proteins salted out with different concentrations of ammonium sulfate. M: protein marker; L1:

wheat extracts; L2: 0-40%; L3: 40-60%; L4: 60-70%; L5: 70-80%; L6: 80-100% of saturation ammonium sulfate. MW: molecular weight;

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Fig. (2). Two-dimensional gel electrophoresis analysis of wheat leaf proteins salted out with different concentrations of ammonium sulfate.

L2: 0-40%; L3: 40-60%; L4: 60-70%; L5: 70-80%; L6: 80-100% of saturation ammonium sulfate. MW: molecular weight; A: 2-DE image;

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proteomic studies to focus on differential expression using

these cataloged proteins as reference proteins, increasing the

throughput of later studies. The results presented here show

the increased feasibility of wheat leaf proteomics and

per-haps, plant proteomics in general.

Identification of Protein Composition in Wheat Leaf

Spots selected from 2-D gels were then digested with

trypsin and the resultant peptides analyzed by MALDI-TOF.

These proteins were identified by searching wheat and

Vir-idiplantae protein sequences from Swiss-Prot and NCBI

da-tabases. In total, 200 spots (wheat proteins) were selected to

be identified, based on reproducibility and relative quantity.

Each spot containing approximately 0.2 g protein, and 123

proteins were putatively identified with an identification

success rate of 61.5%. The peptide mass fingerprint results

were obtained by MALDI-Q-TOF, and the product ion

spec-tra were generated by Q-TOF MS/MS. These identified

pro-teins were assigned with a number and cataloged according

to their pI and molecular weight (Table 2). Furthermore,

among these 123 wheat proteins successfully identified from

2-DE, there were only 47 unique ones. An identification

suc-cess rate of 55% in barrel medic, utilizing both EST and

pro-tein databases which is comparable with the 51%

identifica-tion success rate was observed with the dual protein/EST

search method (Watson et al., 2003). Porubleva et al. (2001)

reported that there was an identification success rate of 72%

in maize leaves, but of the 216 proteins identified, less than

50 proteins were unique. Plant protein databases have grown

substantially in the last few years, yielding higher rates of

successful identifications from mass spectrometric data (Salt

et al., 2005).

As shown in Fig. (2), there were 10, 21, 22, 47 and 23

wheat

proteins

identified

from

L2-L6,

respectively.

We

found

that the same wheat protein precipitated at different

satura-tion levels of AS. According to the results, dehydroascorbate

reductase, ascorbate peroxidase and putative 3-beta

hydrox-ysteroid dehydrogenase were found in L5 and L6. Moreover,

beta-amylase, phosphoglycerate mutase, ribulose

bisphos-phate carboxylase small chain clone 512 and

ferredoxin-NADP(H) oxidoreductase were found in L4, L5,

L3-L6 and L4-L5, respectively. We also found that there were

multiple observations of the same wheat protein on a 2-D

gel. Donnelly et al. (2005) reported that these multiple spots

could be isoforms with different signal or targeted

se-quences, which would cause shifts in pI and molecular

weight. The proteins could be post-translationally modified

where the addition of side chains, phosphate, methyl groups,

etc. affected the pI and molecular weight. Protein

degrada-tion could also be responsible for multiple spots of the same

protein, or as is the case with Rubisco, the protein could be

carbamylated or merely overabundant and streaking. Many

of these same phenomena are also responsible for the

dis-crepancies observed between the experimentally determined

and database observed pI and molecular weights.

Annotation of Wheat Leaf Proteins

The annotations of these identified wheat proteins are

shown in Table 3. This table shows lists of proteins for

bio-logical interpretation in the context of Gene Ontology, which

organizes information according to their molecular function,

biological processes and cellular components. Among 47

identified proteins, 42 proteins had ascertainable functional

annotations. However, the remaining 5 proteins (hypothetical

protein OSJNBb0081B07.22, OSJNBb0048E02.12 protein,

hypothetical protein OJ1007_D04.29, hypothetical protein

and putative hypersensitive-induced reaction protein) did not

have any function annotation. Rostoks et al. (2003) reported

that plant hypersensitive reaction is a defense response to

pathogen infection involving rapid, localized cell death and

the induction of many pathogenesis-related proteins such as

hypersensitive-induced reaction protein. Yahata et al. (2005)

also found several hypothetical proteins and proteins with

unknown function from wheat.

Clark

et al. (2005) reported that the GO project (http://

www.geneontology.org/) produces structured, controlled

vocabularies and gene product annotations. Gene products

were classified according to the cellular locations and

bio-logical process in which they act, and the molecular

func-tions that they carry out. According to the results of GO

an-notation and classifications of proteins (Table 3), wheat

pro-teins expressed different functions such as oxidoreductase,

transferase and kinase activity in wheat leaf. Several wheat

proteins including fructose-bisphosphate aldolase,

phospho-glycerate mutase, malate dehydrogenase, putative malate

dehydrogenase, cytosolic 3-phosphoglycerate kinase, phos-

phoglycerate kinase were involved in glycolysis. Plaxton et

al. (1996) reported that glycolysis is important in plants

be-cause it is the predominant pathway that “fuels” plant

respi-ration. Moreover, a significant proportion of the carbon that

enters the plant glycolytic and TCA cycle pathways is not

oxidized to CO

2

but is utilized in the biosynthesis of

numer-ous compounds such as secondary metabolites, isoprenoids,

amino acids, nucleic acids, and fatty acids. These

annota-tions provided the information to investigate the protein

functions in wheat leaf.

Functional Classifications of Wheat Leaf Proteins

BGSSJ is an XML-based Java application that organizes

information according to biological processes, molecular

function and cellular components (Juan et al., 2006). The

functional classifications of wheat proteins analyzed by

BGSSJ were shown in Fig. (3). Of the 47 identified proteins,

42, 15 and 35 proteins already have information on their

molecular function, cellular component and biological

proc-ess, respectively. These proteins were classified and showed

different functions in wheat leaf. Only five proteins lacked

ascertainable functional annotation and others with an

anno-tation success rate of 89.4%. For molecular function, there

were 37, 9 and 6 proteins with catalytic activity, binding and

antioxidant activity, respectively. The classification of wheat

proteins according to involved biological process, was 35

and 29 proteins with physiological process and cellular

proc-ess respectively. Furthermore, for the classification of wheat

proteins according to their involved cellular component,

there were 12, 15 and 4 proteins with organelle component,

cell and protein complex. Ashburner et al. (2000) reported

that biological process refers to a biological objective to

which the gene or gene product contributes. A process is

accomplished via one or more ordered assemblies of

molecu-lar functions, while processes often involve a chemical or

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Table 2.

Wheat Proteins Identified and Catalogued from the 2-DE

Spot

No. Protein Name Entry Name

Mr (Exp) pI (Exp) Mr (Cal) pI (Cal) Score Sequence

Coverage Mascot Species

1 Ribulose-1,5-bisphosphate carboxylase Q9FRZ3_WHEAT 18000 6.4 19548 8.99 29 33 MS Triticum aestivum

2 Cu/Zn superoxide dismutase Q96123_WHEAT 20000 5.4 20310 5.35 28 11 MS/MS Triticum aestivum

3 Phosphoribulokinase KPPR_WHEAT 24000 4.9 45113 5.72 67 5 MS/MS Triticum aestivum 4 Phosphoribulokinase KPPR_WHEAT 24000 5.0 45113 5.72 40 2 MS/MS Triticum aestivum 5 Phosphoribulokinase KPPR_WHEAT 27000 5.2 45113 5.72 42 2 MS/MS Triticum aestivum

6 Putative hypersensitive- induced

reaction protein Q6L4B0_SOLDE 33000 5.7 32729 5.40 41 25 MS Solanum demissum 7 Fructose-bisphosphate aldolase ALFC_ORYSA 41000 5.6 41980 6.38 33 2 MS/MS Oryza sativa

8 Reversibly glycosylated polypeptide Q9ZR33_WHEAT 45000 5.6 41472 5.82 90 13 MS/MS Triticum aestivum

11 Ribulose bisphosphate carboxylase

small chain clone 512 RBS3_WHEAT 15000 5.4 13046 5.84 25 36 MS Triticum aestivum 12 Nucleoside diphosphate kinase Q9LKM0_LOLPR 18000 6.6 16491 6.30 75 11 MS/MS Lolium perenne

13 Cyclophilin-like protein Q6XPZ4_WHEAT 24000 7.0 25875 9.59 68 7 MS/MS Triticum aestivum

14 2-cys peroxiredoxin BAS1 BAS1_WHEAT 24000 4.4 23312 5.71 62 15 MS/MS Triticum aestivum

21 Alpha 2 subunit of 20S proteasome Q6H852_ORYSA 29000 5.4 25828 5.39 48 7 MS/MS Oryza sativa

22 ADP-glucose pyrophosphorylase

small subunit Q7X9S8_HORVU 30000 5.5 20497 6.23 59 5 MS/MS Hordeum vulgare 23 Fructose-1,6-bisphosphatase F16P1_PEA 45000 5.2 44483 5.06 59 29 MS Pisum sativum

24 Heat shock protein 70 Q9SEW1_WHEAT 52000 4.8 39680 4.56 74 3 MS/MS Triticum aestivum

25 Heat shock protein 70 Q9SEW1_WHEAT 52000 4.9 39680 4.56 72 3 MS/MS Triticum aestivum

26 Phosphoglycerate mutase Q7XYD2_WHEAT 81000 5.3 29558 5.43 138 14 MS/MS Triticum aestivum 27 Phosphoglycerate mutase Q7XYD2_WHEAT 81000 5.5 29558 5.43 262 29 MS/MS Triticum aestivum 28 Phosphoglycerate mutase Q7XYD2_WHEAT 81000 5.6 29558 5.43 173 23 MS/MS Triticum aestivum 29 Phosphoglycerate mutase Q7XYD2_WHEAT 81000 5.7 29558 5.43 152 14 MS/MS Triticum aestivum 30 Beta-amylase AMYB_WHEAT 67000 5.4 56575 5.24 80 7 MS/MS Triticum aestivum 31 Isoprene synthase Q6EJ97_PUELO 67000 5.5 70030 5.60 44 23 MS Pueraria lobata

small chain clone 512 RBS3_WHEAT 15000 5.2 13046 5.84 25 36 MS Triticum aestivum

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(Table 2) contd….

Spot

34 Ferredoxin-NADP(H) oxidoreductase Q8RVZ8_WHEAT 26000 5.2 40206 6.92 27 5 MS/MS Triticum aestivum 35 Ferredoxin-NADP(H) oxidoreductase Q8RVZ8_WHEAT 26000 5.4 40206 6.92 36 5 MS/MS Triticum aestivum 36 Ferredoxin-NADP(H) oxidoreductase Q8RVZ8_WHEAT 26000 5.6 40206 6.92 109 9 MS/MS Triticum aestivum 37 Glutathione transferase Q8RW02_WHEAT 27000 6.4 24996 6.35 132 19 MS/MS Triticum aestivum 38 Malate dehydrogenase Q9SPB8_SOYBN 40000 5.8 36119 8.23 97 3 MS/MS Glycine max

39 Putative malate dehydrogenase Q6F361_ORYSA 40000 6.0 35414 8.22 120 9 MS/MS Oryza sativa

40 Malate dehydrogenase Q9SPB8_SOYBN 40000 6.2 36119 8.23 83 3 MS/MS Glycine max

43 Ferredoxin-NADP(H) oxidoreductase Q8RVZ9_WHEAT 57000 5.7 38782 8.29 120 9 MS/MS Triticum aestivum 44 Alpha-L-arabinofuranosidase Q8W012_HORVU 82000 5.7 81943 5.59 64 4 MS/MS Hordeum vulgare 45 Phosphoglycerate mutase Q7XYD2_WHEAT 93000 5.2 29558 5.43 155 14 MS/MS Triticum aestivum 46 Phosphoglycerate mutase Q7XYD2_WHEAT 93000 5.3 29558 5.43 143 14 MS/MS Triticum aestivum 47 Phosphoglycerate mutase Q7XYD2_WHEAT 93000 5.4 29558 5.43 110 14 MS/MS Triticum aestivum 48 Beta-amylase AMYB_WHEAT 115000 5.1 56575 5.24 134 12 MS/MS Triticum aestivum 49 Beta-amylase AMYB_WHEAT 115000 5.2 56575 5.24 146 15 MS/MS Triticum aestivum 50 Beta-amylase AMYB_WHEAT 115000 5.3 56575 5.24 120 6 MS/MS Triticum aestivum 51 Phosphoglycerate mutase Q7XYD2_WHEAT 110000 5.3 29558 5.43 214 14 MS/MS Triticum aestivum 52 Phosphoglycerate mutase Q7XYD2_WHEAT 110000 5.4 29558 5.43 190 14 MS/MS Triticum aestivum 53 Phosphoglycerate mutase Q7XYD2_WHEAT 110000 5.5 29558 5.43 134 14 MS/MS Triticum aestivum

54 Hypothetical protein

OSJNBb0081B07.22. Q852A3_ORYSA 34000 6.6 27893 6.34 78 9 MS/MS Oryza sativa 55 Peroxidase 4 Q5I3F4_TRIMO 35000 6.7 32925 5.78 223 17 MS/MS Triticum monococcum

56 Triosephosphate isomerase precursor TPIC_SECCE 29000 5.1 31613 6.00 192 14 MS/MS Secale cereale

59 Ascorbate peroxidase O23983_HORVU 29000 5.8 27418 5.85 198 27 MS/MS Hordeum vulgare

60 Ascorbate peroxidase O23983_HORVU 30000 6.2 27418 5.85 73 10 MS/MS Hordeum vulgare

61 Ribulose-5-phosphate-3-epimerase Q8S4X2_PEA 28000 5.5 29880 8.30 182 12 MS/MS Pisum sativum

64 Dehydroascorbate reductase Q84UH6_WHEAT 28000 5.7 23343 5.88 131 15 MS/MS Triticum aestivum

65 Putative 3-beta hydroxysteroid

dehydrogenase Q65XW4_ORYSA 31000 5.6 31256 9.13 53 5 MS/MS Oryza sativa

dehydrogenase Q65XW4_ORYSA 31000 6.0 31256 9.13 149 5 MS/MS Oryza sativa 67 Putative glyoxalase Q75GB0_ORYSA 33000 5.0 29549 4.99 96 12 MS/MS Oryza sativa

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Spot

68 Ferredoxin-NADP(H) oxidoreductase Q8RVZ8_WHEAT 33000 5.1 40206 6.92 267 16 MS/MS Triticum aestivum 69 Ferredoxin-NADP(H) oxidoreductase Q8RVZ8_WHEAT 33000 5.2 40206 6.92 178 9 MS/MS Triticum aestivum 70 Ferredoxin-NADP(H) oxidoreductase Q8RVZ8_WHEAT 35000 5.3 40206 6.92 160 9 MS/MS Triticum aestivum 71 Ferredoxin-NADP(H) oxidoreductase Q8RVZ9_WHEAT 34000 5.6 38782 8.29 226 16 MS/MS Triticum aestivum 72 Ferredoxin-NADP(H) oxidoreductase Q8RVZ8_WHEAT 34000 5.8 40206 6.92 73 5 MS/MS Triticum aestivum 73 Ferredoxin-NADP(H) oxidoreductase Q8RVZ9_WHEAT 37000 6.9 38782 8.29 221 12 MS/MS Triticum aestivum

74 Hypothetical protein

OSJNBb0081B07.22. Q852A3_ORYSA 33000 6.0 27893 6.34 76 9 MS/MS Oryza sativa 75 Peroxidase 5 Q5I3F3_TRIMO 34000 6.0 27533 5.75 61 5 MS/MS Triticum monococcum

76 OSJNBa0042F21.13 protein Q7XRT0_ORYSA 45000 4.7 42218 5.64 148 18 MS/MS Oryza sativa

77 OSJNBa0042F21.13 protein Q7XRT0_ORYSA 45000 4.8 42218 5.64 258 14 MS/MS Oryza sativa

78 Cytosolic 3-phosphoglycerate kinase Q850M3_WHEAT 46000 5.4 31315 4.98 220 19 MS/MS Triticum aestivum

82 HSP70. Q9SAU8_WHEAT 48000 6.0 70986 5.14 140 8 MS/MS Triticum aestivum 83 HSP70. Q9SAU8_WHEAT 48000 6.3 70986 5.14 120 6 MS/MS Triticum aestivum 84 HSP70. Q9SAU8_WHEAT 48000 6.6 70986 5.14 51 2 MS/MS Triticum aestivum

85 UTP-glucose-1-phosphate

uridylyltransferase UGPA_HORVU 63000 4.8 51612 5.20 243 14 MS/MS barley

uridylyltransferase UGPA_HORVU 63000 5.1 51612 5.20 225 14 MS/MS barley 89 Phosphoglycerate kinase PGKY_WHEAT 56000 5.5 42096 5.64 207 15 MS/MS Triticum aestivum

90 Phosphoglycerate kinase PGKY_WHEAT 56000 5.7 42096 5.64 138 15 MS/MS Triticum aestivum

91 Phosphoglycerate kinase PGKY_WHEAT 58000 5.7 42096 5.64 54 4 MS/MS Triticum aestivum

92 Phosphoglycerate mutase Q7XYD2_WHEAT 80000 5.3 29558 5.43 75 11 MS/MS Triticum aestivum 93 Phosphoglycerate mutase Q7XYD2_WHEAT 80000 5.4 29558 5.43 112 11 MS/MS Triticum aestivum 94 OSJNBb0003B01.27 protein Q5JQX8_ORYSA 85000 5.5 89177 6.49 20 2 MS/MS Oryza sativa

95 OSJNBb0003B01.27 protein Q5JQX8_ORYSA 85000 5.7 89177 6.49 13 2 MS/MS Oryza sativa

97 S222. Q9ZTU6_WHEAT 100000 5.3 50111 5.86 46 3 MS/MS Triticum aestivum 98 S222. Q9ZTU6_WHEAT 100000 5.5 50111 5.86 69 3 MS/MS Triticum aestivum 99 S222. Q9ZTU6_WHEAT 100000 5.7 50111 5.86 60 7 MS/MS Triticum aestivum 100 S222. Q9ZTU6_WHEAT 100000 5.9 50111 5.86 31 3 MS/MS Triticum aestivum

(9)

Spot

101 Ribulose-bisphosphate

carboxylase small chain RBS3_WHEAT 17000 5.7 13046 5.84 37 42 MS Triticum aestivum

carboxylase small chain RBS3_WHEAT 18000 5.2 13046 5.84 36 37 MS Triticum aestivum 106 Alcohol dehydrogenase I Q5VLP8_9ORYZ 23000 4.8 20684 6.64 40 8 MS/MS Oryza eichingeri

107 20S proteasome beta 4 subunit Q5XUV7_WHEAT 23000 4.9 23314 5.57 42 57 MS Triticum aestivum

108 OSJNBb0048E02.12 protein Q7XUY5_ORYSA 22000 4.5 17256 4.75 69 17 MS/MS Oryza sativa

109 OSJNBb0048E02.12 protein Q7XUY5_ORYSA 20000 4.5 17256 4.75 60 10 MS/MS Oryza sativa

110 Dehydroascorbate reductase Q84UH6_WHEAT 26000 5.9 23343 5.88 83 8 MS/MS Triticum aestivum 111 Ascorbate peroxidase O23983_HORVU 28000 5.9 27418 5.85 52 15 MS/MS Hordeum vulgare

dehydrogenase Q65XW4_ORYSA 30000 5.9 31256 9.13 155 5 MS/MS Oryza sativa 113 Ascorbate peroxidase O23983_HORVU 28000 6.2 27418 5.85 63 15 MS/MS Hordeum vulgare

114 Peroxidase precursor PER1_WHEAT 34000 5.6 33155 6.06 88 5 MS/MS Triticum aestivum

115 Hypothetical protein OJ1007_D04.29 Q6ZG81_ORYSA 50000 4.6 58281 6.35 40 40 MS Oryza sativa

116 Hypothetical protein Q2QT67_ORYSA 50000 4.7 42105 5.13 56 33 MS Oryza sativa

117 Protein putative laccase LAC5-4 Q5N7B4_ORYSA 97000 4.7 60174 5.28 48 2 MS/MS Oryza sativa

118 Putative Bplo Q9LX04_ORYSA 95000 5.4 65709 6.10 90 2 MS/MS Oryza sativa

physical transformation. Nevertheless, cellular component

refers to the place in the cell where a gene product is active.

The information obtained from our results should be useful

for any future study on the wheat leaf.

Molecular Function of Identified Proteins in Wheat Leaf

Wheat proteins grouped under molecular function were

involved in catalytic activity, binding activity and

antioxi-dant activity (Fig. 3). The catalytic activity of wheat proteins

included oxidoreductase, transferase, hydrolase, lyase and

isomerase activity. Furthermore, phosphoribulokinase,

nu-cleoside diphosphate kinase and heat shock protein 70 had

nucleotide binding activity. We also found ascorbate

peroxi-dase, peroxiperoxi-dase, Cu/Zn superoxide dismutase and 2-cys

peroxiredoxin BAS1 with antioxidant activity. As we know,

active oxygen species such as superoxide and hydroxyl

radi-cals are by-products of normal cell metabolism. These active

oxygen species result in the peroxidation of membrane

lip-ids, breakage of DNA strands and inactivation of enzymes

(Muth et al., 2004). The conditions leading to damage

caused by active oxygen species are referred to as oxidative

stress. Wu et al. (1999) reported that these enzymes with

antioxidant activity in wheat could protect cells from

super-oxide radicals by catalyzing the dismutation of the

superox-ide radical to molecular O

2

and H

2

O

2

.

(10)

Table 3.

Annotation of Identified Wheat Proteins from the 2-DE

Protein Name Spot No. Annotation (Gene Ontology, GO)

Ascorbate peroxidase 59, 60, 111, 113

GO: 0016688; Molecular function: L-ascorbate peroxidase activity. GO: 0016491; Molecular function: oxidoreductase activity. GO: 0006979; Biological process: response to oxidative stress. Ribulose bisphosphate

carboxylase small chain clone 512

11, 32, 33,

101-105 Function: It catalyzes two reactions: the carboxylation of D-ribulose 1,5-bisphosphate.

Phosphoglycerate kinase 89-91 Catalytic activity: ATP + 3-phospho-D-glycerate = ADP + 3-phospho-D-glyceroyl phosphate. Pathway: glycolysis.

Phosphoribulokinase 3-5 Enzyme regulation: Light regulated via thioredoxin by reversible oxidation/reduction of sulfhydryl/ disulfide groups.

Triosephosphate isomerase

precursor 56-58

Catalytic activity: D-glyceraldehyde 3-phosphate = glycerone phosphate. Pathway: Calvin cycle.

Fructose-1,6-bisphosphatase 23 Pathway: The chloroplast isozyme takes part in the regeneration of ribulose bisphosphate in the photosynthetic carbon reduction cycle.

2-cys peroxiredoxin BAS1 14-20

Function: May be an antioxidant enzyme particularly in the developing shoot and photosynthesizing leaf. PTM: The Cys-64-SH group is the primary site of oxidation by H2O2, and the oxidized Cys-64 rapidly

reacts with Cys-185-SH of the other subunit to form an intermolecular disulfide. Beta-amylase 30, 48-50 Catalytic activity: Hydrolysis of 1,4-alpha-D-glucosidic linkages in polysaccharides.

Peroxidase precursor 114 Function: Removal of H2O2, oxidation of toxic reductants, biosynthesis and degradation of lignin, suberization, auxi.

Fructose-bisphosphate aldolase 7 GO:0004332; Molecular function: fructose-bisphosphate aldolase activity. UTP-glucose-1-phosphate

uridylyltransferase 85-88 Function: Plays a central role as a glucosyl donor in cellular metabolic pathways.

Peroxidase 4 Peroxidase 5

55 75

GO:0005506; Molecular function: iron ion binding. GO:0046872; Molecular function: metal ion binding. GO:0016491; Molecular function: oxidoreductase activity. GO:0004601; Molecular function: peroxidase activity. GO:0006979; Biological process: response to oxidative stress.

OSJNBb0003B01.27 protein 94, 95 GO:0004553; Molecular function: hydrolase activity. GO:0005975; Biological process: carbohydrate metabolism.

Protein putative laccase LAC5-4 117

GO:0005507; Molecular function: copper ion binding. GO:0046872; Molecular function: metal ion binding. GO:0016491; Molecular function: oxidoreductase activity.

Alcohol dehydrogenase I 106

GO:0046872; Molecular function: metal ion binding. GO:0016491; Molecular function: oxidoreductase activity. GO:0008270; Molecular function: zinc ion binding.

20S proteasome beta 4 subunit 107

GO:0005829; Cellular component: cytosol.

GO:0005839; Cellular component: proteasome core complex. GO:0043234; Cellular component: protein complex.

GO:0004298; Molecular function: threonine endopeptidase activity. GO:0006511; Biological process: ubiquitin-dependent protein catabolism.

Putative 3-beta hydroxysteroid

dehydrogenase 65, 66, 112

GO:0016853; Molecular function: isomerase activity. GO:0051287; Molecular function: NAD binding.

GO:0009225; Biological process: nucleotide-sugar metabolism.

Isoprene synthase 31 GO:0016829; Molecular function: lyase activity. GO:0008152; Biological process: metabolism.

(11)

Putative malate dehydrogenase 39, 41, 42

GO:0004459; Molecular function: L-lactate dehydrogenase activity. GO:0030060; Molecular function: L-malate dehydrogenase activity. GO:0016491; Molecular function: oxidoreductase activity. GO:0006096; Biological process: glycolysis.

GO:0006108; Biological process: malate metabolism. GO:0006099; Biological process: tricarboxylic acid cycle.

Alpha 2 subunit of 20S proteasome 21

GO:0005829; Cellular component: cytosol.

GO:0005839; Cellular component: proteasome core complex. GO:0043234; Cellular component: protein complex.

GO:0004298; Molecular function: threonine endopeptidase activity. GO:0006511; Biological process: ubiquitin-dependent protein catabolism.

Cyclophilin-like protein 13

GO:0016853; Molecular function: isomerase activity.

GO:0003755; Molecular function: peptidyl-prolyl cis-trans isomerase activity. GO:0006457; Biological process: protein folding.

Putative glyoxalase 67 GO:0004462; Molecular function: lactoylglutathione lyase activity. GO:0005975; Biological process: carbohydrate metabolism.

ADP-glucose pyrophosphorylase small subunit 22

GO:0016779; Molecular function: nucleotidyltransferase activity. GO:0016740; Molecular function: transferase activity.

GO:0009058; Biological process: biosynthesis. GO:0005978; Biological process: glycogen biosynthesis.

OSJNBa0042F21.13 protein 76, 77

GO:0016787; Molecular function: hydrolase activity.

GO:0042578; Molecular function: phosphoric ester hydrolase activity. GO:0005975; Biological process: carbohydrate metabolism.

Phosphoglycerate mutase

26-29, 45-47, 51-53, 92, 93

GO:0005737; Cellular component: cytoplasm.

GO:0030145; Molecular function: manganese ion binding. GO:0004619; Molecular function: phosphoglycerate mutase activity. GO:0006007; Biological process: glucose catabolism.

Dehydroascorbate reductase 64, 110 Ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase.

Cytosolic 3-phosphoglycerate

kinase 78-81, 96

GO:0004618; Molecular function: phosphoglycerate kinase activity. GO:0006096; Biological process: glycolysis.

Ferredoxin-NADP(H) oxidoreductase

34-36, 43, 68-73

GO:0042651; Cellular component: thylakoid membrane. GO:0050660; Molecular function: FAD binding.

GO:0004324; Molecular function: ferredoxin-NADP+ reductase activity. GO:0050661; Molecular function: NADP binding.

GO:0016491; Molecular function: oxidoreductase activity. GO:0006118; Biological process: electron transport.

Glutathione transferase 37 GO:0004364; Molecular function: glutathione transferase activity. GO:0016740; Molecular function: transferase activity.

Ribulose-5-phosphate-3-epimerase 61-63

GO:0016853; Molecular function: isomerase activity.

GO:0004750; Molecular function: ribulose-phosphate 3-epimerase activity. GO:0005975; Biological process: carbohydrate metabolism.

Alpha-L-arabinofuranosidase 44 GO:0004553; Molecular function: hydrolase activity. GO:0005975; Biological process: carbohydrate metabolism.

Cu/Zn superoxide dismutase 2

GO:0009507; Cellular component: chloroplast. GO:0005507; Molecular function: copper ion binding.

GO:0004785; Molecular function: copper, zinc superoxide dismutase activity. GO:0046872; Molecular function: metal ion binding.

GO:0016491; Molecular function: oxidoreductase activity. GO:0008270; Molecular function: zinc ion binding. GO:0006801; Biological process: superoxide metabolism.

(12)

Ribulose-1,5-bisphosphate

carboxylase 1

GO:0009573; Cellular component: ribulose bisphosphate carboxylase complex. GO:0016984; Molecular function: ribulose-bisphosphate carboxylase activity. GO:0015977; Biological process: carbon utilization by fixation of carbon dioxide.

Nucleoside diphosphate kinase 12

GO:0005524; Molecular function: ATP binding. GO:0016301; Molecular function: kinase activity. GO:0000287; Molecular function: magnesium ion binding.

GO:0004550; Molecular function: nucleoside diphosphate kinase activity. GO:0000166; Molecular function: nucleotide binding.

GO:0016740; Molecular function: transferase activity. GO:0006241; Biological process: CTP biosynthesis. GO:0006183; Biological process: GTP biosynthesis. GO:0006228; Biological process: UTP biosynthesis.

Putative Bplo 118-123 GO:0005507; Molecular function: copper ion binding. GO:0016491; Molecular function: oxidoreductase activity.

HSP70. 82-84

GO:0005524; Molecular function: ATP binding. GO:0006457; Biological process: protein folding.

GO:0006986; Biological process: response to unfolded protein.

Heat shock protein 70 24, 25

GO:0005524; Molecular function: ATP binding. GO:0006457; Biological process: protein folding.

GO:0006986; Biological process: response to unfolded protein.

Malate dehydrogenase 38, 40

GO:0004459; Molecular function: L-lactate dehydrogenase activity. GO:0030060; Molecular function: L-malate dehydrogenase activity. GO:0016491; Molecular function: oxidoreductase activity. GO:0006096; Biological process: glycolysis.

GO:0006108; Biological process: malate metabolism. GO:0006099; Biological process: tricarboxylic acid cycle.

Reversibly glycosylated

polypeptide 8-10

GO:0009505; Cellular component: cell wall. GO:0005794; Cellular component: Golgi apparatus.

GO:0047210; Molecular function: alpha-1,4-glucan-protein synthase activity. GO:0030244; Biological process: cellulose biosynthesis.

S222. 97-100 GO:0008889; Molecular function: glycerophosphodiester phosphodiesterase activity. GO:0006071; Biological process: glycerol metabolism.

Cellular Component and Biological Process of Identified

Proteins in Wheat Leaf

Of those identified wheat proteins, 15 identified proteins

were involved in cellular component of which 13 proteins

were intracellular proteins. All of these intracellular proteins

were in cytoplasm, while 2 wheat proteins including alpha 2

subunit of 20S proteasome and 20S proteasome beta 4

subunit were also found in the nucleus. Moreover, reversibly

glycosylated polypeptide and ferredoxin-NADP(H)

oxi-doreductase were the components of cell wall and

mem-brane, respectively. Dhugga et al. (1997) indicated that

re-versibly glycosylated polypeptide was possibly involved in

plant cell wall synthesis. Matthijs et al. (1986) reported that

the reduction of NADP

+

by ferredoxin:NADP

+

oxidoreduc-tase is the terminal step in the electron transport chain of the

thylakoid, and the point at which the reductant is delivered to

the stromal compartment. In addition, total of 35 wheat

pro-teins were grouped in biological process of which 5 propro-teins

were clustered in photosynthesis. These were identified as

ribulose-1,5-bisphosphate carboxylase, phosphoribulokinase,

ribulose bisphosphate carboxylase small chain clone 512,

fructose-1,6-bisphosphatase and triosephosphate isomerase.

Whitney et al. (2003) suggested that all plants depend on the

photosynthetic CO

2

-fixing enzyme (ribulose-1,5-biphosphote

carboxylase, Rubisco) to supply them with combined carbon.

Rubisco of tobacco with the dimeric version from the

bacte-rium, Rhodospirillum rubrum, resulted in fully autotrophic

and reproductive tobacco plants that required high CO

2

con-centrations to grow.

CONCLUSION

In this study, we used two-dimensional electrophoresis,

mass spectrometry and bioinformatic software to investigate

wheat leaf proteins’ composition and function. Compared

with previous publications (Bahrman et al., 2004; Donnelly,

et al. 2005), our results show that the proteins could be

salted out by ammonium sulfate and separated

electropho-retically in the 2D gels. A total of 123 wheat proteins were

(13)

putatively identified with an identification success rate of

61.5%. These wheat protein maps generated will also enable

future proteomic studies to focus on differential expression

by using the identified proteins as reference proteins. In

ad-dition, the annotations and classifications of the identified

proteins by bioinformatic software were also completed. It

shows lists of proteins for biological interpretation in the

context of Gene Ontology, which organizes information

ac-cording to their molecular function, biological processes and

cellular components. This information should be useful for

any future study on the wheat leaf and perhaps, other plants

in general.

ABBREVIATIONS

2-DE

=

Two-Dimensional gel electrophoresis

ACN =

Acetonitrile

AS =

Ammonium

sulfate

-Me =

-Mercaptoethanol

BGSS

=

Bulk Gene Search System

IPG

=

Immobilized pH gradient

MS =

Mass

spectrometry

SDS-PAGE =

Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis

TCA =

Trichloroacetic

acid

TFA =

Trifluoroacetic

acid

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