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Proteins associated with cork formation in Quercus suber L. stem tissues Cândido P.P. Ricardo a, b, , Isabel Martins a, 1 , Rita Francisco a, 1 , Kjell Sergeant c , 1 , Carla Pinheiro a , Alexandre Campos a , Jenny Renaut c , Pedro Fevereiro a, d a Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157 Oeiras, Portugal b Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal c Centre de Recherche Public-Gabriel Lippmann, Department Environment and Agrobiotechnologies, 41, rue du Brill, L-4422 Belvaux, Luxembourg d Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal ARTICLE INFO ABSTRACT Available online 12 February 2011 Cork (phellem) formation in Quercus suber stem was studied by proteomic analysis of young shoots of increasing age (Y0, Y1 and Y4) and recently-formed phellem (Y8Ph) and xylem (Y8X) from an 8-year-old branch. In this study 99 proteins were identified, 45 excised from Y8X and 54 from Y8Ph. These ones, specifically associated with phellem, are of carbohydrate metabolism(28%), defence(22%), protein folding, stability and degradation(19%), regulation/ signalling(11%), secondary metabolism(9%), energy metabolism(6%), and membrane transport(2%). The identification in phellem of galactosidases, xylosidases, apiose/xylose synthase, laccases and diphenol oxidases suggests intense cell wall reorganization, possibly with participation of hemicellulose/pectin biosynthesis and phenol oxidation. The identification of proteasome subunits, heat shock proteins, cyclophylin, subtilisin-like proteases, 14-3-3 proteins, Rab2 protein and enzymes interacting with nucleosides/nucleic acids gives additional evidence for cellular reorganization, involving cellular secretion, protein turnover regulation and active control processes. The high involvement in phellem of defence proteins (thioredoxin-dependent peroxidase, glutathione-S-transferase, SGT1 protein, cystatin, and chitinases) suggests a strong need for cell protection from the intense stressful events occurring in active phellem, namely, desiccation, pests/disease protection, detoxification and cell death. Identically, highly enhanced defence functions were previously reported for potato periderm formation. © 2011 Elsevier B.V. All rights reserved. Keywords: Cork Phellem formation Suberisation Proteomics Quercus suber stem 1. Introduction Quercus suber (cork oak), an evergreen tree of South-Western Europe (mostly Portugal and Spain) and North Africa (Morocco to Tunisia), is well adapted to the Mediterranean climate [1]. In this region it is an important and often dominant forest species that covers an estimated area above 2 million ha [2]. The cork oak stands are generally managed as agro- forestry systems (the Portuguese montadoand the Spanish dehesa) of high ecological, economic and social importance, JOURNAL OF PROTEOMICS 74 (2011) 1266 1278 Corresponding author at: ITQB, UNL, Av. da República, EAN, 2780-157 Oeiras, Portugal. Fax: +351 214433644. E-mail address: [email protected] (C.P.P. Ricardo). 1 These authors contributed equally to this paper. 1874-3919/$ see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.02.003 available at www.sciencedirect.com www.elsevier.com/locate/jprot

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Proteins associated with cork formation in Quercus suber L.

stem tissues

Cândido P.P. Ricardoa,b,⁎, Isabel Martinsa,1, Rita Franciscoa,1, Kjell Sergeantc,1,Carla Pinheiroa, Alexandre Camposa, Jenny Renautc, Pedro Fevereiroa,d

aInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157 Oeiras, PortugalbInstituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, PortugalcCentre de Recherche Public-Gabriel Lippmann, Department Environment and Agrobiotechnologies, 41, rue du Brill,

L-4422 Belvaux, LuxembourgdFaculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal

A R T I C L E I N F O A B S T R A C T

Available online 12 February 2011 Cork (phellem) formation in Quercus suber stem was studied by proteomic analysis of young

shoots of increasing age (Y0, Y1 and Y4) and recently-formed phellem (Y8Ph) and xylem (Y8X)

froman8-year-oldbranch. In this study99proteinswere identified, 45excised fromY8Xand54

fromY8Ph.Theseones, specificallyassociatedwithphellem,areof “carbohydratemetabolism”

(28%), “defence” (22%), “protein folding, stability and degradation” (19%), “regulation/

signalling” (11%), “secondary metabolism” (9%), “energy metabolism” (6%), and “membrane

transport” (2%).

The identification in phellem of galactosidases, xylosidases, apiose/xylose synthase,

laccases and diphenol oxidases suggests intense cell wall reorganization, possibly with

participation of hemicellulose/pectin biosynthesis and phenol oxidation. The identification

of proteasome subunits, heat shock proteins, cyclophylin, subtilisin-like proteases, 14-3-3

proteins, Rab2 protein and enzymes interacting with nucleosides/nucleic acids gives

additional evidence for cellular reorganization, involving cellular secretion, protein turnover

regulation and active control processes.

The high involvement in phellem of defence proteins (thioredoxin-dependent peroxidase,

glutathione-S-transferase, SGT1 protein, cystatin, and chitinases) suggests a strong need for

cell protection from the intense stressful events occurring in active phellem, namely,

desiccation, pests/disease protection, detoxification and cell death. Identically, highly

enhanced defence functions were previously reported for potato periderm formation.

© 2011 Elsevier B.V. All rights reserved.

Keywords:

Cork

Phellem formation

Suberisation

Proteomics

Quercus suber stem

1. Introduction

Quercus suber (cork oak), an evergreen tree of South-WesternEurope (mostly Portugal and Spain) and North Africa (Moroccoto Tunisia), is well adapted to theMediterranean climate [1]. In

this region it is an important and often dominant forestspecies that covers an estimated area above 2 million ha [2].

The cork oak stands are generally managed as agro-forestry systems (the Portuguese “montado” and the Spanish“dehesa”) of high ecological, economic and social importance,

J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 6 6 – 1 2 7 8

⁎ Corresponding author at: ITQB, UNL, Av. da República, EAN, 2780-157 Oeiras, Portugal. Fax: +351 214433644.E-mail address: [email protected] (C.P.P. Ricardo).

1 These authors contributed equally to this paper.

1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.jprot.2011.02.003

ava i l ab l e a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te / j p ro t

currently oriented towards the production of cork, a materialof high commercial value [3]. Cork (or phellem), the product ofa specialized cambium (the phellogen), protects the tree fromadverse environmental conditions (such as fire) and self-regenerates after damaging or peeling-off, without apparentinjury to the tree. The agricultural removal of cork is initiatedin 25–30 years-old trees and continues at periodic intervals(usually of 9-years), as a sustainable exploitation that the treecanwithstand formany decades [4]. Preserving and improvingthe quality of cork is of great economical relevance and manystudies have focused on cork oak in vitro regeneration in aneffort to overcome the difficulties of vegetative propagation ofselectedmature trees that producehigh-quality cork (e.g., [9–11]).However, due to lack of biochemical and genetic information it isnot possible, at present, to predict corkquality at anearly stageoftree development.

Cork owes its unique features to a highly complexcomposition [5–7]. Suberin, the main component (40–58%)that greatly determines cork physical properties, is a hetero-geneous polymer generally accepted to have two distinctdomains: a polyaliphatic one (formed by very long chain offatty acids and alcohols, hydroxyacids and diacids) and alignin-like polyphenolic one (formed by hydroxycinnamicacids derivatives) cross-linked to each other through theesterification of glycerol [8]. However, the other majorcomponents of cork, polysaccharides (cellulose and hemi-celluloses), lignin, extractives (mainly waxes, isoprenoids, andtannins) and minerals, also greatly influence cork character-istics and quality.

Given the complexity of Q. suber cork, few studies haveaddressed the metabolic pathways involved in its formation[12]. A simpler system, the potato tuber, has, however,received more attention and been used as a model to studysuberization [13–16]. Wounding of the potato tuber results inthe rapid formation of uniform suberin layers and, therefore, itwas possible to identify a few genes/proteins related tosuberin biosynthesis and deposition [15,16]. In Q. suber therehas been a notorious lack of data on these mechanisms, but,recently, Soler et al. [17] used molecular biology tools toapproach suberin biosynthesis in Q. suber, identifying genespotentially involved in cork formation.

Proteins are the agents that ultimately determine metab-olism and, consequently, proteomics has become a veryimportant field of research. The powerful tools of proteomics,permitting to analyze complex mixtures of proteins, allow thestudy of differences in protein abundance between distinctmetabolic states, thereby providing a global view of theimportant functions required to perform the biosyntheticactivities linked to a specific tissue [18]. Proteomic techniqueshave already been successfully applied in the study of cellularprocesses of woody tissues [19–21]. However, there is a lack ofproteomic andbiochemical studies on cork oak and, so, it is notsurprising that very few proteins from this species arerepresented in publicly accessible databases. Nonetheless,the economical importance of cork formation by the cork oaktree justifies that, in addition to the studies on suberindeposition in the potato tuber model, this process is studiedin this tree. Making use of 2-DE and mass spectrometry wehave extracted, visualized and identified proteins putativelyinvolved in cork formation from the cork oak stem. We

compared young stems with increasing capacity to form corkand the recently-formedphellemand xylemcomponents froma branch with thick cork. The results here presented consti-tute, to our knowledge, the first proteomic study of corkformation in the cork oak.

2. Materials and methods

2.1. Plant material

Q. suber samples were collected during the period of activegrowth (May–June) from a tree at Tapada da Ajuda, Lisbon (T1tree in [22]). Young shoots formed during the year of sampling(Y0) and of increasing age (one-year-old, Y1 and four-years-old, Y4), and eight-years-old branches (Y8) were sampled(Fig. 1). The young shoots, free of any visible symptoms ofdisease or damage, were surface washed in running deionisedwater, frozen in liquid nitrogen and stored at −80 °C until use.From the Y8 branches, cork was stripped off so that twoseparate samples could be collected by scraping, the newlyformed phellem (Y8Ph), at the subero-phellogenic transitionand the newly formed xylem (Y8X), at the libero-woodtransition [23]; these samples were immediately frozen forsubsequent analysis.

2.2. Microscopic observations

The young shoots, after dehydration in ethanol, wereimbedded in polyethyleneglycol. Sections (about 10 μm thick)were cut utilizing a rotating LEICA RM 2155 microtome(Wetzlar, Germany), and stained using Sudan IV [24]. Theobservations were made with a Nikon Microphot lightmicroscope (Nikon Corporation, Tokyo, Japan).

Fig. 1 – Representation of the plant material utilised for the

proteomic studies. Young shoots of increasing age: formed

during the year of sampling (Y0), one-year-old (Y1) and

four-years-old (Y4). Tissue components of eight-year-old

branches: thenewly formedphellem, at the subero-phellogenic

transition (Y8Ph) and the newly formed xylem, at the

libero-wood transition (Y8X).

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2.3. Protein extraction

For each tissue, two distinct biological samples were processedand from each of them two technical replicates were subse-quently analysed. The initial amounts of plantmaterial used areindicated in Table 1. The samples were pulverized in liquidnitrogen, homogenised in a cold solution of 10% (w/v) trichlor-oacetic acid inacetone, containing0.6 Mdithiothreitol (DTT), andincubated overnight at −20 °C. The homogenatewas centrifugedat 15,000×g for 15 min at 4 °C and the pellet recovered andwashed with a solution of acetone and DTT (0.6 M) for 1 h(−20 °C). After centrifugation, the pellet was dried under vacuumand the proteins were resolubilized by constant stirring in abuffer containing 7 M urea, 2 M thiourea, 0.4% (v/v) Triton X-100,4% (w/v) CHAPS, 2% (w/v) PVPP and 1% (v/v) IPG ampholite-containing buffer pH 3–10 (120min incubation at 25 °C). Theproteincontent of the sampleswasquantifiedusing theBradfordassay, modified by Ramagli [25], and shown in Table 1.

2.4. Two dimensional gel electrophoresis (2-DE)

2-DE was performed with extracts of the five Q. suber samples:the Y0, Y1 and Y4 young shoots and the Y8Ph and Y8Xcomponents of eight-year old branches. Isoelectric focusing(IEF) was performed in 24 cmgel strips, linear pHgradient of 3 to10 (IPG strips — GE Healthcare, Uppsala, Sweden), loaded with500 μg of total proteins. The isoelectric focusing was carried outat 30 V for 12 h, followed by 200 V for 1 h, 500 V for 1.5 h, 1000 Vfor 1.5 h, and 8000 V for 6.5 h, in a IPGphor unit (GE Healthcare,Uppsala, Sweden), at 20 °C. Following IEF, the proteins wereequilibrated in a solution of 50 mM Tris, pH 8.8, 6 M urea,30% [v/v] glycerol, 2% [w/v] SDS supplemented (i) with 1%(v/v) DTT for their reduction and (ii) then alkylated with theequilibration solution containing 2.5% (v/v) iodoacetamide andsubsequently separatedbySDS-PAGE, in25mm×20mm×1mmacrylamide gels (%T=12% and %C=3.3%). The proteinmigrationtook place at constant voltage of 10 V per gel, for 17 h, at 15 °C.Thegelswere stainedusingsilvernitrate [26], for imageanalysis,or colloidal blue coloration [27], for spot excision.

2.5. 2-DE gel image analysis

The gels were scanned using the Dynamics 300S densitometer(Molecular Dynamics, Sunnyvale, CA, USA) and the images

were analysed using the ImageMaster-Platinum v5.0 software(GE Healthcare, Uppsala, Sweden). The analysis consisted indetermining the spots present in each gel, their normalizedvolume, and comparing the profiles obtained. Four gels weremade for each type of tissue (two protein extractions fromdistinct samples and two technical replicates from each). Toensure that the spots were representative of each sample, thesoftware built synthetic gels that included only the spotspresent in at least three of the replicate gels. The normalizedvolume (spot volume/sum of volume of every spot in the gel)of each spot represents the average of the normalized volumein the replicate gels. By comparing the synthetic gels built forthe five types of studied tissues, it was possible to find, foreach tissue type, the spots that were only present in thatparticular tissue and those that were common to other tissues.

2.6. Multivariate analysis

Unsupervised multivariate analysis was used to explore therelationships existing between the several sampled tissues(Y0, Y1, Y4, Y8Ph, and Y8x). Principal component analysis(PCA) was carried out using the R software (version 2.10.1) andthe ade4 package [28], after missing values imputation usingthe tool SeqKnn (K=10) [29].

2.7. Protein identification and database search

Spots were excised from colloidal Coomassie Blue stained gelsof the cork-forming tissue Y8Ph and of the cork-non-formingtissueY8X. The spotswere processed according to a previouslypublished protocol [30]. An Ettan Spot Handling Workstation(GE-Healthcare, Uppsala, Sweden)was used for destaining anddigestion of the gel-separated proteins. Since modules forfurther sample handling and spotting on MALDI-target platesare integrated in the same instrument, a completely hand-freeapproachwasperformed.After spotting of the samples, amassspectrometric analysis in MS and MS/MS-mode using a 4800MALDI TOF/TOF, externally calibrated as outlined by themanufacturer (Applied Biosystems, Foster City, CA, USA), wasdone. Per spot one MS- and 8 MS/MS-spectra of the mostintense peaks were acquired.

The acquired spectra of each spot were submitted as asingle file in database searches against a protein and an ESTdatabase, downloaded from theNCBi-server (http://www.ncbi.

Table 1 – Q. suber tissues analysed, the total material and the samples utilized in the experiments, their protein content andthe number of spots visualized in the corresponding 2-DE gels.

Tissue Sample weight(g)

Total amount(g)

Protein content(mg protein g−1 FW)

Number spots

Min Max Average

Y0 3.9 7.5 0.672±0.14 665 735 6993.6

Y1 3.6 5.6 0.703±0.070 681 764 7162.0

Y4 4.2 6.9 0.492±0.065 630 725 6932.7

Y8Ph 6.6 13.2 0.898±0.090 592 683 6436.6

Y8X 6.6 13.2 0.463±0.039 601 648 6196.6

1268 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 6 6 – 1 2 7 8

nlm.nih.gov), limited to the taxonomic class of the viridiplan-tae using the Applied Biosystems GPS-software and an in-house MASCOT-platform. The results of these searches werecombined, thereby increasing the number of proteins thatcould be identified with significance. Because of the limitedbiomolecular characterization of cork oak all identificationswere manually verified. For spots that did not result in asignificant identification, more peptides were manuallyselected for fragmentation and, if required, peptide sequenceswere determined manually and used for homology searcheswith the FASTS-algorithm (http://www.fasta.bioch.virginia.edu/fasta_www/cgi/) [31]. During de novo sequence determina-tions, all mass increments of 113 Da were arbitrarily desig-nated as Ile, because it is impossible to discern the isobaricamino acids Ile and Leu. Similarly, mass increments of 128 Dawere always denoted as Gln, unless at the C-terminal positionof a peptide when Lys was used. To avoid haphazardidentifications, an E-value threshold of 1.0×10−4 was rigor-ously used and a proteinwas only considered to be identified ifthe majority of proteins with a significant search score had anidentical function [32].

3. Results

3.1. Comparison of the 2-DE polypeptide patterns

To analyze the changes in the proteome of the stem, linked tostem development, 2-DE gels were run from young shoots ofincreasing age: shoots of the year (Y0) with no visible signs ofsuberin deposition (Fig. 2), Y1 shoots already with a continu-ous suberin layer, and Y4 shoots with a thicker suberin layerexposed at the surface. These structural differences of theyoung shoots are reflected in the complexity of the polypep-tide patterns of the 2-DE gelswhich increase from the Y0 to theY4 shoots (Fig. 3).

Given the differences observed in the proteome of theyoung stems, when aiming at specifically identifying proteinsinvolved in cork formation in the cork oak tree, the age of thecork-non-forming tissue had to be identical to the age of thesampled cork-forming tissue. Therefore, cork was strippedfrom 8-year old branches in order to collect from the samebranch samples of two contrasting tissues: recently formedphellem (Y8Ph) and xylem (Y8X). The protein extracts from

these tissues were also subjected to 2-D gel electrophoresis.The 2-DE polypeptide patterns obtained (Fig. 3) are quitedistinct from each other indicating the existence of markeddifferences in the proteins expressed by the two tissues.

Although the protein patterns of phellem, xylem andyoung shoots were different, an image analysis comparisonallowed to detect similarities, when grouping the representa-tive gel spots in classes according to their presence/absence inthe several tissues (Fig. 4). Two hundred and fifty spots werepresent in all samples, 95 spots were only present in thosetissues not producing cork (namely Y0 and Y8X), and 121 spotswere present in all cork forming tissues (Y1, Y4 and Y8Ph).

In order to visualize the relationships existing between theseveral studied tissues we performed a principal componentanalysis, which is represented in Fig. 5.Whenwe compared allthe tissues together (Y0, Y1, Y4, Y8Ph and Y8X), we found thatthey are all clearly separated from each other (Fig. 5A).Interestingly, the phellem tissue (Y8Ph) and the young shootthat produces more phellem (Y4) localize very close to eachother. This observation reinforces the idea that polypeptidespots associated with cork formation are already present inthe young shoots. When comparing only the tissues that formphellem (Fig. 5B) the first axis discriminates the young shootthat forms less phellem (Y1) from Y4 to Y8Ph, which arefurther separated in the second axis.

3.2. Proteins associated with cork formation

To identify by MS, polypeptides implicated in cork formation,spots were excised from the colloidal blue stained gels of thetwo contrasting tissues of the Y8 branches, the phellem (Y8Ph)and the xylem (Y8X), as labelled in Fig. 3. From Y8Ph we chosespots only detected in this tissue and not in Y8X and thusconsidered to be more specifically associated with phellemformation. In order to increase the proteome knowledge of theY8Ph tissue some spots common to Y8Xwere excised from theY8X-gels as well. The main characteristics of the Y8X andY8Ph identified proteins are shown in Tables 2 and 3,respectively, and Supplementary data are presented as TablesS1 and S2. It is observed that although different proteins wereidentified from the two tissues most of them are grouped insimilar functional classes (Fig. 6). In phellem, the 54 proteinsthat were identified are classified as involved in: “carbohy-drate metabolism” (28%), “defence” (22%), “protein folding,

Fig. 2 – Microscopic observation of transversal sections of young Q. suber shoots stained with Sudan IV to evidence suberin

deposition at the periderm cell walls. The polyaliphatic domain of suberin coloured red is clearly shown from the 1st year

onwards. Y0, shoots formed in the current year; Y1, one-year-old shoots; and Y4, four-years-old shoots. Amplification: 100×.

1269J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 6 6 – 1 2 7 8

Fig. 3 – 2-DE analysis of proteins (500 μg) isolated from Q. suber shoots of the current year (Y0), 4-years-old (Y4) and from

recently formed phellem (Y8Ph) and xylem (Y8X) of an 8-years-old branch. The pH range of protein separationwas 3–10 and the

gels were silver stained. The spots excised for MS identification are numbered.

Fig. 4 – Comparison of the number of representative polypeptide spots present in the 2-DE gels ofQ. suber shoots of increasing age

(Y0, currentyear; Y1, one-year-old; andY4, four-years-old) andof recently formedphellem (Y8Ph) andxylem (Y8X) of an8-years-old

branch. Each tissue is indicated with the numbers of total spots, of spots common to all tissues and of spots only present in that

specific tissue. Only spots present in at least 3 replicate gels were considered for this analysis.

1270 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 6 6 – 1 2 7 8

stability and degradation” (19%), “regulation/signalling” (11%),“secondary metabolism” (9%), “energy metabolism” (6%) and“membrane transport” (2%). The spots picked from the xylemgels and present in both gels resulted in the identification of 45proteins, 27% of which belong to “protein folding, stability anddegradation”, whereas “defence”, “carbohydrate metabolism”

and “cellular structure” represent, respectively, 18%, 16% and11%, “secondary metabolism” and “energy metabolism” cor-respond to 9%, each, and “lipid metabolism” and “nitrogenmetabolism” to 4%, each.

4. Discussion

This article is a first report on the identification of proteinsrelated to cork formation inQ. suber branches.We identified twosets of proteins: 1) those excised from the phellem (Y8Ph) andnot detected in the xylem (Y8X) (Tables 3 and S2), that might bespecifically associatedwith cork formation; and 2) those excisedfrom the xylem but also detected in the phellem (Tables 2 andS1). These proteins could participate in processes with similar-ities in the two tissues, such as cell wall thickening and celldeath, and be less specifically associated with cork formation.

Cork is a very complexmaterial, that is the result of severalcellular processes (cell commitment, expansion, stressresponses, senescence, and death), requiring the participationof numerous biochemical pathways (carbohydrates, pheno-lics, lipids, isoprenoids, and respiration), signalling mecha-nisms and a coherent integration of the distinct processes. Inthe following section, most of the detected proteins involvedin cork formation will be discussed.

4.1. Most relevant proteins identified in phellem cells

The identified phellem proteins are discussed following theirdistribution in the different functional classes. It must benoted that many of the identified proteins have multiple anddiverse attributed functions and in these cases the most

prevalent function was used for classification. For instance,stress/defence is a dual function shared by many proteins,indicating that stress and senescence mechanisms have greatprevalence during cork formation.

4.1.1. Carbohydrate metabolism/cell wall differentiation

In spite of its high content of suberin (around 50%), the corkcell wall still has about 20% of carbohydrate constituents [5],which is highlighted by the high proportion of proteinsidentified in the phellem related to carbohydrate metabolism(ca. 30%).

The identification of galactosidases, xylosidases and anapiose/xylose synthase, is indicative of an intense hemicellu-lose/pectin biosynthesis in the differentiating phellem. Hemi-celluloses, for instance, have been suggested to have animportant participation in the regulation of the nanoscalearchitecture of cell wall constituents [33]. On the other hand,the identification of a glycine-rich protein (a cell wall proteinclassed under Defence) could express the existence of profoundcell wall alterations in developing phellem cells, similar to whatwas reported for the protoxylem cell wall [34]. The typicalpolysaccharide-rich primary cell wall of living and elongatingcells is progressivelymodified and finally replaced by a protein-rich cell wall in the dead and passively stretched protoxylemelements in which glycine-rich protein participates.

The detection of proteins of dual function (mainly stressresponses), is an interesting observation. Of the glycolyticenzymes, enolase has been implicated in, among other roles,cold responses [35] and glyceraldehyde 3-phosphate dehydro-genase may mediate reactive oxygen species (ROS) signallingin plants [36]. Phosphomannomutases (PMMs), in addition totheir role in GDP-mannose biosynthesis (formation of struc-tural carbohydrates) are also known to participate in thebiosynthesis of ascorbic acid or in N-glycosylation processes[37]. Protein glycosylation is a basic cellular process andascorbic acid participates in stress responses, senescence andsignalling [38,39]. Cytoplasmic aconitate hydratase (CAH) alsoparticipates in senescence and is an important component of

Fig. 5 – Principal component analysis bi-plots of the protein profiles of all the Q. suber tissues studied (A) and of only the

cork-producing tissues (B). Y0, Y1 and Y4: shoots of increasing age, of the current year, one-year-old and four-years-old,

respectively; Y8Ph and Y8X: recently formed phellem and xylem, of an 8-year-old branch, respectively.

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Table 2 – Xylemproteins identified byMS/MS analysis (see Supplementary Table S1). Spot numbering according to Fig. 3; theproteins are grouped by their functional similarity.

Spot ID Protein identification Functional category Cellularlocalization

Species

Carbohydrate metabolism128 NAD-dependent malate

dehydrogenaseTCA cycle Cytosol Quercus petraea

131 Enolase Glycolysis/gluconeogenesis Cytosol Q. robur

174 Enolase Glycolysis/gluconeogenesis Cytosol Coffea canephora

176 Enolase Glycolysis/gluconeogenesis Cytosol Lycopersicon esculentum

132 Polygalacturonase(pectinase) family protein

Pentose and glucuronateinterconversions

Endomembranesystem

Arabidopsis thaliana

8 Alpha-amylase precursor Starch and sucrosemetabolism

Q. robur

170 Alpha-amylase precursor Starch and sucrosemetabolism

Vigna mungo

Energy metabolism196 Putative ATP synthase

beta subunitATP synthesis Helianthus tuberosus

197 ⁎ Vacuolar H+-ATPase Proton transport H. tuberosus

12 Precursor protein ofoxygen-evolving complex

Photosynthesis Chloroplast Solanum tuberosum

160 Oxygen-evolving complexof photosystem II

Photosynthesis Chloroplast Brassica napus

Nitrogen metabolism134 Glutamate dehydrogenase Glutamate metabolism H. paradoxus

Secondary metabolism101 Laccase2 Phenol oxydation Apoplast Juglans hindsii x J. regia

107 Laccase2 Phenol oxydation Apoplast J. hindsii x J. regia

127 Isoflavone reductase-likeprotein Bet v 6.0102

Flavonoid biosynthesis Q. suber

138 Cinnamyl alcohol dehydrogenase Phenylpropanoidbiosynthesis

Lolium perenne

Lipid metabolism161 Allene oxide cyclase Alpha-linolenic acid

metabolismChloroplast Medicago truncatula

183 Putative palmitoyl-proteinthioesterase

Sphingolipid metabolism Oryza sativa

Protein folding, stability and degradation171 Putative mitochondrial

processing peptidasealpha subunit

Proteasome Mitocondrion O. sativa

147 Ubiquitin-conjugatingenzyme UBC2

Proteasome Mesembryanthemum

crystallinum

167 20S proteasome alphasubunit A

Proteasome Cytosol Q. robur

163 ⁎ Proteasome subunitbeta type 1

Proteasome O. sativa

126 WD-40 repeat protein Folding, sorting anddegradation

Chloroplast; cytosolicribosome

A.thaliana

162 ⁎ Chloroplast chaperonin 21 Chaperone activity Cytosol Q. robur

191 Disulfide isomerase Chaperone activity ER lumen Cucumis sativus

44 Heat shock protein 17.4 Chaperone activity Q. suber

150 Heat shock protein 17.4 Chaperone activity Cytosol Q. petraea

154 HSP19 class II Chaperone activity Citrus x paradisi

192 HSP70,chloroplast Chaperone activity Chloroplast H. tuberosus

197 ⁎ HSP70, mitochondrialprecursor

Chaperone activity Chloroplast Carthamus tinctorius

Cellular structure169 Actin Cytoskeleton Cytosol, cytoskeleton Gossypium hirsutum

190 Beta tubulin Cytoskeleton Cytosol, cytoskeleton Glycine max

193 Beta tubulin Cytoskeleton Cytosol, cytoskeleton Medicago sativa

194 Alpha tubulin Cytoskeleton Cytosol, cytoskeleton H. paradoxus

195 Alpha tubulin Cytoskeleton Cytosol, cytoskeleton G. barbadense

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the glyoxylate bypass for the remobilisation of macromole-cules [40]. So, PMMs and CAH could be additionally involved instress responses or senescence processes of the phellem cells.

The identificationof lactoylglutathione lyase (or glyoxalase I)also seems relevant, since this enzymecatalyses the first step ofa critical two-step detoxification system ofmethylglyoxal (MG),a highly toxic and common by-product of carbohydratemetabolism. It has been shown that MG increases several-foldunder salinity and other abiotic stresses and that overexpres-sion of glyoxalase decreases MG levels [41].

4.1.2. Defence

The high proportion of proteins related to cellular defencepathways that we detected could be needed in associationwith processes likely to occur in the phellem, namely,desiccation, cell death, temperature stress, protection frompests and disease. Since ROS are produced in both unstressedand stressed cells, the detection in phellem of proteins relatedto oxidative stress protection and associated with detoxifyingprocesses is important. Thioredoxin-dependent peroxidasehas an antioxidant function towards ROS [42], while glutathi-one S-transferase and flavodoxin-like quinone reductase arepossibly involved in detoxification against auxin-inducedoxidative stress [43].

The SGT1 protein is highly conserved in eukaryots and isrelated to HSPs, since it binds specifically to the molecularchaperone HSP90; it possibly regulates disease resistanceconferred by resistance proteins and developmentalresponses to auxin [44]. Cystatin (a cysteine protease inhibitor)was shown to be active against phytopathogenic fungi [45] andthe expression of its gene in potato confers partial resistanceto the potato-cyst nematode [46]. However, a possible functionin the regulation of proteolysis should also be envisaged.

Concerning the several chitinases identified, there is muchcontroversy about the role of these enzymes in plants. Inaddition to defence they may be involved in many more

aspects of the plant life cycle [47], including the biogenesis ofthe cell wall.

A defence role can also be attributed to some of theproteins detected in phellem, but ascribed to other functionalclasses. Examples are the heat shock proteins, cyclophylin andsubtilisin-like proteases (Protein folding, stability and degra-dation) and Rab2 GTP-binding protein (Regulation/Signalling),as discussed later. Some proteins of basic metabolism couldalso be implicated in defence/stress processes as, for instance,the proteins referred above from Carbohydrate metabolism.So, many proteins involved in defence/stress responses seemto be needed during phellem formation in the cork oak stem, asituation that is similar to what was recently reported for thepotato native periderm [48,49], and that we further observed inthe wound-reconstructed periderm [49].

4.1.3. Protein folding, stability and degradation

The functioning of proteins in the cell depends on adequatefolding and stabilisation of proteins and, hence, on the strictregulation of protein turnover. In phellem this class ofproteins is very important, comparing in number to that ofproteins classified as being involved in defence. Furthermore,most of the proteins grouped under this heading are alsoimplicated in defence mechanisms and/or developmentalprocesses.

Some proteins are associated with the proteasome or havea chaperonin or protein folding function, like the numerousheat shock proteins (HSPs), which are known to be alsoexpressed during development and in associationwith severalstress conditions [50]. Also important are: a protein disulfideisomerase, that contains a thioredoxin domain [51], a proteinthat participates in regulation of rDNA transcription (WD40-repeat protein) [52] and cyclophylin, which catalyze theisomerisation of peptide bonds, facilitating protein foldingand suggested to have crucial roles during both plantdevelopment and stress responses [53,54].

Table 2 (continued)

Spot ID Protein identification Functional category Cellularlocalization

Species

Defence163 ⁎ Germin-like protein Abiotic/biotic stress

responseApoplast Solanum tuberosum

151 Pathogenesis-relatedprotein family 10

Abiotic/biotic stressresponse

Q. robur

155 Hypothetical protein Abiotic/biotic stressresponse

Vitis vinifera

158 Cyanate hydratase Detoxification Arabidopsis thaliana

157 Cu/Zn superoxidedismutase

Superoxide metabolicprocess

H. annuus

162 ⁎ Manganese superoxidedismutase

Superoxide metabolicprocess

J. hindsii x J. regia

15 Glutathione peroxidase 5 Glutathione metabolism Coffea canephora

165 Glutathione S-transferase Oxidative stressprotection

Cytosol Q. robur

Unclassified172 Hypothetical protein Unclassified Cytosol Glycine max

177 Hypothetical protein Unclassified Centaurea solstitialis

⁎ Two polypeptides identified in the same spot.

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Table 3 – Phellem proteins identified by MS/MS analysis (see Supplementary Table S2). Spot numbering according to Fig. 3;the proteins are grouped by their functional similarity.

Spot ID Protein identification Functional category Cellular localization Species

Carbohydrate metabolism133 UDP-glucose pyrophosphorylase Starch synthesis Amorpha fruticosa

173 UDP-D-apiose/UDP-D-xylosesynthase 2

Apiose synthesis Apoplast; cytosol B. rapa

129 Malate dehydrogenase TCA cycle Cichorium endivia

109 Cytoplasmic aconitate hydratase TCA cycle and Glyoxylatemetabolism

Mitocondrion Musa acuminata

166 Phosphomannomutasefamily protein

Fructose and mannosemetabolism

Cytosol B. napus

111 Alpha-D-xylosidase Xylose metabolism Tropaeolum majus

119 Alpha-D-xylosidase Xylose metabolism T. majus

122 ⁎ Beta-D-galactosidase Galactose metabolism Carthamus tinctorius

124 Putative beta-galactosidase Galactose metabolism Lycopersicon

esculentum

137 Alpha-galactosidase 1 Galactose metabolism Cyamopsis

tetragonoloba

123 Glyceraldehyde 3-phosphatedehydrogenase

Glycolysis/gluconeogenesis Cytosol Prunus persica

125 Glyceraldehyde 3-phosphatedehydrogenase-like protein

Glycolysis/gluconeogenesis Quercus robur

136 Enolase Glycolysis/gluconeogenesis Cytosol Gossypium barbadense

130 Triosephosphate isomerase Glycolysis/gluconeogenesis Q. robur

164L Triosephosphate isomerase Glycolysis/gluconeogenesis Chloroplast Helianthus ciliaris

Energy metabolism112 ATP synthase subunit alpha,

mitochondrialATP synthesis Mitocondrion Glycine max

141 ⁎ Oxygen-evolving enhancerprotein 2

Photosynthesis Chloroplast Pisum sativum

148 Ribulose-bisphosphatecarboxylase

Photosynthesis Chloroplast Platanus x acerifolia

Secondary metabolism100 Laccase2 Phenol oxidation Apoplast Juglans hindsii x J. regia

105 Laccase2 Phenol oxidation Apoplast J. hindsii x J. regia

104 Laccase2 Phenol oxidation Apoplast J. hindsii x J. regia

102 Diphenol oxidase Phenol oxidation Apoplast Acer pseudoplatanus

114 Diphenol oxidase Phenol oxidation Apoplast J. hindsii x J. regia

Regulation/signalling181 14-3-3 protein Signal transduction Nicotiana tabacum

182 14-3-3 protein Signal transduction Arachis hypogaea

141 ⁎ AT-RAB2; GTP binding Signal transduction Membrane; PM;vacuole

A thaliana.

144 Nucleoside diphosphate kinase 1 Nucleic acid metabolism Q. robur

117 Nucleoid DNA-binding protein-related

Nucleic acid metabolism Apoplast B. napus

118 ⁎ Nucleoid DNA-binding protein-related

Nucleic acid metabolism Apoplast B. napus

122 ⁎ Nucleoid DNA-binding protein-related

Nucleic acid metabolism Apoplast B. napus

Protein folding, stability and degradation164 20S proteasome subunit PAB1 Proteasome Cytosol; nucleous G. max

20 Subtilisin-like protease C1 Proteolysis G. max

108 Subtilisin-like protease C1 Proteolysis G. max

110 Subtilisin-like protease C1 Proteolysis G. max

149 Cyclophylin Chaperone activity Digitalis lanata

143 Heat shock protein 17.4 Chaperone activity Q. suber

152 Heat shock protein 17.3kDa class II

Chaperone activity Cytosol Lycopersicon

peruvianum

153 Heat shock protein 17d Chaperone activity Q. suber

189 Heat shock cognate 70-1 Chaperone activity Cytosol A. thaliana

158L Heat shock protein 17.4 Chaperone activity Q. petraea

Membrane transport120 Porin I, 36 K Ion channel Solanum tuberosum

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Equally important is the identification of several subtilisin-like proteases. In plants these proteases have been associatedwith developmental processes and stress responses. One suchprotease, detected in soybean, appears to be secreted to theextracellular matrix where it may function in the reorganiza-tion of the cell wall components [55].

4.1.4. Secondary metabolism

The identification of laccases and diphenol oxidases inphellem-specific spots illustrates the importance of phenoloxidation during phellem formation. Laccase, in addition toperoxidase, is considered to be involved in lignin biosynthesis[56,57]. The down-regulation of laccase gene in poplar affectsphenolic metabolism and cell wall structure [58].

4.1.5. Regulation/signalling

We identified a significant number of proteins of this class(11%), namely 14-3-3 proteins, Rab2 protein and enzymes that

interact with nucleosides/nucleic acids. The 14-3-3 proteinsare important regulators of a wide diversity of targets, viadirect protein–protein interactions, generally mediated byphosphorylation. It is now recognized that an increasingnumber of plant signalling proteins mediating developmentaland stress responses interact with 14-3-3 proteins [59]. Rab2proteins are small GTP-binding proteins that are key players invesicle-mediated protein transport [60]. They have beenshown to be important in secretion of membrane and cellwall materials during pollen tube growth and seed germina-tion [61,62], but are furthermore implicated in desiccationtolerance and damage repair [63]. Nucleoside-diphosphatekinases are enzymes that catalyze the exchange of phosphategroups between different nucleoside diphosphates and are asource of GTP. In animals they were suggested to regulatesynaptic vesicle internalization [64] and in plants severalisoforms have been described in the cytoplasm, the mito-chondria and the chloroplast, with possible functions in

Table 3 (continued)

Spot ID Protein identification Functional category Cellular localization Species

Defence178 Endochitinase Abiotic/biotic stress response Musa acuminata

142 Glycine-rich protein 2b Abiotic/biotic stress response B. napus

188 SGT1 (suppressor of the G2allele of skp1)

Abiotic/biotic stress response Nicotiana benthamiana

14 Putative chitinase Abiotic/biotic stress response Musa acuminata

32 Chitinase class III-1; MtChitIII-1 Abiotic/biotic stress response Medicago truncatula

121 Acidic endochitinase precursor Abiotic/biotic stress response Apoplast Cucumis sativus

180 Basic chitinase Abiotic/biotic stress response Oryza sativa

145 Cystatin Abiotic/biotic stress response Q. robur

159 Flavodoxin-like quinonereductase 1

Oxidative stress protection Membrane; PM; vacuole B. napus

153L Thioredoxin-dependent peroxidase Oxidative stress protection Plantago major

139 Glutathione S-transferase Oxidative stress protection Cytosol M. truncatula

168 Lactoylglutathione lyase Detoxification Chloroplast Arabidopsis thaliana

Unclassified113 Hypothetical protein Unclassified Ricinus communis

118 ⁎ Hypothetical protein Unclassified A. thaliana

⁎ Two polypeptides identified in the same spot.

Fig. 6 – Functional class distribution of themajor proteins identified from recently formed phellem (Y8Ph) and xylem (Y8X) of an

8-year-old Q. suber branch.

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processes of signal transduction in addition to a housekeepingrole (to provide nucleotide-triphosphates) [65].

In an attempt to understand the significance of thedetected chloroplast nucleoid DNA-binding proteins in thephellem, we performed a Blast search by sequence homology,which showed that several homologous proteins had alreadybeen identified (in Arabidopsis, castor bean, poplar and rape),some of them with an ascribed aspartic protease activity. It isinteresting that for the Arabidopsis homologue F21M12.13(At1g09750) an extracellular localization in the apoplast wassuggested. A protein of similar type, showing proteolyticactivity, was detected in cultured tobacco cells in associationwith non-photosynthesizing, actively growing plastids [66].

So, these observations give indications on the existence inthe newly emerging phellem cells of important regulatingmechanisms (through signalling proteins, phosphorylationsand protein–protein interactions) that participate in thecomplex developmental processes leading to cork formation.

4.2. Analogies with previous suberization studies

Several of the proteins identified in the presentworkwere alsodetected during studies on the suberization processes ofpotato tuber tissues, both the native tuber periderm [48,49]and thewound-reconstructed peridermof potato slices [49], asindicated in Table 4. In the slices their expression markedlyincreases in the last stages of slice healing, when the wound-periderm is being strengthened. These observations reinforcethe significance of the cork specific proteins identified in thisstudy and their involvement in the suberization process, asthey are detected in the distinct suberizing systems of twoplants. It is interesting that some of the proteins are of basicmetabolism, but have a dual function in the cell (e.g., enolase),in most cases associated with stress responses.

When our proteomic results are compared with the tran-scriptomicanalysis recently reported for thephellemofcorkoak[17] little resemblance is found. We did not detect proteinsinvolved in the synthesis of the lipidic components of cork,possibly because thoseproteinsare associatedwithmembranesor other insoluble cell components that cannot be solubilizedwith the extraction procedure used in this study. Conversely,many proteins we detected in association with cork formationwere not brought into evidence in the transcriptomic analysis.For instance, stress proteins identified in the current study, andalso previously detected during the formation of the native andthe wound-induced potato tuber periderms [48,49], are missingfrom the transcriptomic data. The fact that not all the identifiedproteins were utilised as probes in the construction of themicroarray used by Soler et al. [17], seems a partial explanationfor the discrepancy. Therefore, the two methodologies givecomplementary insights into phellem cell functioning and corkformation.

5. Concluding remarks

The proteins associated with phellem formation that weredetected in this work give evidence for the functioning in thecork oak stem tissues of various processes, not only of basic andsecondary metabolism but also related to secretion of cellular

components and cell wall reorganization, and indicate theexistence of active control mechanisms dependent on severalregulation/signalling pathways. Furthermore, the similarity ofresults with previous observations on the suberization process-es of the potato tuber tissues reinforces the significance for corkformation of the proteins here described.

The gathered information thus provide a starting point forthe further study on the role of these proteins in the formationof cork, as the observations strongly suggest the occurrence inthe newly emerging phellem cells of complex, highly regulat-ed, developmental processes (through signalling proteins,phosphorylation and protein–protein interaction) that includeproteasome-controlled proteolysis, vesicle-mediated secre-tion of membrane and wall materials, and reorganization ofthe cell wall components. The subsequent analysis of theparticipation of these several types of proteins in suchcomplex processes will contribute to understand the basiccellularmechanisms of cork formation and, eventually, lead tothe identification/definition of parameters important to

Table 4 – Q.suber stem proteins also previously identifiedin suberizing potato tuber tissues.

Peel Slices

Barelet al.

Chaveset al.

Chaveset al.

Carbohydrate metabolism

Malate dehydrogenase +Enolase +Glyceraldehyde-3-P dehydrogenase +Triose-P isomerase +Alpha-galactosidase +

Energy metabolism

ATP synthase, mitochondrial +

Secondary metabolism

Phenol oxidases + +

Protein folding, stability and degradation

Proteasome subunits + + +Disulfide isomerase +Cyclophilin +Heat shock proteins +WD-40 repeat protein +

Membrane transport

Porin + +

Cellular structure

Actin + +Tubulin +

Defence

Chitinases + + +PR-10 + + +Cu/Zn SOD +Mn SOD +Peroxidases + + +GlutathioneS-transferase

+

Glycine-rich protein +Cystatin +

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evaluate specific properties of the cork material and of itstechnological quality.

Supplementary materials related to this article can befound online at doi:10.1016/j.jprot.2011.02.003.

Acknowledgements

We thank Prof. José Graça (Instituto Superior de Agronomia,Lisbon) for guidance on the microscopic observations. Thiswork was supported by the FCT project POCTI/AGR/39011/2001.

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1278 J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 2 6 6 – 1 2 7 8

Sample Protein* Organism* Gi Accession EST ° Gi Accession Prot° Experim. Mr Calc. Mr Mass Δ MC Pept. score Sequences MW a

pIa

MW b

pIb

8 Alpha-amylase precursor Vigna mungo 62974924 113781 1083.5784   1083.4985   0.0799  0   43   K.TEIGFDGWR.F + kynurenin (W) 46888.8 5.45 63730 5.48

1404.8794   1404.8201   0.0594  1   63   K.GIKAVADIVINHR.T

1444.8306   1444.7674   0.0633  0   61   K.GILQAAVEGELWR.L + kynurenin (W)

1472.8271   1472.7623   0.0648  0   (50)  K.GILQAAVEGELWR.L + Double Ox (W)

1567.7654   1567.7089   0.0565  0   49   R.GIWCIFEGGTPDGR.L + kynurenin (W)

12 33kDa precursor protein of oxygen-evolving complex Solanum tuberosum 809113 963.5775   963.5793   -0.0018  0   49   R.VPFLFTIK.Q 35257.7 5.86 32565 4.27

2309.1248   2309.1314   -0.0066  0   102   R.LTYTLDEIEGPFEVSPDGTVK.F

2424.1365   2424.1379   -0.0014  0   94   K.GTGTANQCPTIDGGVDSFAFKPGK.Y

58245995 1251.6580   1251.6459   0.0121  1   55   K.RLTYDEIQSK.T

118554186 2631.3240   2631.3279   -0.0038  0   42   K.TKPETGELIGVFESLQPSDTDLGAK.T

15 glutathione peroxidase 5 Populus trichocarpa x Populus deltoides119324971 125976395 2080.3594   2079.9207   0.4387  0   88   K.CGFTNSNYTELNELYQK.Y 19412.9 4.78 13392 4.50

2669.5540   2671.3533   -1.7992  2   58   R.FKSEFPIFDKIEVNGENSAPIYK.F

117708410 1699.5843   1699.7994   -0.2151  0   29   K.WGIFGDDIQWNFAK.F + kynurenin (W)

2080.3594   2079.8877   0.4717  0   104   K.CGMTNSNYTELNQLYEK.Y

151201869 1906.0800   1904.9884   1.0917  1   70   R.YYPTTSPLTLEHDIKK.L

44 heat shock protein 17.4 Quercus suber 4456758 966.5141   966.4770   0.0371  0   70   R.ETTAFATAR.I 17398.8 6.34 13392 7.57

973.5703   973.5345   0.0359  1   29   R.FRLPENAK.V

1616.8399   1616.8198   0.0201  1   62   R.IDWKETPEAHIFK.A + kynurenin (W)

1725.8325   1725.8183   0.0143  3   48   R.SKEHEEKNDKWHR.V + kynurenin (W)

2047.9939   2047.9917   0.0022  1   44   K.ANMENGVLTVMVPKEEQK.K

2128.0745   2128.0647   0.0098  1   88   K.EEVKVEVEDGNVLQISGER.S

2256.1634   2256.1596   0.0038  2   116   K.KEEVKVEVEDGNVLQISGER.S

2950.5340   2950.5610   -0.0270  3   51   K.ADLPGLKKEEVKVEVEDGNVLQISGER.S

101 laccase2 Toxicodendron vernicifluum 133867518 23503483 1680.7747   1680.8848   -0.1101 1   72   K.YGVTIHWHGVKQPR.N + kynurenin (W) 58751.9 7.34 90136 7.29

FASTS score VSVDGQFPGIPIR

1.2E-07 VTIHWHGVK

DHPNSV

107 laccase2 Toxicodendron vernicifluum 133867518 23503483 1299.5707   1299.6724   -0,1016  0   60   K.YGVTIHWHGVK.Q + kynurenin (W) 58751.9 7.34 80065 6.75

1680.7861   1680.8848   0.0987 1   18   K.YGVTIHWHGVKQPR.N + kynurenin (W)

133866816 1475.6771   1475.7442   0.0671  0   43   R.HASWGMSTVIIVK.N + Double Ox (W)

126 WD-40 repeat protein Arabidopsis thaliana 2289095 1893.9344   1893.9472   -0.0128  0   69   R.FSPNTLQPTIVSASWDK.T + kynurenin (W) 35779.6 7.61 35553 7.18

2004.0302   2004.0428   -0.0126  1   91   R.FVGHTKDVLSVAFSLDNR.Q

3276.5818   3276.5323   0.0496  0   8   R.LTGHSHFVEDVVLSSDGQFALSGSWDGELR.L + Double Ox (W)

3023857 1333.7185   1333.7969   -0.0784  2   22   R.DKSIIVWKLTK.D + kynurenin (W) 35723.5 8.06

127 allergenic isoflavone reductase-like protein Bet v 6.0102 Betula pendula 124567508 10764491 1067.5862   1067.5876   -0.0013  0   54   K.AGHPTFALVR.E 38899 6.95

1444.6260   1444.6259   0.0002  0   71   R.FYPSEFGNDVDR.V

1740.8952   1740.8934   0.0019  1   112   K.AIFNKEEDIGTYTIK.-

2036.0729   2036.0690   0.0039  0   136   K.NLGVTLVHGDLYDHGSLVK.A

2076.1585   2076.1578   0.0007  0   109   K.QVDVVISTVGHLQIVDQVK.I

2262.0718   2262.0705   0.0014  1   62   R.FYPSEFGNDVDRVHAVDPAK.T

133863378 2678.2476   2679.2339   -0.9863  0   77   K.GDHTNFEIEPSFGVEASELYPDVK.Y

128 NAD-dependent malate dehydrogenase Prunus persica 34316528 15982948 1874.9793   1874.9560   0.0233  1   80   K.EFAPSIPEKNITCLTR.L 35497.8 6.6 38559 6.75

2301.1389   2301.1389   0.0000  0   79   R.ELVADDAWLHGEFIATVQQR.G + kynurenin (W)

2455.1651   2455.1880   -0.0229  0   64   K.NAIIWGNHSSTQYPDVNHATVK.T + kynurenin (W)

2483.1599   2483.1829   -0.0230  0   (37)  K.NAIIWGNHSSTQYPDVNHATVK.T + Double Ox (W)

2926.4685   2926.5295   -0.0610  0   12   R.GVMLGPDQPVILHMLDIPPAAEALNGVK.M

131 enolase Medicago truncatula 62976157 140063641 1227.5130   1227.6070   -0.0940  0   9   K.MGVEVYHHLK.A 45053.2 5.79 53353 6.44

1678.9257   1678.9518   -0.0261  1   79   K.KIPLYQHIANLAGNK.T

1803.9207   1803.9366   -0.0159  0   83   R.AAVPSGASTGIYEALELR.D

2104.1416   2104.1428   -0.0012  0   103   K.TLVLPVPAFNVINGGSHAGNK.L

2320.2307   2320.2062   0.0245  1   36   K.YNQLLRIEEELGPAAVYAGSK.F

224482647 976.4550   976.5130   -0.0580  1   36   K.FRAPVQPY.- 47803.4 5.7

132 polygalacturonase (pectinase) family proteinArabidopsis thaliana 15233124 FASTS score IDGINPDSCTNVR 51939.1 5.85 56238 6.73

2.6E-11 DAVGGFDQISASNK

134 Glutamate dehydrogenase Arabidopsis thaliana 15238762 1080.5645   1080.5386   0.0260  0   17   R.MGAFTLGVNR.V 44524.1 6.39 45457 6.73

1185.6757   1185.6968   -0.0211  0   66   K.IVAVSDITGAIK.N

1283.6461   1283.6146   0.0316  0   61   K.DDGTLASFVGFR.V

2111.0882   2111.0357   0.0525  1   51   K.VECTIPKDDGTLASFVGFR.V

15228667 1597.8858   1597.8463   0.0395  0   78   R.GVLFATEALLNEHGK.T 44527.8 5.75

125471198 1158.5853   1160.6189   -0.0336  0   69   K.TAVANIPYGGAK.G

1911.0752   1911.1556   -0.0804 2   22   R.LLGLDSKLEKGLLIPFR.E

138 cinnamyl alcohol dehydrogenase Lolium perenne 19849246 1511.8614   1511.8935   -0.0321  0   104   K.HLGVVGLGGLGHVAVK.F 43093.4 8.55 36904 5.99

224138470 1490.6640   1490.6902   -0.0262  0   83   R.DQSGHLSPFNFSR.R 38944.7 6.23

147 ubiquitin-conjugating enzyme UBC2 Mesembryanthemum crystallinum 5762457 2040.0669   2040.0462   0.0207  0   65   K.VFHPNINSNGSICLDILK.E 16466 7.71 9552 8.48

2269.1731   2269.1888   -0.0157  1   43   R.TKVFHPNINSNGSICLDILK.E

150 heat shock protein 17.4 Quercus suber 75975700 4456758 966.4568   966.4770   -0.0202  0   51   R.ETTAFATAR.I 17398.8 6.34 13333 6.70

973.5149   973.5345   -0.0195  1   30   R.FRLPENAK.V

1616.8135   1616.8198   -0.0063  1   71   R.IDWKETPEAHIFK.A + kynurenin (W)

2110.0371   2110.0541   -0.0170  1   (10)  K.EEVKVEVEDGNVLQISGER.S + Pyro-glu (N-term E)

2128.0764   2128.0647   0.0117  1   47   K.EEVKVEVEDGNVLQISGER.S

2256.1726   2256.1596   0.0130  2   105   K.KEEVKVEVEDGNVLQISGER.S

2950.5801   2950.5610   0.0191  3   77   K.ADLPGLKKEEVKVEVEDGNVLQISGER.S

151 ypr10 Castanea sativa 62974982 16555781 1112.6035   1112.5502   0.0534  0   81   K.ITFGEGSQFK.Y 17561 5.75 10405 6.44

1240.6977   1240.6451   0.0526  1   85   K.KITFGEGSQFK.Y

1300.7412   1300.7027   0.0386  0   59   K.AFVLDGDNLIPK.V

154 HSP19 class II Citrus x paradisi 30575570 1684.9921   1684.8631   0.1290  0   73   K.VQVEDDNVLLISGER.K 11141 8.01 12053 5.48

2185.1358   2185.1226   0.0133  1   133   K.SGDIKVQVEDDNVLLISGER.K

75279028 1699.0014   1698.8788   0.1227  0   48   K.VQVEEDNVLLISGER.K 17265 6.32

1854.9849   1854.8862   0.0988  0   56   K.EYPNSYVFVVDMPGLK.S + Pyro-glu (N-term E)

1872.9671   1872.8967   0.0704  0   (46)  K.EYPNSYVFVVDMPGLK.S

2199.1455   2199.1382   0.0073  1   39   K.SGDIKVQVEEDNVLLISGER.K

155 hypothetical protein Vitis vinifera 147776053 FASTS score IVEEYIIANPNVYVY 15455.7 5.41 11236 5.23

1.8E-19 SIEFIEGDGGVGSIKK

FFEGSPFK

IIEGDVIGDEIESISYEVK

AMIIDSHNIFPK

AVTTTFEENYTT

156 cyanate hydratase Arabidopsis thaliana 15229458 FASTS score YSDPNIIQEPTVYR 18592.2 5.49 28611 5.03

5.5E-16 AVVTLDGKFLPYTEQK

157 Cu/Zn superoxide dismutase Helianthus annuus 50978416 1333.7810   1333.7717   0.0093  0   (25)  K.QIPLIGGQSIIGR.A + Pyro-glu (N-term Q) 15417.3 5.61 9510 6.08

1350.8043   1350.7983   0.0060  0   26   K.QIPLIGGQSIIGR.A

2043.0481   2043.0384   0.0097  1   128   R.AVVVHADPDDLGKGGHELSK.S

160 oxygen-evolving complex of photosystem IISinapis alba 150895089 21133 1350.6875   1350.6931   -0.0056  0   93   -.AYGEAANVFGKPK.K 27925 6.83 18816 6.22

161 allene oxide cyclase Medicago truncatula 40644132 1033.6154   1033.5305   0.0850  1   25   R.DRGSPAYLR.L 27977.6 9.25 19747 6.22

1093.6616   1093.5848   0.0768  0   38   K.LFYTFYLK.G

1475.8133   1475.7619   0.0514  0   49   K.SVNSLGDLVPFSNK.L

118551237 1127.7124   1127.6491   0.0633  0   58   K.LHQIVFPFK.I

162 chloroplast chaperonin 21 Vitis vinifera 62975565 50660327 1148.6688   1149.5778   -0.909 0   48   K.YTSNKPLGDR.V 26396.4 8.96 20052 6.36

3101.6023   3101.6608   -0.0585  2   75   K.HLILKDDDIVGILETDDVKDLKPLNDR.V

manganese superoxide dismutase Gossypium hirsutum 133865041 3219353 1387.6939   1387.6633   0.0307  0   83   K.HHQAYITNYNK.A 22098.2 8.55

1616.8146   1616.7847   0.0299  0   74   K.FNGGGHINHSIFWK.N + kynurenin (W)

2257.1695   2257.1755   -0.0060  1   48   K.LQSAIKFNGGGHINHSIFWK.N + kynurenin (W)

163 Proteasome subunit beta type1 Oryza sativa (japonica cultivar-group) 115477445 1622.7683   1622.7034   0.0649  0   93   K.GCVFTYDAVGSYER.T 23817 5.82 23639 5.71

2022.9889   2022.9726   0.0163  0   92   R.FFPYYAFNVLGGLDSEGK.G

2357.1345   2357.1572   -0.0227  0   74   R.VGYSSQGSGSTLIMPFLDNQLK.S

germin-like protein Solanum tuberosum 3171251 1740.9210   1740.8696   0.0515  0   72   R.IDYAPGGINPPHTHPR.A 23234.9 8.79

165 glutathione S-transferase Pisum sativum 62974787 37051105 1902.9437   1902.9363   0.0074  1   129   K.KQFAEDLIAYTDTFNK.T 26710.4 4.97 31650 5.71

2257.1602   2257.1531   0.0071  0   63   R.FQIVFSALWNYDITAGRPK.L + Double Ox (W)

167 20S proteasome alpha subunit A Glycine max 62975240 12229897 1143.6098   1143.5964   0.0134  0   57   R.LFQVEYAFK.A 27392.1 5.83 26322 5.99

1155.6225   1155.6036   0.0189  0   51   R.HITIFSPEGR.L

1467.7501   1467.7391   0.0110  0   21   K.YLGLLATGMTADAR.T

1718.8385   1718.8337   0.0048  1   14   R.FRYGYEMPVDVLSK.W

1904.9226   1904.9128   0.0098  1   19   R.GSGGGYDRHITIFSPEGR.L

2279.2893   2279.2889   0.0004  2   74   K.KVPDKLLDQTSVTHLFPITK.Y

169 actin Gossypium hirsutum 32186896 1518.7832   1518.7368   0.0465  0   51   K.IWHHTFYNELR.V + kynurenin (W) 41733.8 5.44 42748 4.95

1773.9248   1773.8897   0.0352  0   78   K.NYELPDGQVITIGAER.F

1953.0845   1953.0571   0.0275  0   79   R.VAPEEHPVLLTEAPLNPK.A

2198.0921   2198.0677   0.0244  0   30   K.DLYGNIVLSGGSTMFPGIADR.M

3150.6157   3150.6349   -0.0191  0   37   R.TTGIVLDSGDGVSHTVPIYEGYALPHAILR.L

170 Alpha-amylase precursor Vigna mungo 113781 1083.5670   1083.4985   0.0685  0   54   K.TEIGFDGWR.F + kynurenin (W) 46888.8 5.45 46465 5.17

1444.8202   1444.7674   0.0529  0   63   K.GILQAAVEGELWR.L + kynurenin (W)

1472.8120   1472.7623   0.0497  0   (32)  K.GILQAAVEGELWR.L + Double Ox (W)

1567.7539   1567.7089   0.0450  0   50   R.GIWCIFEGGTPDGR.L + kynurenin (W)

3265.5369   3265.6214   -0.0845  0   7   K.DSNGKPPGLIGIKPENSVTFIDNHDTGSTQK.L

3363.5781   3363.6669   -0.0888  0   6   K.NSIPDLANAGITHVWLPPASQSVAPQGYMPGR.L + kynurenin (W)

3506.7156   3506.8004   -0.0848  1   5   R.LKDSNGKPPGLIGIKPENSVTFIDNHDTGSTQK.L

171 putative mitochondrial processing peptidase alpha subunitOryza sativa (japonica cultivar-group) 55168176 FASTS score YGDVISVPSYESVSR 41838.3 5.38 45857 5.31

2.2E-16 SVYVGGDYRR

TENYTAPR

172 hypothetical protein Vitis vinifera 151397711 147836469 1835.9043   1835.9087   -0.0044  0   101   R.LTLADALVYACNQGAEK.I 60782.3 7.03 51625 5.48

manual IHTIAQANIGITYPAGIEAPK db-entry XHTLARATLGLTHPSNVEPPK

174 Enolase Medicago truncatula 119327087 140063641 1802.5014   1803.9366   -1.4352  0   88   R.AAVPSGASTGIYEALELR.D 45053.2 5.79 48020 5.71

1898.8181   1899.9474   -1.1292  0   60   K.LAMQEFMILPVGASSFK.E

2002.3404   2003.0951   -0.7547  0   109   K.LVLPVPAFNVINGGSHAGNK.L

2698.2554   2696.3292   1.9262  1   17   R.AAVPSGASTGIYEALELRDGGSDYLGK.G

58238214 1825.5113   1826.8686   -1.3572  0   103   R.IEEELGSEAVYAGASFR.K

22226198 2251.2796   2251.1219   0.1577  0   150   R.SGETEDTFIADLSVGLATGQIK.T

176 Enolase (2-phosphoglycerate dehydratase)Lycopersicon esculentum 119354 1803.9602   1803.9366   0.0236  0   31   R.AAVPSGASTGIYEALELR.D 47798.3 5.68 54897 5.94

1826.8881   1826.8686   0.0196  0   29   R.IEEELGSEAVYAGASFR.K

2003.1067   2003.0951   0.0116  0   38   K.LVLPVPAFNVINGGSHAGNK.L

33415263 1899.9575   1899.9474   0.0102  0   31   K.LAMQEFMILPVGASSFK.E

177 hypothetical protein Vitis vinifera 124689928 147859325 1097.4439   1097.5076   -0.0637  0   41   R.CGFVQFANR.A 47731.4 6.16 51399 5.91

1190.5484   1190.6043   -0.0559  0   56   R.LNWATLGAGER.R + kynurenin (W)

133862314 1049.4134   1049.4777   -0.0643  0   47   R.FGDEGEQLR.A

183 putative palmitoyl-protein thioesterase Oryza sativa 12597876 1240.6565   1240.6339   0.0226  0   61   K.LYTEDWIGLK.A + kynurenin (W) 31999.2 5.52 39762 4.27

1268.6474   1268.6288   0.0186  0   (46)  K.LYTEDWIGLK.A + Double Ox (W)

190 Tubulin beta chain (Beta tubulin) Glycine max 1351202 1138.6549   1138.6862   -0.0313  0   42   K.LAVNLIPFPR.L 45751.1 5.63 55259 4.50

1341.6068   1341.6313   -0.0244  0   92   R.INVYYNEASGGR.Y

2100.0911   2100.0851   0.0061  1   65   K.GHYTEGAELIDSVLDVVRK.E

191 disulfide isomerase Cucumis sativus 11559422 1605.9779   1605.8726   0.1053  0   52   K.AASVLSSHDPPITLAK.V 37271.8 5.07 63173 4.38

15223975 1720.8981   1720.8155   0.0826  0   51   K.LDATANDIPSDTFDVK.G

FASTS score ANEEVNQEIATEFEVK

2.3E-11 GYTPIYFK

DEIFVDSQDFH

192 chloroplast HSP70 Cucumis sativus 125441207 124245039 1375.4948   1372.7198   2.7751  0   98   K.DIDEVILVGGSTR.I 75396.1 5.18 68666 4.50

1463.2923   1460.7623   2.5300  0   74   K.QFAAEEISAQVLR.K

125414994 1736.5895   1734.9052   1.6843  1   23   R.QAVVNPENTFFSVKR.F

6746592 1581.9727   1579.7994   2.1733  0   70   K.AVITVPAYFNDSQR.Q 77105.6 5.13

119331002 2148.3416   2148.1062   0.2354  1   81   R.DAKLSFNDIDEVILVGGSTR.I

193 beta-tubulin Medicago sativa subsp. falcata 14331109 1138.6962   1138.6862   0.0100  0   44   K.LAVNLIPFPR.L 48115 4.73 53353 4.81

1327.6328   1327.6156   0.0172  0   65   R.VNVYYNEASGGR.Y

1699.8308   1699.8205   0.0103  0   62   K.NSSYFVEWIPNNVK.S + kynurenin (W)

2100.1040   2100.0851   0.0190  1   70   K.GHYTEGAELIDSVLDVVRK.E

194 alpha tubulin 1 Pseudotsuga menziesii var. menziesii 125425369 56481443 1398.4031   1395.7510   2.6521  0   51   R.QLFHPEQLISGK.E 49642 4.93 55991 5.03

1716.6304   1714.9141   1.7163  0   81   R.AIFVDLEPTVIDEVR.T

1809.3252   1807.9178   1.4074  0   96   R.IHFMLSSYAPVISAEK.A

1977.7173   1976.8751   0.8422  0   118   K.TVGGGDDAFNTFFSETGAGK.H

125935371 1702.6575   1700.8984   1.7591  0   54   R.AVFVDLEPTVIDEVR.T

90674877 2383.5447   2385.1349   -1.5901  0   125   R.QLFHPEQLISGEEDAANNFAR.G

195 alpha-tubulin Gossypium barbadense 118552662 37789885 1716.6632   1714.9141   1.7491  0   109   R.AIFVDLEPTVIDEVR.T 42268.1 6.14 55746 5.17

1809.3502   1807.9178   1.4324  0   106   R.IHFMLSSYAPVISAEK.A

2383.5230   2384.1872   -0.6642  1   107   R.QLFHPEQLISGKEDAANNFAR.G

118552392 1885.0607   1884.9403   0.1204  0   55   K.CGINYQPPTVVPGGDLAK.V

62532421 2407.4348   2408.2012   -0.7663  0   26   R.FDGALNVDITEFQTNLVPYPR.I

196 putative ATP synthase beta subunit Oryza sativa Japonica Group 125448130 56784991 1389.6942   1389.6788   0.0154  0   110   K.AHGGFSVFAGVGER.T 45908.3 5.33 61531 5.23

1863.9358   1863.9366   -0.0008  0   106   R.DAEGQDVLLFIDNIFR.F

2060.0279   2060.0426   -0.0146  0   88   R.QISELGIYPAVDPLDSTSR.M

2185.1189   2185.1378   -0.0189  0   98   R.IPSAVGYQPTLATDLGGLQER.I

125437504 1172.6769   1172.6553   0.0216  0   64   K.VVDLLAPYQR.G

125436753 1722.9044   1722.9086   -0.0042  0   45   R.LVLEVAQHLGENMVR.T

197 Heat shock 70 kDa protein, mitochondrial precursorPisum sativum 125379169 585272 1565.9785   1563.8045   2.1740  0   42   K.AVITVPAYFNDAQR.Q 72300.8 5.81 68817 5.23

2311.8906   2312.2851   -0.3945  2   45   R.TTPSVVAFNQKGELIVGTPAKR.Q

70kD vacuolar H+-ATPase Daucus carota 125425369 137460 1734.5122   1732.8671   1.6451  0   43   R.EASIYTGITIAEYFR.D 68835.5 5.29

1812.2456   1810.8559   1.3897  0   18   R.LAEMPADSGYPAYLAAR.L

manual NIIHFFNIANQAVER db-entry NIIHFYNLANQAVER

manual YSGAIESFYDQFDPDFINIR db-entry YSTALESFYDQFDPDFINIR

a theoretical

b experimental

Sample Protein * Organism * Gi Accession EST ° Gi Accession Prot° Experim. Mr Calc. Mr Mass Δ MC Pept. score Sequences MW a

pIa

MW b

pIb

14 putative chitinase Musa acuminata 17932710 FASTS score AGNTTNIIDGENECGIGYDQR 33517.4 6.77 17890 3.93

5.0E-11 DIIGVTYGENIDCYTEKTIWITR

20 subtilisin-like protease C1 Glycine max 37548634 FASTS score QFEGVSINTFDIK 78964.2 7.86 64151 6.13

7.6E-05 SWDFIGIPK

32 chitinase class III-1; MtChitIII-1 Medicago truncatula 37959340 1114.6590   1114.5229   0.1361  0   43   K.YGGVMLWNR.R + kynurenin (W) 33260.5 8.44 25470 8.02

FASTS score QVFIGIPAATAAA

9.0E-11 NFDNGYSAISK

ISIGGGDGSYSISSA

100 Laccase2 Castanea mollissima 133867518 209420826 1680.8759   1680.8848   -0.0089  1 54 K.YGVTIHWHGVKQPR.N + kynurenin (W) 62646.9 6.49 84765 7.40

1153.5862   1153.6131   -0.0269  0 64 -.IDILQAYYR.S

133867050 manual YGISIHWHGVKQPR db-entry YGITIHWHGVKQPR

102 Diphenol oxidase Acer pseudoplatanus 529353 FASTS score IIDPPEVNTFGVPK 62547.5 5.45 82381 6.95

2.1E-08 EFFFAIAHHK

DHPISVPTGVDER

104 Laccase2 Toxicodendron vernicifluum 133867518 23503483 1327.6027   1327.6673   -0.0646  0   55   K.YGVTIHWHGVK.Q + Double Ox (W) 58751.9 7.34 73822 7.29

529353 FASTS score MNEEMFFAIAHHK 62547.5 5.45

2.2E-15 DHPISVPTGVDER

IIDPPEVNTFGVPK

GMNTVIIVK

105 Laccase2 Toxicodendron vernicifluum 133867518 23503483 1680.7327   1680.8848   -0.1521  1   61   K.YGVTIHWHGVKQPR.N + kynurenin (W) 58751.9 7.34 74147 7.18

FASTS score LINAGMNEEMFFAIAHHK

2.7E-08 NIIDPFV

MDVSVDGQFPGIPIR

VIDER

108 subtilisin-like protease C1 Glycine max 37548634 FASTS score QFEGVSINTFDIK 78964.2 7.86 67916 6.33

8.4E-11 SWDFIGIPK

ASIAAGNIVSAASIEGIG

109 cytoplasmic aconitate hydratase Arabidopsis thaliana 146224356  22531152 2118.7388   2119.0805   -0.3417  0   31   R.VLLQDFTGVPAVVDLACMR.D 108201 6.72 80065 6.08

2357.7598   2356.2790   1.4808  1   18   K.IIDWENTSPKLVEIPFKPAR.V + kynurenin (W)

2408.0117   2407.2681   0.7436  0   45   R.NMLVVPPGSGIVHQVNLEYLGR.V

118458443 2453.1831   2453.2266   -0.0435  0   84   R.FDTEVELAYFDHGGILPYVIR.N

118409257 2357.7598   2359.2535   -1.4937  1   47   K.IIDWENSSVKQVEIPFKPAR.V + kynurenin (W)

110 subtilisin-like protease C1 Glycine max 37548634 FASTS score KDTFAPYIAAFSSR 78964.2 7.86 65718 6.08

5.5E-10 PNANVISFTSSR

NINGQSFFEQR

SNAAGNIVSAASNQGIG

SWDFIGIPK

111 alpha-D-xylosidase Tropaeolum majus 5725356 1121.5271   1121.4890   0.0381  0   50   R.DHANYYSPR.Q 104937 5.41 79365 5.85

1502.7498   1502.7194   0.0305  0   39   R.WIEVGAFYPFSR.D + Double Ox (W)

1698.8074   1698.7849   0.0225  0   68   R.QELYQWESVAESAR.N + kynurenin (W)

1726.8069   1726.7798   0.0271  0   (43)  R.QELYQWESVAESAR.N + Double Ox (W)

112 ATP synthase subunit alpha, mitochondrialGlycine max 231585 1437.8620   1437.8415   0.0205  0   38   R.GIRPAINVGLSVSR.V 55330.6 6.23 52655 6.08

1518.7466   1518.7255   0.0211  0   (53)  R.EAFPGDVFYLHSR.L + Pyro-glu (N-term E)

1536.7548   1536.7361   0.0188  0   87   R.EAFPGDVFYLHSR.L

1719.8206   1719.8138   0.0069  0   79   R.DNGMHALIIYDDLSK.Q

1966.1182   1966.1139   0.0044  1   73   R.LTEVLKQPQYAPLPIEK.Q

2156.0484   2156.0459   0.0026  0   85   R.VYGLNEIQAGEMVEFASGVK.G

2307.1563   2307.1494   0.0069  0   90   R.EVAAFAQFGSDLDAATQALLNR.G

113 hypothetical protein Trifolium pratense 117361423 84453222 1050.4777   1050.5022   -0.0244  0   65   K.SYYFSGSLK.L 46443 7.89 79365 5.71

1206.6145   1206.6761   -0.0615  0   33   R.FVQPIYTAIR.D

151014229 1033.5316   1033.5556   -0.0240  0   70   R.ILFDVANSR.L

125318759 1192.6011   1192.6240   -0.0229  0   20   R.FAQPVYEAIR.D

1738.8721   1739.8631   -0.9909  1   32   R.FAQPVYEAIRDEFR.K

1866.9721   1867.9580   -0.9859  2   41   R.FAQPVYEAIRDEFRK.Q

114 diphenol oxidase Acer pseudoplatanus 133866816 529353 1475.7660   1475.7442   0.0218  0   30   R.HASWGMSTVIIVK.N + Double Ox (W) 62547.5 5.45 96480 5.48

FASTS score VMNEEMFF

4.5E-09 HNEASYGVTIHW

117 chloroplast nucleoid DNA-binding protein-relatedArabidopsis thaliana 151014229 18391062 1033.4867   1033.5556   -0.0689  0   79   R.ILFDVANSR.L 47661.1 7.48 48020 9.07

117361423 1050.4335   1050.5022   -0.0686  0   63   K.SYYFSGSLK.L

manual INAFGSTGSGTVIDSGTVISR db-entry LLAFNPSTGAGTIIDSGTVITR

118 chloroplast nucleoid DNA-binding protein-relatedArabidopsis thaliana 125318760 18391062 1192.5020   1192.6240   -0.1220  0   17   R.FAQPVYEAIR.D 47661.1 7.48 47600 8.87

1739.8129   1739.8631   -0.0501  1   27   R.FAQPVYEAIRDEFR.K

1867.9252   1867.9580   -0.0328  2   44   R.FAQPVYEAIRDEFRK.Q

manual INAFGSTGSGTVIDSGTVISR db-entry LLAFNPSTGAGTIIDSGTVITR

119 alpha-D-xylosidase Tropaeolum majus 5725356 1121.5637   1121.4890   0.0747  0   56   R.DHANYYSPR.Q 104937 5.41 31304 8.70

1502.7779   1502.7194   0.0586  0   35   R.WIEVGAFYPFSR.D + Double Ox (W)

1698.8303   1698.7849   0.0454  0   72   R.QELYQWESVAESAR.N + kynurenin (W)

120 porin I, 36K Solanum tuberosum 629728 1438.8026   1438.7681   0.0346  0   49   K.ASALIQHEWRPK.S + kynurenin (W) 29364.1 7.78 23953 8.53

1466.7942   1466.7630   0.0312  0   (28)  K.ASALIQHEWRPK.S + Double Ox (W)

1547.8806   1547.8558   0.0248  1   114   K.KGELFLADVSTQLK.N

121 Acidic endochitinase precursor Cucumis sativus 167515 FASTS score FDYDWVQFYNNR 30774.4 4.46 21701 8.56

3.1E-20 HIGPAATAAAPSG

IFDNGYSASIK

ANYIWNTYIG

ISNGGGDGSYSIS

122 beta-D-galactosidase Pyrus pyrifolia 125482162 61162203 1277.6740   1277.6000   0,0740  0   24   K.GEAWVNGESIGR.Y + kynurenin (W) 92044.2 7.74 27443 8.48

1655.8769   1655.8202   0.0567  0   76   K.NCGKPSQTLYHVPR.S

90487062 1052.5500   1052.5291   0.0209  0   28   K.NQPLTWYK.A + kynurenin (W)

FASTS score ESSGDTIVIFEEIGGDPTK

3.6E-09 QPSQTIYFVPR

chloroplast nucleoid DNA-binding protein-related Arabidopsis thaliana 151014229 18391062 1033.5856   1033.5556   0.0300  0   79   R.ILFDVANSR.L 47661.1 7.48

125318759 1739.9003   1739.8631   0.0372  1   25   R.FAQPVYEAIRDEFR.K

1867.9991   1867.9580   0.0411  2   31   R.FAQPVYEAIRDEFRK.Q

123 glyceraldehyde 3-phosphate dehydrogenase Antirrhinum majus 115594542 1345501 1497.8732   1497.8402   0.0330  0   96   R.VPTVDVSVVDLTVR.I 36553.8 8.34 29310 8.48

1796.7938   1796.7642   0.0297  0   39   K.LVSWYDNEWGYSSR.V + Double Ox (W); kynurenin (W)

2048.0725   2048.0612   0.0114  0   72   R.FGIVEGLMTTVHSITATQK.T

2158.0159   2158.0065   0.0094  0   138   K.GILGYTEDDVVSTDFIGDSR.S

2290.2351   2290.2354   -0.0003  1   6   K.LTGMAFRVPTVDVSVVDLTVR.I

2645.3709   2645.3846   -0.0137  1   23   K.VINDRFGIVEGLMTTVHSITATQK.T

125385888 2170.0337   2170.0429   -0,0092  0   71   K.GILGYIDEDVVSTDFIGDSR.S

124 putative beta-galactosidase Lycopersicon esculentum 7939623 1396.8697   1396.7939   0.0759  1   36   R.RVLISGSIHYPR.S 93243.4 6.8 30424 8.22

2362.1565   2362.1593   -0.0028  0   42   K.DGGLDVIETYVFWNLHEPVR.N + kynurenin (W)

2390.1441   2390.1542   -0.0101  0   (39)  K.DGGLDVIETYVFWNLHEPVR.N + Double Ox (W)

2605.2300   2605.2812   -0.0512  1   4   K.SKDGGLDVIETYVFWNLHEPVR.N + Double Ox (W)

125 glyceraldehyde 3-phosphate dehydrogenase-like protein Solanum tuberosum 62974693 82400130 1768.7901   1768.7692   0.0209  0   52   K.LVSWYDNEWGYSSR.V + 2 kynurenin (W) 36675 6.34 30224 8.05

1796.7847   1796.7642   0.0206  0   (29)  K.LVSWYDNEWGYSSR.V + Double Ox (W); kynurenin (W)

2158.0186   2158.0065   0.0121  0   138   K.GILGYTEDDVVSTDFIGDSR.S

129 malate dehydrogenase Glycine max 125346411 5929964 1320.5598   1317.6928   2.8670  0   97   R.DDLFNINAGIVK.A 36141.7 8.23 36421 6.61

1578.0365   1575.8984   2.1382  0   91   K.ALEGADVVIIPAGVPR.K

1795.4937   1794.0436   1.4501  0   87   K.VAVLGAAGGIGQPLALLMK.L

125482740 1377.5030   1374.7983   2.7047  1   58   K.RLFGVTTLDVVR.A

3011.3611   3014.5357   -3.1746  1   17   K.YCPHALVNMISNPVNSTVPIAAEVFKK.A

130 triosephosphate isomerase Glycine max 62976185 77540216 957.3711   957.4708   -0.0997  0   60   K.FFVGGNWK.C + kynurenin (W) 27204.1 5.87 31099 6.61

1694.8344   1694.8740   -0.0395  0   62   K.VATPAQAQEVHFELR.K

1714.8408   1714.8791   -0.0382  0   77   K.WFHANISPEVAATIR.I + kynurenin (W)

1742.8368   1742.8740   -0.0372  0   (31)  K.WFHANISPEVAATIR.I + Double Ox (W)

1822.9409   1822.9689   -0.0280  1   70   K.VATPAQAQEVHFELRK.W

2071.0376   2071.0421   -0.0045  0   34   K.NLLRPDFHVAAQNCWVK.K + kynurenin (W)

133 UDP-glucose pyrophosphorylase Amorpha fruticosa 17026394 1678.9181   1678.8889   0.0292  1   102   K.TLADVKGGTLISYEGR.V 51584.2 6.07 55624 6.95

2467.2757   2467.2846   -0.0088  0   112   K.ATSDLLLVQSDLYTLEDGFVIR.N

136 enolase Gossypium barbadense 33415263 1227.6001   1227.6070   -0.0069  0   20   K.MGVEVYHHLK.S 47731.4 6.16 55746 6.22

1803.9535   1803.9366   0.0169  0   54   R.AAVPSGASTGIYEALELR.D

2251.1209   2251.1219   -0.0010  0   123   R.SGETEDTFIADLSVGLATGQIK.T

223944349 2320.1975   2320.2426   -0.0451  1   28   K.YNQLLRIEEELGAIAVYAGAK.F 48114.6 5.59

6996529 1678.9717   1678.9882   -0.0165  2   72   K.KIPLYKHIANLAGNK.K 47784.2 5.14

137 alpha-galactosidase 1 Pisum sativum 117898294 53747927 1126.6028   1126.6168   -0.0140  0   57   K.APLLLGCDIR.N 44963.6 6.52 38985 5.94

FASTS score IDSGYYTCSR

5.9E-17 EDIGIPENSVVVAR

IDANDVYADYAR

DNCYNDGTQPTVR

139 glutathione S-transferase Medicago sativa 16416392 FASTS score FISINPFGQVPAIEH 24368 5.67 20860 7.35

7.6E-16 DVSIVDG

LVWELGIKP

EYADQGTQII

141 Oxygen-evolving enhancer protein 2, chloroplast precursor (OEE2)Pisum sativa 131390 1386.7063   1386.6667   0.0397  1   19   R.KFVEDTASSFSVA.- 28047.5 8.28 14684 8.22

1571.7665   1571.7355   0.0311  0   48   K.SITDYGSPEEFLSK.V

1839.9412   1839.9326   0.0086  1   16   R.TADGDEGGKHQLITATVK.D

124940950 1350.7252   1350.6931   0.0321  0   94   -.AYGEAANVFGKPK.T

AT-RAB2; GTP binding Arabidopsis thaliana 15235981 1691.8051   1691.7903   0.0148  0   26   R.ETFNHLASWLEDAR.Q + kynurenin (W) 23164.3 6.96

1769.8556   1769.8471   0.0085  0   73   K.IQDGVFDVSNESYGIK.V

1784.9330   1784.9209   0.0121  0   68   R.FQPVHDLTIGVEFGAR.M

142 Glycine-rich protein 2b Arabidopsis thaliana 150041632 17366505 2392.1475   2393.1287   -0.9812  0   151   K.GFGFITPDDGGEDLFVHQSSIR.S 19077.2 6.29 14556 7.43

manual SLGEGETVEYQIESGNDGR db-entry (14779139)LGEGETVEFQIVLGEDGR

143 heat shock protein 17.4 Quercus suber 4456758 1616.8584   1616.8198   0.0386  1   56   R.IDWKETPEAHIFK.A + kynurenin (W) 17398.8 6.34 12677 7.35

973.5885   973.5345   0.0541  1   31   R.FRLPENAK.V

144 Nucleoside diphosphate kinase 1 Glycine max 62975153 2498078 1346.8137   1346.7557   0.0580  1   50   R.GLVGEIISRFEK.K 16442.8 5.93 9427 7.09

962.5562   962.4821   0.0741  0   47   R.GDFAVEIGR.N

145 cystatin Castanea sativa 62975975 4150974 1466.7545   1466.7113   0.0432  0   99   K.GHENSLQIDDLAR.F 11389 6.91 7704 7.09

2046.0626   2046.0534   0.0092  2   85   R.FAVDDHNKKENTLLQFK.K

2174.1465   2174.1483   -0.0018  3   80   R.FAVDDHNKKENTLLQFKK.V

148 ribulose-bisphosphate carboxylase Platanus x acerifolia 133865265 110224770 996.6010   996.5029   0.0982  0   36   K.TYPNAYIR.C 8410.72 9.15 8963 8.56

1124.6909   1124.5978   0.0931  1   55   K.KTYPNAYIR.C

115597468 973.4923   973.3963   0.0960  0   28   R.MPGYYDGR.Y

119003543 2037.0840   2037.0670   0.0171  1   26   K.KFETLSYLPPLSDESIAK.E

149 cyclophylin Digitalis lanata 1563719 1096.6592   1096.5553   0.1040  1   57   K.FADENFVKK.H 18127.8 8.52 13217 9.46

2706.1468   2706.2053   -0.0585  0   57   R.VIPGFMCQGGDFTAGNGTGGESIYGAK.F

170440 1671.9177   1671.8653   0.0524  0   19   K.HVVFGQVVEGMDVIK.K 17910.5 8.83

2749.2373   2749.2951   -0.0578  0   14   K.HTGPGILSMANAGPGTNGSQFFICTAK.T

829119 1510.7949   1510.7238   0.0711  0   45   K.VFFDMTIGGQPAGR.I 18159.7 8.36

152 17.3 kDa class II heat shock protein (Hsp17.3) (Hsp20.2)Lycopersicon peruvianum 75279028 1698.9213   1698.8788   0.0426  0   54   K.VQVEEDNVLLISGER.K 17265 6.32 11256.7 5.60

1854.9297   1854.8862   0.0436  0   54   K.EYPNSYVFVVDMPGLK.S + Pyro-glu (N-term E)

2199.1419   2199.1382   0.0037  1   97   K.SGDIKVQVEEDNVLLISGER.K

30575570 1684.9036   1684.8631   0.0405  0   79   K.VQVEDDNVLLISGER.K 11141 8.01

2185.1277   2185.1226   0.0052  1   112   K.SGDIKVQVEDDNVLLISGER.K

153 heat shock protein 17d Quercus suber 15558864 1557.9051   1557.8514   0.0537  2   65   R.FRLPENAKVEEVK.A 12546.8 5.44 11714 5.60

1573.8452   1573.7888   0.0564  1   48   K.VDWKETPNAHVFK.A + kynurenin (W)

1662.7760   1662.7234   0.0527  1   97   R.SQEQEEKSDTWHR.V + kynurenin (W)

2124.1621   2124.1425   0.0196  3   87   K.ADVPGLKKEEVKVEIEEGR.V

153L thioredoxin-dependent peroxidase Plantago major 115439131 1875.9567   1875.9651   -0.0084  0   87   K.GVDDILLVSVNDPFVMK.A 17290.9 5.59 13333 5.65

224143583 1805.9762   1805.9749   0.0013  0   68   K.VILFGVPGAFTPTCSLK.H 17416.1 5.55

158L heat shock protein 17.4 Quercus suber 75975966 4456758 966.5911   966.4770   0.1141  0   36   R.ETTAFATAR.I 17398.8 6.34 13421 6.08

973.6482   973.5345   0.1138  1   33   R.FRLPENAK.V

2256.1648   2256.1596   0.0052  2   51   K.KEEVKVEVEDGNVLQISGER.S

2950.4717   2950.5610   -0.0893  3   21   K.ADLPGLKKEEVKVEVEDGNVLQISGER.S

119004323 1724.8842   1724.7866   0.0976  2   23   R.HVEKEDKNDTWHR.V + Double Ox (W)

159 flavodoxin-like quinone reductase 1Arabidopsis thaliana 150881148 15239652 1237.6741   1237.6091   0.0650  0   55   K.AFLDATGGLWR.T + Double Ox (W) 21795.8 5.96 22975 5.94

1455.6945   1455.6378   0.0567  0   102   K.GGSPYGAGTFAGDGSR.Q

1682.8448   1682.8011   0.0437  1   125   K.VKGGSPYGAGTFAGDGSR.Q

1753.8968   1752.8795   1.0174  0   38   R.QPTQLELEQAFHQGK.Y

1765.8705   1765.8385   0.0321  0   48   K.VYIVYYSMYGHVEK.L

164 20S proteasome subunit PAB1 Arabidopsis thaliana 120534400 3421075 1555.2484   1554.9133   0.3351  1   49 R.LYKEPIPVTQLVR.E 25701.3 5.53 30158 5.31

1979.8791   1978.9887   0.8904  1   62   K.FRVLTPAEIDDYLAEVE.-

2121.5261   2121.1793   0.3468  0   87   K.LVQIEHALTAVGSGQTSLGIK.A

2192.2061   2192.1299   0.0762  0   12   K.IQLLTPNIGVVYSGMGPDFR.V

3152562 1849.8627   1849.8370   0.0257  0   129   M.GDSQYSFSLTTFSPSGK.L + Acetyl (N-term)

164L triosephosphate isomerase Arabidopsis thaliana 125407461 7839391 953.6223   957.4708   -3.8485  0   51   K.FFVGGNWK.C + kynurenin (W) 33345.8 7.67 31999 5.31

1281.5218   1284.6536   -3.1318  1   50   K.TFDVCFKQLK.A

1482.2383   1484.7623   -2.5240  1   58   R.HVIGEDDQFIGKK.A

125932133 1432.0523   1434.7354   -2.6831  0   52   K.GGAFTGEISVEQLK.D

1821.5395   1820.9341   0.6054  0   41   K.AAYALSEGLGVIACIGEK.L

166 eukaryotic phosphomannomutase family proteinArabidopsis thaliana 150993646 15225896 1869.0609   1869.0359   0.0250  0   80   K.KPGVIALFDVDGTLTAPR.K 27761.6 5.33 29633 5.99

168 lactoylglutathione lyase Arabidopsis thaliana 15220397 1088.6250   1088.5913   0.0337  1   40   R.RMLHVVYR.V 39167.1 6.97 36262 4.95

1512.7527   1512.7428   0.0099  0   17   R.GPTPEPLCQVMLR.V

1697.8344   1697.8260   0.0084  0   91   K.GNAYAQIAIGTDDVYK.T

157890952 1129.5233   1129.5590   -0.0356  1   15   K.FYEKALGMR.L 31912.4 5.37

223542315 1464.7332   1464.7249   0.0084  0   75   K.DPDGYIFELIQR.G 31547.1 7.63

115475151 1697.8344   1697.8260   0.0084  0   35   K.GNAYAQVAIGTEDVYK.S 32553.4 5.51

173 UDP-D-apiose/UDP-D-xylose synthase 2 Arabidopsis thaliana 126365555 18390863 1703.5597   1701.8032   1.7566  0   69   R.MDFIPGIDGPSEGVPR.V 43790.2 5.58 44470 5.60

1951.9002   1950.9659   0.9343  0   132   R.ANGHIFNVGNPNNEVTVR.Q

1966.8627   1965.9656   0.8971  0   38 R.ANGHIFNVGNPNNEVTVR.Q + Me-ester (DE)

178 endochitinase Musa acuminata 15705988 FASTS score SSEGPNCPNGE 24692.4 7.66 41546 3.82

1.3E-06 GITTNIIDGENEC

ANTYGENLDCYTEQPSI

180 basic chitinase Oryza sativa 227845 FASTS score VPGYGITTNIIDGENECG 35565.6 6.05 39937 3.99

2.8E-13 IIGVTYGENIDCYTQQ

181 14-3-3 protein Nicotiana tabacum 42491254 1832.9269   1832.9380   -0.0111  0   70   K.SAQDIANTELAPTHPIR.L 29025.5 4.72 36904 3.99

2123.0528   2123.0647   -0.0119  0   34   K.AYQSATTAAEAELPPTHPIR.L

182 14-3-3 protein Vigna angularis 149216123 13928452 1829.3136   1828.9431   0.3705  0   93   K.SAQDIANAELPPTHPIR.L 29202.7 4.66 35397 4.10

2163.1194   2162.9499   0.1695  1   12   K.LAEQAERYEEMVEFMEK.V

119429327 1829.3136   1829.9635   -0.6499  0   53   K.SAQDIALSDLPPTHPIR.L

188 SGT1 Nicotiana benthamiana 58760268 1482.7348   1482.7354   -0.0006  0   32   K.LNYFTEAVVDANK.A * 41238.7 5.24 50505 4.10

2382.2364   2382.2371   -0.0007  1   25   K.YRHEFYQKPEEVVVTIFAK.G

manual AEPLQWASLEYAK db-entry AEPLHWTSLEYTR

* although this is matched to the correct protein the actual sequence of this peptide is LNYFTEAVADANR

189 heat shock cognate 70-1 Arabidopsis thaliana 450880 1373.7221   1373.6033   0.1188  0   29   K.NALENYAYNMR.N 70071.4 5.11 45757 4.44

1555.8446   1555.7453   0.0994  1   15   R.ARFEELNMDLFR.K2657.1848   2657.2608   -0.0760  0   98   K.EQVFSTYSDNQPGVLIQVYEGER.A

a theoretical

b experimental