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Plant Science 193–194 (2012) 120–129

Contents lists available at SciVerse ScienceDirect

Plant Science

j our nal ho me p age: www.elsev ier .com/ locate /p lantsc i

omparative proteomic study between tuberous roots of light orange- andurple-fleshed sweetpotato cultivars

eung Joo Leea,1, Kee Woong Parkb,1, Youn-Sig Kwaka, Jae Young Ahna, Young Hak Jungc,yung-Hyun Leec, Jae Cheol Jeongd, Haeng-Soon Leed, Sang-Soo Kwakd,∗

Department of Applied Biology, IALS, Gyeongsang National University, Jinju 660-751, Republic of KoreaDepartment of Crop Science, Chungnam National University, Daejeon 305-764, Republic of KoreaDivision of Applied Life Science (BK21 Program), IALS, PMBBRC, Gyeongsang National University, Jinju 660-751, Republic of KoreaEnvironmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea

r t i c l e i n f o

rticle history:eceived 18 January 2012eceived in revised form 5 June 2012ccepted 6 June 2012vailable online 13 June 2012

eywords:omparative proteomicsight orange-fleshed cultivarurple-fleshed cultivarweetpotatouberous root

a b s t r a c t

This study compares the differences in proteomes expressed in tuberous roots of a light orange-fleshedsweetpotato (Ipomoea batatas (L.) Lam. cultivar Yulmi) and a purple-fleshed sweetpotato cultivar (Shin-jami). More than 370 protein spots were reproducibly detected by two-dimensional gel electrophoresis,in which 35 spots were up-regulated (Yulmi vs. Shinjami) or uniquely expressed (only Yulmi or Shin-jami) in either of the two cultivars. Of these 35 protein spots, 23 were expressed in Yulmi and 12 wereexpressed in Shinjami. These protein spots were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry. Fifteen proteinsin Yulmi and eight proteins in Shinjami were identified from the up-regulated (Yulmi vs. Shinjami) oruniquely expressed (only Yulmi or Shinjami) proteins, respectively. In Yulmi, �-amylase and isomeraseprecursor–like protein were uniquely expressed or up-regulated and activities of �-amylase, monode-hydroascorbate reductase, and dehydroascorbate reductase were higher than in Shinjami. In Shinjami,peroxidase precursor and aldo–keto reductase were uniquely expressed or up-regulated and peroxi-

dase and aldo–keto reductase activities were higher than in Yulmi. PSG-RGH7 uniquely expressed onlyin Shinjami and the cultivar was evaluated more resistant than Yulmi against the root-knot nematode,Meloidogyne incognita (Kofold and White, 1919) Chitwood 1949 on the basis of shoot and root growth. Eggmass formation was 14.9-fold less in Shinjami than in Yulmi. These results provide important clues thatcan provide a foundation for sweetpotato proteomics and lead to the characterization of the physiologicalfunction of differentially expressed proteins.

. Introduction

Of the crops that have underground storage organs, there aretem tubers and root tubers. The underground stem tuber suchs potato tuber is the developed, enlarged and thickened stolonshich acts as a storage organ. Root tubers such as sweetpotato,

assava, and yam are modified lateral root, enlarged to function as atorage organ. Sweetpotato (Ipomoea batatas (L.) Lam.) ranks as theeventh-most important food crop in the world [1]. The tuberousoots of sweetpotato are produced for the human consumption,

nimal feed, and industrial products. Its wide adaptability onarginal lands and rich nutritional content provide great potential

or preventing malnutrition and enhancing food security in devel-

∗ Corresponding author. Tel.: +82 42 860 4432; fax: +82 42 860 4608.E-mail address: sskwak@kribb.re.kr (S.-S. Kwak).

1 These authors contributed equally to this work.

168-9452/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.plantsci.2012.06.003

© 2012 Elsevier Ireland Ltd. All rights reserved.

oping countries [2]. Although it is an important food crop, the studyof sweetpotato genomics has not been actively conducted. Thelength of the genome is estimated to be approximately 3000 Mb.Currently, approximately 23,982 ESTs of sweetpotato are regis-tered in NCBI, whereas 261,791 ESTs are registered in the potatodatabase (www.ncbi.nlm.nih.gov, as of January 16, 2012). To date,no large-scale systematic studies have been undertaken to identifythe proteins present in sweetpotato. The lack of genomic and pro-teomic studies is probably because the chromosome is hexaploid(2n = 6× = 90), the size of genome is large, and sweetpotato hasbeen cultivated primarily in developing countries where oppor-tunities to fund such research may be scarce. Several previousresearches were conducted in storage tube crops with proteomics.In cassava, many fibrous and tuberous unique proteins were

revealed [3]. According to a recent potato proteomic study, nearly100 proteins in the tube have been identified and most of themwere related to tuber development and maturation [4]. However,large number of differently expressed proteins may have similar

dx.doi.org/10.1016/j.plantsci.2012.06.003http://www.sciencedirect.com/science/journal/01689452http://www.elsevier.com/locate/plantscimailto:sskwak@kribb.re.krhttp://www.ncbi.nlm.nih.gov/dx.doi.org/10.1016/j.plantsci.2012.06.003

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iological function, because some of the proteins were isoforms ofhe similar enzymes. [5,6]. Interestingly, comparative proteomicnalysis between genetically modified (GM) and non-GM lineotatoes, there is differentiation between the lines [7].

Proteomics, the systematic study of the proteome, allows quali-ative and quantitative measurements of a large number of proteinshat directly influence cellular biochemistry at the organelle, cell,rgan, and tissue levels, and thus can provide an accurate analysis ofhe cellular state or system changes during growth, development,nd response to environmental factors [8]. Proteomic analysis alsoffers molecular information for the classification of cultivars ineveral cereal crops such as rice, barley, and wheat [9].

The present work was conducted to identify differentlyxpressed proteins in the tuberous roots of two sweetpotato culti-ars by proteomic approaches. Two-dimensional electrophoresis2-DE) was applied for protein separation and expression anal-sis, and proteins were identified by matrix-assisted laseresorption/ionization-time of flight mass spectrometry (MALDI-OF MS) and electrospray ionization tandem mass spectrometryESI-MS/MS). The two sweetpotato cultivars that are used in thistudy are Yulmi and Shinjami; Yulmi has a light orange flesh colornd Shinjami has a purple flesh color. Orange-fleshed sweetpotatoultivars primarily contain carotenoids, whereas purple-fleshedweetpotato cultivars primarily contain anthocyanins [10,11].

This study characterized quantitative and qualitative differ-nces in protein expression between Yulmi and Shinjami, andetected 35 protein spots that were up-regulated (Yulmi vs. Shin-

ami) or uniquely expressed (only Yulmi or Shinjami). Of these5 spots, 18 were identified as proteins with unique physiolog-

cal roles. Several enzyme activities and their expression levelsere compared in the two cultivars. This study also investigated

he resistance of the two cultivars to the root-knot nematode,eloidogyne incognita (Kofold and White, 1919) Chitwood 1949,

o determine the role of up-regulated or uniquely expressed pro-eins in nematode resistance. To the best of our knowledge, this ishe first report on sweetpotato proteomics.

. Materials and methods

.1. Plant materials

Two sweetpotato cultivars, the light orange-fleshed Yulmi andhe purple-fleshed Shinjami, were grown in a randomized field plotccording to standard agricultural practices in 2009 at the Bioen-rgy Crop Research Center, National Crop Research Institute, RDA,epublic of Korea. The experiment was arranged in a randomizedomplete block design with four replicates and each plot has thirtylants, all materials were harvested for further analysis. The tubersith 5–6 cm in central diameter were selected and each replicateas three individual tubers. The tubers were peeled then sliced to

mm thickness with only parenchyma tissues. The sliced tubersere grounded to a fine powder with liquid nitrogen and stored at80 ◦C until further analysis.

.2. Protein extraction and 2-DE analysis

Total protein was isolated from tuberous roots of Yulmind Shinjami according to a modified phenol-based procedure12]. The frozen powder (approximately 10 g) was suspendedirectly in 20 ml of extraction buffer (0.7 M sucrose, 2% [v/v] 2-ercaptoethanol, and 1 mM PMSF, 0.5 M Tris–HCl, pH 8.3), and

omogenized by vigorous vortexing. The sample was incubated at◦C for 10 min, then an equal volume of Tris-saturated phenol (pH.3) was added, then the sample was homogenized by vigorous vor-exing. Total protein was precipitated from the phenol phase with

3–194 (2012) 120–129 121

methanol containing 0.1 M ammonium acetate and washed with80% acetone. The acetone-washed protein pellet was dried at roomtemperature and dissolved in lysis buffer (9 M urea, 2% CHAPS, 0.2%(w/v) Pharmalyte, pH 3–10, 50 mM DTT) immediately prior to iso-electric focusing (IEF) according to the manufacturer’s instructions(Bio-Rad). Protein concentration was measured according to theBradford procedure [13].

IEF was performed using Protein IEF Cell (Bio-Rad). Four hun-dred micrograms of total protein extract was loaded on Bio-Rad17 cm immobilized pH gradient (IPG) gel strips (pH 4–7) duringovernight strip rehydration. IEF conditions were as follows: 250 Vfor a conditioning step of 15 min, followed by a slow ramping stepto 10,000 V for 3 h, and finally 10,000 V for 9 h. After IEF, the IPGstrips were equilibrated according to the manufacturer’s protocol(Bio-Rad). Two-dimensional SDS-PAGE gels (13% total monomer,with 0.8% crosslinker) were electrophoresed using a PROTEAN IIxi Cell (Bio-Rad) at 60 V for 15 min, 80 V for 1 h, 100 V for 1 h, andthen at 120 V for 18 h at 20 ◦C. The 2-DE gels were stained withcolloidal Coomassie [14]. All 2-DE analyses were repeated 3 timesas replications and each replicate has three individual tubers. Gelimages were acquired using a GS-800 Calibrated Imaging Densito-meter (Bio-Rad) and analyzed with PDQuest software (Version 7.2;Bio-Rad, Hercules, CA, USA). The intensities of the up-regulated oruniquely expressed protein spots in the two cultivars were nor-malized to a relative intensity, and mean values calculated fromtriplicate data were compared. Only protein spots with a signifi-cant and reproducible increase over 1.5 times were considered to beup-regulated or uniquely expressed proteins, and the spots show-ing a statistical level of p < 0.05 by Student’s t test were selected foridentification.

2.3. MALDI-TOF MS and ESI-MS/MS analysis

The differentially up-regulated and/or uniquely expressed pro-tein spots were excised from the CBB-stained gels and subjectedto in-gel digestion with trypsin as described previously [15].The peptides were extracted twice with 5% trifluoroacetic acid(TFA) in 50% acetonitrile (ACN), lyophilized, dissolved in 0.1%TFA [16], and desalted with a Zip-Tip �-C18 (Millipore, Bed-ford, MA, USA), according to the manufacturer’s protocol. Analiquot of the formic acid containing the resulting peptideswas lyophilized again and then dissolved in a matrix solu-tion containing 50% ACN, 0.5% �-cyano-4-hydroxycinnamic acid(CHCA), and 0.1% TFA [17]. MS analysis was carried out with aVoyger-DE STR MALDI-TOF mass spectrometer (Applied Biosys-tems, Framingham, MA, USA), and spectra were obtained inthe reflection/delayed extraction mode [15]. Peptide peaks wereselected in the mass range from 800 to 3000 Da. The spectra wereinternally calibrated using angiotensin II (m/z 1046.5423), neu-rotensin (m/z 1672.9175), and adrenocorticotropic hormone (m/z2465.1989) (Sigma–Aldrich). Monoisotopic peptide masses wereanalyzed with MoverZ (http://www.proteomics). Peptide massfingerprints (PMFs) were searched in the NCBI database usingMASCOT (http://www.matrixscience.com) and Protein Prospec-tor (http://prospector.ucsf.edu). The following parameters wereused for database searches: mass tolerance of 50 ppm, one missedcleavage, oxidation of methionine, and alkylation of cysteine byiodoacetamide [18].

For proteins that were not identified by MALDI-TOF, tan-dem MS/MS analysis was performed on a QSTAR pulsar-iMS system (AB/MDS Sciex, Toronto, Canada) equipped with anano-electrospray ion source (MDS Protana, Odense, Denmark),

according to the method described previously [19]. BioAnalyst(Applied Biosystems) was used for data processing and inter-pretation. The resulting peptide sequences were submitted toProteinInfo (http://prowl.rockefeller.edu/prowl/proteininfo.html)

http://www.proteomics/http://www.matrixscience.com/http://prospector.ucsf.edu/http://prowl.rockefeller.edu/prowl/proteininfo.html

122 J.J. Lee et al. / Plant Science 193–194 (2012) 120–129

Fig. 1. High-resolution 2-DE of total proteins extracted from tuberous roots of sweetpotato in (A) the light-orange fleshed cultivar Yulmi and (B) the purple-fleshed cultivarS GE oni

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hinjami. The proteins were separated on IPG strips (pH 4–7), followed by SDS-PAndicate proteins that were up-regulated in Yulmi or Shinjami, respectively.

nd MASCOT to compare with the NCBI database for identifica-ion. The sequences were further queried with the NCBInr databasey BLAST homology and similarity searches. MS/MS spectra wereearched with peptide and fragment ion mass tolerances of 50 ppm.arbamidomethylation of cysteines and oxidation of methioninesere allowed during the search of the peptides, and one missing

rypsin cleavage was allowed [19].

.4. Enzyme assays

The frozen fine powder (approximately 2 g) homogenized withce-cold 50 mM potassium phosphate buffer (pH 7.0) containing

mM ethylenediaminetetraacetic acid (EDTA) and 10% insolubleolyvinylpolypyrrolidone (PVP) (w/w) for the determination ofuaiacol-type peroxidase (POD; EC 1.11.1.7), monodehydroascor-ate reductase (MDHAR; EC 1.6.5.4), and dehydroascorbateeductase (DHAR; EC 1.8.5.4) activity [20]. The homogenate wasentrifuged at 12,000 × g for 20 min at 4 ◦C, and the super-atant was used as crude enzyme for the determination of eachnzyme activity. Activity of guaiacol POD was assayed by mon-toring the increase in absorbance at 470 nm due to guaiacolxidation (E = 26.6 mM−1 cm−1) [21]. Activities of MDHAR andHAR were assayed by monitoring the decrease in absorbancet 340 nm due to NADPH oxidation (E = 6.2 mM−1 cm−1) andhe increase in absorbance at 290 nm due to ascorbate reduc-ion (E = 2.8 mM−1 cm−1), respectively [22]. Alpha-amylase (EC.2.1.1)was extracted from 2 g of the fine powder with 200 mMhosphate buffer (pH 6.9) containing 1% NaCl, and the activity wasssayed by following the increase in absorbance at 540 nm due tohe production of reducing sugar [23]. Aldo–keto reductase (AKR)as extracted from 2 g of the fine powder using an extraction buffer

ontaining 25 mM Tris–HCl (pH 7.4), and the activity was deter-ined by monitoring the decrease in absorbance at 340 nm due toADPH oxidation as described previously [24]. All enzyme assaysere done with four replicates.

.5. Examination of tolerance to root-knot nematode

Cuttings (approximately 10 cm) with three leaves of Yulmi andhinjami were transplanted in a plastic pot (24 cm width, 18 cm

13% polyacrylamide gels. Gels were stained with colloidal CBB. The spot numbers

length, and 12 cm height) filled with sandy loam infested withM. incognita at a density of 1154 ± 176 second-stage juveniles per300 g soil. For the control, the soil was steam-sterilized at 115 ◦Cand 1.5 atmospheric pressure for 20 min. The sweetpotato cuttingswere grown in a controlled-environment growth chamber (16 hphotoperiod, 30/22 ◦C day/night temperatures, and 70% relativehumidity) with a light intensity of 200 �mol s−1 m−2 provided byfluorescent and metal halide lamps. The plants were harvested for50 days after planting the cuttings, washed with tap water, andthen the fresh weight of the shoot and root were measured. Eggmass formed by M. incognita was dyed with Phloxin B solution andcounted [25]. The experiment was done in a completely random-ized design with 10 replicates. The data were subjected to analysisof variance (ANOVA), and means were separated using Duncan’smultiple range test (p < 0.05).

3. Results

3.1. 2-DE analysis of sweetpotato tuberous root proteomes

Total proteins extracted from the tuberous roots of Yulmi andShinjami were electrophoresed and compared by 2-DE. The repre-sentative 2-DE gels of high quality and reproducibility are shownin Fig. 1. More than 370 protein spots were reproducibly detectedacross three replicate gels from two sweetpotato cultivars, as deter-mined by visual inspection. The pIs of the spots ranged from pH 4to 7, and the molecular masses of most of the spots ranged from 10to 70 kDa. To investigate protein expression patterns in the twocultivars, protein spots of all replicate gels were compared andquantified using PDQuest software. Although the general patternof tuberous root proteins remained largely unchanged in the twosweetpotato cultivars, total of 35 protein spots showed significantand reproducible up-regulation (>1.5 times) or unique expressionin either Yulmi or Shinjami (Fig. 2). Of these 35 protein spots, twelve(1, 5, 7, 19, 21, 24–26, 28, 31, 32, and 34) were expressed onlyin Yulmi, whereas eight (8, 10, 11, 16, 17, 23, 27, and 35) were

expressed only in Shinjami. Eleven spots (2, 3, 6, 9, 12, 13, 15, 20,22, 30, and 33) were up-regulated in Yulmi, and four spots (4, 14, 18,and 29) were up-regulated in Shinjami. The 35 spots were excisedfrom the CBB-stained gels and subjected to further analysis.

J.J. Lee et al. / Plant Science 193–194 (2012) 120–129 123

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Fig. 2. Enlarged views of 2-DE maps of up-regulated or uniquely expre

.2. Identification of the up-regulated or uniquely expressedroteins

The excised protein spots were digested with trypsin, andhe peptides were extracted and analyzed by PMF using MALDI-OF MS, or by sequence tag using ESI-MS/MS. Twelve spots (1,, 5, 9, 11, 22–24, 27, 30, 33, and 34) out of the 35 spotsere unidentified, whereas 23 were identified (Table 1 and

upplemental Fig 1). Eleven proteins were identified in Yulmi,ncluding catechol oxidase (COD; EC 1.10.3.1, spot 2), putativexalyl-CoA decarboxylase (EC 4.1.1.8, spot 3), �-amylase (spot 7),emialdehyde dehydrogenase family protein (spots 12 and 13),isulfide-isomerase (EC 5.3.4.1) precursor-like protein (spot 15),nionic POD swpa2 (spot 20), putative beta-1,3-glucanase (EC.2.1.39) precursor (spot 21), cysteine proteinase inhibitor (spot5), amino acid transporter (spot 26), sporamin A precursor (cloneIM 0335) (spot 28), and sporamin B (spot 32). Five proteinsere identified in Shinjami, including resistance protein PSH-RGH7

spot 8), POD precursor (spot 10), AKR (spot 14), flavonone 3-ydroxylase (F3H; EC 1.14.11.9, spot 16), and sporamin B precursor

spots 29 and 35). Five of the identified proteins were anno-ated either as unknown proteins (spots 6, 17, 18, and 31) or asn unnamed protein product (spot 19) without a specific func-ion.

proteins marked in Fig. 1. (A) cultivar Yulmi and (B) cultivar Shinjami.

3.3. Activities of several up-regulated or uniquely expressedenzyme proteins

Activities of several enzyme proteins that were up-regulated inYulmi and Shinjami were investigated. �-Amylase was expressedonly in Yulmi (spot 7 in Fig. 2), and the enzyme activity was 1.7times higher in Yulmi than in Shinjami (Fig. 3A). POD precursorwas expressed only in Shinjami, and anionic POD swpa2 was up-regulated in Yulmi compared to Shinjami (spots 10 and 20 in Fig. 2).The activity of guaiacol-type POD in Shinjami was 11.9 times higherthan in Yulmi (Fig. 3B). AKR was up-regulated in Shinjami (spot 14in Fig. 2), the enzyme activity was 2.7 times higher in Shinjami thanin Yulmi (Fig. 3C). Activities of MDHAR and DHAR in Yulmi, in whichdisulfide-isomerase precursor-like protein was up-regulated (spot15 in Fig. 2), were 2.2 and 5.1 times higher, respectively, than thosein Shinjami (Fig. 3D and E).

3.4. Cultivar-specific tolerance to root-knot nematode

PSH-RGH7 has a nematode resistance function and it is

expressed in Shinjami only. Therefore resistance to M. incognitawas compared between Shinjami and Yulmi. When the plants weregrown in soils infested with M. incognita, shoot and root growth ofShinjami was inhibited by 14.3% and 18.2%, respectively, whereas

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Table 1Analysis of up-regulated or uniquely expressed proteins in the tuberous roots of the sweetpotato cultivars Yulmi and Shinjami by MALDI-TOF MS and ESI-MS/MS.

Spot no. MALDI-TOF ESI-MS/MS Protein name Organism Accession no. Varietyd Abundance ratioe Theoretical Observed

S.C. (%)a Sequenceb Identity(%)c Mr f pIg Mr f pIg

1 Unidentified Y 2.33 ± 0.29 71.0 6.42 23 Catechol oxidase I Ipomoea batatas Q9ZP19 Y 1.57 ± 0.17 55.0 5.8 64.1 5.93 LVGTPDEL 100 Putative oxalyl-CoA decarboxylase Vitis vinifera AAP69814 Y 2.58 ± 0.65 17.3 5.2 62.0 6.24 Unidentified S 2.28 ± 0.25 55.7 5.75 Unidentified Y 2.15 ± 0.10 48.2 4.86 (K)GVGTLL 100 Unknown protein Zea mays ACF78604 Y 3.08 ± 0.25 83.2 9.6 45.0 6.27 LVAALHD 100 �-Amylase Ipomoea nil BAC02435 Y 2.33 ± 0.29 47.1 4.9 44.0 5.88 LDHLNP 100 Resistance protein PSH-RGH7 Solanum tuberosum ABY61746 S 2.17 ± 0.29 107.1 5.4 44.0 5.99 Unidentified Y 2.23 ± 0.10 43.0 5.9

10 FYSST 100 Peroxidase precursor Solanum tuberosum CAA64413 S 3.14 ± 0.75 35.9 6.9 42.0 4.711 Unidentified S 2.50 ± 0.43 41.0 4.812 LVQQT 100 Semialdehyde dehydrogenase family protein Arabidopsis thaliana NP 172934 Y 2.83 ± 0.49 40.7 6.5 40.0 5.613 LVQQT 100 Semialdehyde dehydrogenase family protein Arabidopsis thaliana NP 172934 Y 2.46 ± 0.29 40.7 6.5 40.0 5.714 GLSEASAATL 100 Aldo/keto reductase Medicago truncatula ABN08782 S 3.83 ± 0.15 30.7 6.1 38.0 6.215 VLTEENFE 100 Disulfide-isomerase precursor-like protein Solanum tuberosum ABB02620 Y 2.00 ± 0.03 39.4 5.6 38.0 6.016 13 Fravanone 3-hydroxyrase Ipomoea batatas BAA75307 S 2.03 ± 0.29 41.2 5.8 36.0 6.317 NYANAG – Unknown protein – – S 1.58 ± 0.14 35.0 4.718 NYANAG – Unknown protein – – S 1.78 ± 0.03 34.3 4.719 SFSSEDLH 100 Unnamed protein product Vitis vinifera CAO45840 Y 2.05 ± 0.12 36.4 5.9 34.3 5.420 SDQQLM 100 Anionic peroxidase swpa2 Ipomoea batatas AAF00093 Y 1.83 ± 0.29 38.5 4.7 34.4 6.721 DADPNVLNAL 90 Putative beta-1,3-glucanase precursor Oryza sativa BAC80125 Y 8.33 ± 2.02 53.8 5.7 32.2 5.422 Unidentified Y 2.32 ± 0.11 31.5 5.723 Unidentified S 1.93 ± 0.29 30.8 5.224 Unidentified Y 2.24 ± 0.10 30.8 4.825 38 Cysteine proteinase inhibitor Ipomoea batatas AAD13812 Y 2.07 ± 0.17 28.6 5.8 26.4 6.126 ELENGDVQ 100 Amino acid transporter Solanum tuberosum CAA70968 Y 1.58 ± 0.14 51.6 8.3 26.4 6.427 Unidentified S 17.33 ± 1.33 25.0 4.528 PVLDLNGD 100 Sporamin A precursor (Clone PIM0335) Ipomoea batatas P14715 Y 6.00 ± 0.40 24.0 5.8 24.0 5.529 ASDVLVS 100 Sporamin B precursor Ipomoea batatas ABB90968 S 6.33 ± 0.58 23.9 5.4 18.4 5.330 Unidentified Y 2.41 ± 0.15 18.0 5.631 LTVTVP, SDVLVS – Unknown protein – – Y 2.27 ± 0.33 18.0 5.932 38 Sporamin B Ipomoea batatas P10965 Y 2.30 ± 0.19 23.9 5.4 17.2 4.533 Unidentified Y 4.46 ± 0.22 16.4 4.834 Unidentified Y 1.67 ± 0.29 17.3 5.935 25 Sporamin B precursor Ipomoea batatas ABB90968 S 2.42 ± 0.20 23.9 5.4 15.8 5.2a Sequence coverage by PMF.b The sequence of the matching peptide.c Percentage of identities for sequence tags.d Y, Yulmi; S, Shinjami.e The abundance ratios of each spot were measured using a densitometer (Bio-Rad) and then compared to Yulmi and Shinjami.f Molecular weight.g Isoelectric point.

J.J. Lee et al. / Plant Science 193–194 (2012) 120–129 125

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ig. 3. Enzyme activities of several up-regulated or uniquely expressed proteins eroxidase, (C) aldo–keto reductase, (D) monodehydroascorbate reductase, and (E)

hoot and root growth of Yulmi was inhibited by 76.9 and 64.0%,espectively (Fig. 4A–C). Egg mass formed by M. incognita waspproximately 15 times greater in Yulmi than in Shinjami (Fig. 5And B).

. Discussion

Although sweetpotato is the seventh-most important staplerop in the world, its proteome has not been studied. In this work,e conducted a comparative proteomic study in the tuberous roots

f two sweetpotato cultivars, the light orange-fleshed Yulmi andhe purple-fleshed Shinjami. Using phenol extraction of total pro-eins, approximately 370 protein spots were visualized on 2-DE gelsith colloidal CBB staining (Fig. 1). The protein spots expressed inulmi and Shinjami were compared by careful analysis, and 35 pro-ein spots were found to be up-regulated or uniquely expressed inhe two cultivars (Fig. 2). The spots were analyzed by MALDI-TOF

S and ESI-MS/MS, and 18 were identified as proteins with specificunctions (Table 1). Interestingly enough, all spots showed highlyimilarity with a single peptide. Based on their putative physiolog-cal functions, the potential roles of the identified proteins werenvestigated.

In Yulmi, the up-regulated protein spots (2, 3, 12, 13, 15, and0) and the uniquely expressed protein spots (7, 21, 25, 26, 28,nd 32) were identified as proteins with known physiologicaloles. For example, spot 2 was identified as a COX, which is a

biquitous plant enzyme that catalyzes the formation of quinonesrom phenols [26]. These o-quinones are so highly reactive thathey auto-polymerize to polyphenolic catechol melanins thatre believed to protect plants from pathogens or insects [27].

ed from tuberous roots of Yulmi and Shinjami. (A) �-Amylase, (B) guaiacol-typeroascorbate reductase. Data represent means ± SE of four replicates.

The oxidation of plant phenolic substrates by COX also causesenzymatic browning of many fruits, vegetables, and food crops[28]. Spot 3 was identified as a putative oxalyl-CoA decarboxylase,which catalyzes the irreversible decarboxylation of oxalyl-CoA inthe formation of formyl-CoA from glyoxylic acid [29]. This enzymeis reported to play a major role in the catabolism of highly toxiccompounds in animals, as it induces the formation of calciumoxalate stones in the kidney (urolithiasis), renal failure, car-diomyopathy, and cardiac conductance disorders [30]. Spot 7 wasidentified as an �-amylase, which is responsible for the hydrolysisof �-1,4-glucan bonds as the major starch hydrolase in plants [31].Spots 12 and 13 were identified as semialdehyde dehydrogenasefamily proteins. The two spots have same Mr, but different pI,it may be due to post-translational modification of the protein.The proteins, asparte semialdehyde dehydrogenase (EC 1.2.1.11)catalyses the formation of l-aspartate-�-semialdehyde at the firstbranch point from l-aspartic acid in the biosynthetic pathwayof the essential amino acids lysine, isoleucine, methionine, andthreonine in bacteria, fungi, and the higher plants [32,33]. Spot15 was matched to disulfide-isomerase precursor-like protein.Disulfide-isomerase family proteins are thought to have key rolesin the endoplasmic reticulum in the folding of nascent polypeptidesand the formation of disulfide bonds [34]. Spot 20 was identified asan anionic POD swpa2. Most higher plants have a number of PODisoenzymes, which are commonly divided into anionic, neutral,and cationic groups, based on their isoelectric points [35]. Ten

secretory class III plant POD cDNAs, including anionic POD swpa2cDNA, were isolated from cell cultures of sweetpotato [36]. Amongthe POD genes, swpa2 was highly expressed in response to variousenvironmental stresses such as wounding, chilling, stress-related

126 J.J. Lee et al. / Plant Science 193–194 (2012) 120–129

Fig. 4. Effects of the root-knot nematode, M. incognita, on the growth of Yulmi and Shinjami. (A) Growth inhibition of Yulmi and Shinjami by M. incognita. The plants werep nd nop were n

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hotographed 50 days after planting cuttings into steam-sterilized sand (control) aer 300 g soil. (B) Shoot fresh weight. (C) Root fresh weight. The fresh weights (g)on-sterilized sand (treatment). Data represent means ± SD of 10 replicates.

hemicals, and pathogen infection [35–37]. Spot 21 was identifieds a putative beta-1,3-glucanase precursor. Beta-1,3-glucanases inhe fungal cell wall catalyze the endo-type hydrolytic cleavage of,3-beta-d-glucosidic linkages in beta-1,3-glucans [38]. In plants,hese enzymes are thought to be involved in both constitutive andnduced defense mechanisms against pathogenic fungi [39]. Spot5 was identified as a cysteine proteinase inhibitor (phytocystatin).

n many plant species, phytocystatins are proposed to protect plantells from inappropriate proteolysis, or could be involved in theontrol mechanism responsible for the breakdown of proteins40,41]. Spot 26 was identified as an amino acid transporter,hich is an integral membrane protein that catalyzes H+-coupled

mino acid uptake, playing a major role in the transportation ofmino acids synthesized in the plant leaves to other parts suchs apices, newly developing tissues, and reproductive organs42,43]. In potato tubers, reduced expression of this specific

n-sterilized sand (treatment) with 1154 ± 176 M. incognita second-stage juvenilesmeasured 50 days after planting cuttings into steam-sterilized sand (control) and

amino acid transporter gene leads to a reduction of amino acidcontent [44].

In Shinjami, the up-regulated protein spots (14 and 29) andthe uniquely expressed spots (8, 10, 16, and 35) were identifiedas proteins with known physiological roles. Spot 8 was identi-fied as a resistance protein PSH-RGH 7. Plants have evolved aninnate surveillance system consisting of a large set of resistance(R) genes to defend against a wide range of pathogens, insects andnematodes [45–47]. PSH-RGH7 is one of nine R gene homologues(RGHs) identified in two diploid potato clones (SH and RH) [45]. Todate, no R genes have been cloned in sweetpotato. Spot 10 wasidentified as a POD precursor. Plant POD is a secretory class III

enzyme that has been implicated in a broad range of physiologi-cal processes such as lignification, suberization, auxin metabolism,cross-linking of cell wall compounds, and defense against biotic andabiotic stresses [48]. Spot 14 was identified as a AKR. In various

J.J. Lee et al. / Plant Science 193–194 (2012) 120–129 127

Fig. 5. Cultivar-specific differences of egg masses formed by M. incognita on the roots of Yulmi and Shinjami. (A) Egg masses formed by M. incognita. Egg masses werephotographed 50 days after planting cuttings in non-sterilized sand with M. incognita. (B) The number of egg masses formed by M. incognita. Egg masses were counted 50d re nom

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ays after planting cuttings in non-sterilized sand with M. incognita. Egg masses weeans ± SD of ten replicates.

rganisms, AKRs metabolize a wide range of substrates includ-ng aliphatic aldehydes, monosaccharides, steroids, prostaglandins,olycyclic aromatic hydrocarbons, isoflavonoid phytoalexins, andenobiotics [49]. In plants, aldose/aldehyde reductase, a member ofKRs, increases tolerance to abiotic stresses such as paraquat, heavyetals, and drought [50]. Chalcone reductase, one of the AKR super-

amily, plays key roles in the de novo synthesis of phytoalexins,hich have antimicrobial activity [51]. Spot 16 was identified as a

3H. This is the enzyme which catalyzes the hydroxylation of narin-enin to form dihydrokaempferol, the common precursor for threeajor classes of 3-hydroxy flavonoids, flavonols, anthocyanins, and

roanthocyanidins [52]. A wide variety of flavonoid compoundsncluding anthocyanins play important roles in defense against var-ous biotic and abiotic stressors, in addition to the coloration ofowers, fruits, and underground organs [53,54]. Anthocyanins arebundant in sweetpotato cultivars with a purple flesh color, but arecarce in cultivars with a white, creamy orange, or yellow flesh color11,55]. These results suggest that uniquely expressed F3H in Shin-ami may critically involve in purple root pigment accumulation inhe tubers.

Polypeptides consisting of sporamin were differentially up-egulated or uniquely expressed in both of the sweetpotatoultivars. In Yulmi, the uniquely expressed spots 28 and 32 weredentified as sporamin A precursor and sporamin B, respectively. Inhinjami, the up-regulated spot 29 and the uniquely expressed spot5 were identified as sporamin B precursors. Sporamin consists ofolypeptides that are encoded by a multigene family composedf two major subfamilies, sporamins A and B [56]. In sweetpotatoubers, up to 80% of soluble proteins are stored in the form of spo-amins [57]. Sporamin also strongly inhibits trypsin activity andunctions as an endogenous insecticide in pest resistance [58–60].urrounding the spots 28, 32, 29 and 35, there were several spotsxisted. The spots showed similar Mr and pI value with sporamin.owever the spots were not identified by either MALDI-MS orSI-MS/MS. The proteins may be isoforms of sporamin or similarroteins. Many other sporamin in both cultivars may express with

imilar expression level. In this study, we only identified differentlyxpressed and uniquely expressed proteins. Therefore, it is diffi-ult to conclude that relationship between cultivar and sporaminxpression level.

t formed in the sweetpotato cultivars cultured in the sterilized soil. Data represent

In this study, it was assumed that the up-regulation or uniqueexpression of the proteins was related to an increase in enzymeactivities. The results show that the increased activities of �-amylase, GP, and AKR correlated with the up-regulation or uniqueexpression in Yulmi or Shinjami (Fig. 3A–C). In addition to addingdisulfide bonds to target proteins, disulfide-isomerase is knownto have a similar activity as the antioxidant enzymes DHAR andMDHAR [61]. Disulfide-isomerase precursor-like protein (spot 15)was up-regulated in Yulmi compared to Shinjami (Fig. 2), and theactivities of MDHAR and DHAR were also higher in Yulmi than inShinjami (Fig. 3D and E). These results suggest that the increase inprotein expression detected with 2-DE is closely correlated withthe increase in enzyme activities.

Plants have a large set of resistance genes to defend against awide range of pathogens and pests including nematodes [45–47].This study showed that resistance protein PSH-RGH 7 (spot 8) wasuniquely expressed in Shinjami (Fig. 2). Growth inhibition and eggmass formation by infestation of M. incognita were much lower inShinjami than in Yulmi (Figs. 4 and 5). This result suggests that theexpression of resistance protein PSH-RGH 7 may have some rolesin root-knot nematode resistance in sweetpotato. Currently, theresearch is progress to isolate the genes for PSH-RGH 7, with a long-term goal of molecular breeding for the enhanced performance ofsweetpotato.

Up to now, there is no previous proteomic study in sweet-potato. The entire genome of sweetpotato also has not beensequenced and number of EST is limited. Therefore, identificationof proteins through proteomic analysis also has some limitation.In this comparative proteomic analysis, of the 370 total proteinspots, 23 protein spots in light orange-fleshed sweetpotato cultivarYulmi and 12 protein spots in purple-fleshed sweetpotato cultivarShinjami were significantly up-regulated or uniquely expressed,respectively. Of the differently expressed spots, 12 spots in Yulmiand 6 spots in Shinjami were identified with known physiolog-ical functions. These identified proteins could become primaryinformation about protein expression and variability between the

cultivars of sweetpotato. Although transcriptome provides a deepcoverage of gene expression, proteins expression level not sig-nificantly correlated with the level of their transcripts [62,63]. Inmost case the proteins are final product of the genes and have

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28 J.J. Lee et al. / Plant Scien

iological functions. High-throughput RNA sequencing resultsevealed that biotic and abiotic stresses resistance genes includ-ng nematode resistance-like protein and PSH-RGH7 in onlyurple sweetpotato [64]. The result suggested that the purpleweetpotato may have tolerant to various stresses. In addition,everal chalcone isomerase genes and flavonoid-3-hydroxylaseenes that play important roles in synthesizing substrates ofnthocyanin pigments, were only detected in the purple sweetotato or the expression of the genes was significantly higher

n the purple sweet potato than that in the yellow sweetpotato64]. In this study, Shinjami (purple-fleshed) uniquely expressedSH-RGH 7 protein and showed much more tolerance to theoot-knot nematode. Also F3H, another key enzyme participat-ng in the pathway of anthocyanin biosynthesis, was uniquelyxpressed in the purple sweetpotato. Thus the combined vali-ated proteomic and transcriptomic approaches will provide moreeliable informative data about the purple fleshed tuber and itshysiology.

. Conclusions

To our knowledge, this is the first proteomic study in sweet-otato tuberous root. In this study, the differences in proteomesxpressed in tuberous roots of the light orange-fleshed cultivarulmi and the purple-fleshed cultivar Shinjami were examined by-DE analysis. Thirty-five proteins that were up-regulated and/orniquely expressed were identified, and 23 of them were charac-erized by MALDI-TOF MS and tandem ESI-MS/MS analysis. Theseroteins were found to be involved in several metabolic processeshat exhibited distinct properties in each sweetpotato cultivar.n both cultivars, stress-related proteins were up-regulated orniquely expressed, including COX, resistance protein PSH-RGH, POX precursor, AKR, disulfide-isomerase precursor-like protein,3H, anionic POX swpa2, putative beta-1,3-glucanase precursor,ysteine proteinase inhibitor, and sporamins. The increased expres-ion of these proteins could confer resistance to biotic and/orbiotic stresses in sweetpotato tuberous roots. The results indi-ated that an increase in enzyme activities correlated to thep-regulation or unique expression of proteins in the two culti-ars. The resistance protein PSH-RGH 7 was uniquely expressedn Shinjami, which was more resistant to M. incognita than Yulmi.urther studies on unidentified or unknown proteins will provideore information for comparative proteomics and the analysis of

ultivar-specific differences in sweetpotato. Because the genomeequence database for sweetpotato has not been constructedet, the identification of proteins by proteomics in sweetpotatoequires complex analysis. This study provides new insights intohe differences and functions of proteins expressed in sweetpotatoultivars and a starting point for further studies aimed at producingore vigorous sweetpotato varieties using molecular or classical

reeding.

cknowledgments

This research was partially supported by grants from theext-Generation BioGreen 21 Program (SSAC, grant nr. PJ008119),

he Rural Development Administration of the Republic of Korea,he KRIBB Initiative Program, and sabbatical research project

011–2012 from Gyeongsang National University. We are gratefulo Dr. Mi-Nam Chung, Bioenergy Crop Research Center, Nationalnstitute of Crop Science, RDA for the supply of sweetpotato mate-ials.

[

3–194 (2012) 120–129

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2012.06.003.

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http://dx.doi.org/10.1007/s00425-012-1591-4

Comparative proteomic study between tuberous roots of light orange- and purple-fleshed sweetpotato cultivars1 Introduction2 Materials and methods2.1 Plant materials2.2 Protein extraction and 2-DE analysis2.3 MALDI-TOF MS and ESI-MS/MS analysis2.4 Enzyme assays2.5 Examination of tolerance to root-knot nematode

3 Results3.1 2-DE analysis of sweetpotato tuberous root proteomes3.2 Identification of the up-regulated or uniquely expressed proteins3.3 Activities of several up-regulated or uniquely expressed enzyme proteins3.4 Cultivar-specific tolerance to root-knot nematode

4 Discussion5 ConclusionsAcknowledgmentsAppendix A Supplementary dataAppendix A Supplementary data