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REGULAR ARTICLE
The proteome of maritime pine wood forming tissue
Jean-Marc Gion1*, Céline Lalanne1*, Grégoire Le Provost1*, Hélène Ferry-Dumazet2,Jorge Paiva1, 3, Phillipe Chaumeil1, Jean-Marc Frigerio1, Jean Brach1, Aurélien Barré2,Antoine de Daruvar2, 4, Stéphane Claverol5, Marc Bonneu5, Nicolas Sommerer6,Luc Negroni7 and Christophe Plomion1
1 UMR 1202 BIOGECO, INRA, Equipe de Génétique, Cestas, France2 Centre de Bioinformatique de Bordeaux, Université V. Segalen Bordeaux 2, Bordeaux, France3 Plant Cell Biotechnology Lab. IBET/ITQB, Oeiras, Portugal4 UMR 5162, Génomique Fonctionnelle des Trypanosomatides, CNRS – Université Bordeaux 2, Bordeaux, France5 Pôle Protéomique, Plateforme Génomique Fonctionnelle Bordeaux, Université V. Segalen Bordeaux 2,
Bordeaux, France6 Unité de Recherches Protéomique, UR 1199, INRA, Montpellier, France7 UMR de Génétique Végétale, INRA/UPS/CNRS/INA-PG, Gif-sur-Yvette, France
Wood is one of our most important natural resources. Surprisingly, we know hardly anything aboutthe details of the process of wood formation. The aim of this work was to describe the main proteinsexpressed in wood forming tissue of a conifer species (Pinus pinaster Ait.). Using high resolution2-DE with linear pH gradient ranging from 4 to 7, a total of 1039 spots were detected. Out of the240 spots analyzed by MS/MS, 67.9% were identified, 16.7% presented no homology in the data-bases, and 15.4% corresponded to protein mixtures. Out of the 57 spots analyzed by MALDI-MS,only 15.8% were identified. Most of the 175 identified proteins play a role in either defense (19.4%),carbohydrates (16.6%) and amino acid (14.9%) metabolisms, genes and proteins expression (13.1%),cytoskeleton (8%), cell wall biosynthesis (5.7%), secondary (5.1%) and primary (4%) metabolisms. Asummary of the identified proteins, their putative functions, and behavior in different types of woodare presented. This information was introduced into the PROTICdb database and is accessible athttp://cbib1.cbib.u-bordeaux2.fr/Protic/Protic/home/index.php. Finally, the average protein amountwas compared with their respective transcript abundance as quantified through ESTcounting in acDNA-library constructed with mRNA extracted from wood forming tissue.
Received: September 6, 2004Revised: November 17, 2004
Accepted: December 14, 2004
Keywords:
Mass spectrometry / Pinus pinaster Ait. / Proteome analysis / Wood
Proteomics 2005, 5, 3731–3751 3731
1 Introduction
In perennial plants, the successive addition of secondaryxylem tissue differentiated from the vascular cambium givesrise to a unique tissue called wood. Wood is composed of
non-conducting and conducting elements implicated in thelong distance transport of water and nutriments in trees. Inconifers, wood is comprised of two main cell types: tracheidsand ray parenchyma. This simplicity hides the fact that it isalso a highly variable raw material. Field experiments haveshown genetic factors can influence the activity of the vas-cular cambium and the differentiation of newly divided cells,ultimately influencing wood and end-use properties [1–3].The ageing process constitutes another important source ofvariation affecting the characteristics of secondary xylem(reviewed in Zobel and Sprague [4]). Wood derived from ayoung cambium is referred as juvenile wood (JW), while
Correspondence: Dr. Christophe Plomion, UMR 1202 BIOGECO,INRA, Equipe de Génétique, 69 route d’Arcachon, F-33610 CestasCédex, FranceE-mail: [email protected]: 133-5-5712-2881
Abbreviations: SAM-S, S-adenosylmethionine synthetase; JW,juvenile wood; MW, mature wood; EW, early wood; LW, latewood; OW, opposite wood; CW, compression wood * These authors contributed equally.
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
DOI 10.1002/pmic.200401197
3732 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751
wood formed by an older cambium is referred as maturewood (MW). In fast growing pine trees, the transition be-tween JW and MW occurs around 10 years of age (Fig. 1A)and is accompanied by drastic changes in many wood prop-erties. Seasonal and gravitational effects are, among envi-ronmental factors, the most significant external sources ofvariation affecting wood characteristic. In temperate zones,climatic variation during the annual course of the vascularcambium give rise to early wood (EW) formed early duringthe growing season, and late wood (LW) formed in late sum-mer (Fig. 1B). The major changes are in the structure of thetracheids, which affect their ability to transport water underwet and dry conditions. A change in the orientation of aconifer tree stem stimulates the formation of a specializedtype of wood at the underside of a bent tree, termed com-pression wood (CW) (Fig. 1C). It serves to reorient the stemto a vertical position. CW differs anatomically in its chemicalcomposition, compared to opposite wood (OW) formed atthe other side of the leaning stem (reviewed in Timell [5]).The formation of these six types of wood is the result of pro-found molecular changes during xylogenesis, triggered byexternal (e.g., temperature, photoperiod [6, 7]) and/or endog-enous factors (e.g., phytohormones, sugars [8]). The con-siderable plasticity in anatomical, chemical and physicalwood properties provides a unique opportunity to dissect themolecular and biochemical mechanisms responsible forsuch differences.
Wood formation (xylogenesis) includes four major steps:cell division, cell expansion, secondary cell wall thickeningand programmed cell death (reviewed by Lachaud et al. [7]
and Plomion et al. [9]). It is a complex phenomenon driven bythe coordinate expression of numerous genes especiallyinvolved in the biosynthesis and the assembly of poly-saccharides, lignins, and cell wall proteins [8, 10]. Up to now,the study of molecular mechanisms involved in the develop-ment of wood has mainly taken a transcriptomic approach,combining expressed sequence tag (EST) sequencing andtranscript profiling [11–18]. Comparatively, there has beenno large-scale project to identify proteins from differentiat-ing secondary xylem, and only few studies have reported on ahandful of proteins in wood forming tissue [19–24]. Theobjective of the present work was to partially fill this gap andprovide for the first time in a forest tree species an overviewof the proteome expressed in this highly specialized tissue,and to serve as a basis for future proteome comparisons ofenvironmentally challenged trees, and in the course of theirdevelopment.
Maritime pine (Pinus pinaster Ait.), a conifer of greateconomic and ecological interest in Southwestern Europe(where it covers 4 millionhectares), was chosen as a modelspecies. A reference map was first obtained using high reso-lution 2-DE with proteins extracted from differentiatingxylem associated to the six types of wood mentioned above. Atotal of 300 spots were then excised from the gels and ana-lyzed by LC ESI-MS/MS, MALDI-TOF MS or internal se-quencing. The identified proteins are discussed and classi-fied based on their putative function and their behavior inthe six types of wood. Finally, the expression levels of 95 pro-teins quantified by 2-DE was compared with mRNA levelsquantified by EST counting.
Figure 1. The six types of wood typically found in a conifer tree. (A) Juvenile wood (JW) vs. mature wood (MW),(B) early wood (EW) vs. late wood (LW), and (C) compression wood (CW) vs. opposite wood (OW). (D) Upper andlower part of a leaning stem of a 4 months bent tree showing the red wood phenotype of CW immediately afterdebarking, 14-year-old tree.
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
Proteomics 2005, 5, 3731–3751 Plant Proteomics 3733
2 Material and methods
2.1 Sampling differentiating secondary xylem tissue
To take into account the natural variability found in the woodof an adult conifer tree, differentiating xylem tissues weresampled: (i) at the base (breast height) and at the top of thestem of a 30-year-old maritime pine tree, corresponding toxylem associated to MW (formed by a 25-year-old cambium)and JW (formed by a 3-year-old cambium), respectively.Samples were taken in April (27.04.01). (ii) in April(25.04.00) and August (23.08.00) of two 14-year-old maritimepine trees belonging of the same clone (accession #4015),corresponding to xylem associated to EW and LW, respec-tively. (iii) in the upper and lower side of a 14-year-old mar-itime pine genotype (accession #105), whose grafted copieswere bent to a 157 angle by tying their trunk to neighbortrees, and sampled after 2 years of mechanical bending, cor-responding to xylem associated to OW and CW, respectively.Samples were taken in August (23.08.00).
After that, bark, phloem and cambium were peeled fromthe stem, scrapings were taken from exposed differentiatingxylem, immediately frozen in liquid N2 and stored at 2807Cuntil used for protein extraction.
2.2 Protein extraction and quantification
Starting from 500 mg fresh tissue, total protein of each of thesix samples described above was extracted following the pro-cedure described by Damerval et al. [25], with the followingmodifications: (i) for protein resolubilization, the “UKS”buffer was replaced by “TCT” buffer (urea 7 M, thiourea 2 M,Triton X-100 0.4%, CHAPS 4%, DTT 10 mM, IPG buffer 1%),(ii) samples were then centrifuged (4 min, 2000 rpm, 207C)and the supernatant was transferred to a new Eppendorftube. This step was added in order to insure that all cellularfragments were removed from the extract. Proteins werestored at 2807C. Three extractions were completed for eachsample and pooled for protein quantification. The resultingmix was quantified over six replicated assays, using the pro-tocol described by Ramagli et al. [26]. The mean concentra-tion was then calculated and used to load 300 mg of proteinson each IPG strip.
2.3 2-DE
2-DE [27] was used to analyze total protein from the xylemsamples following the procedure of Bahrman et al. [28]adapted for the IPGphor system (Amersham Biosciences,Uppsala, Sweden). For the IEF, 24 cm strips were used with alinear pH gradient ranging from 4 to 7. Proteins were mixedwith a strip rehydration solution (urea 7 M, thiourea 2 M, Tri-ton X-100 0.4%, CHAPS 4%, DTT 10 mM, IPG buffer 1%).The IPGphor system was then programmed for 12 h at 30 V(active rehydration), 1 h at 200 V, 1 h at 500 V, 1 h at 1000 V,
30 min from 1000 V to 8000 V and finally 8000 V per hour toachieve a total of 74 000 Vh. After approximately 15 h of IEF,strips were equilibrated (SDS saturation) with a 10 mL ofequilibration solution (Tris-HCl pH 8.8 50 mM, urea 6 M,glycerol 30%, SDS 2%, bromophenol blue). Equilibrationwas performed in two steps, with DTT (65 mM) in the firstequilibration, and iodoacetamide (135 mM) in the secondequilibration (without DTT). SDS-PAGE was performed bybatches of 15 gels run in a buffer (Tris 25 mM, glycine 0.2 M
and SDS 0.1 M) at 110 V for 17 h. To ensure gel reproduci-bility, five replicates were performed for each sample, result-ing in a total of 30 gels from which the four best were select-ed with the help of the image analysis software.
2.4 Gel staining
CBB G-250 (Bio-Rad, Hercules, CA, USA) was used for gelstaining. Gels were fixed for 2 h in a solution containing2% phosphoric acid and 50% ethanol. After three waterwashings of 30 min each, the gels were placed in an incuba-tion solution (methanol 34%, ammonium sulfate 17%,phosphoric acid 2%) for 1 h, and then immersed in a stain-ing solution (methanol 34%, ammonium sulfate 17%, phos-phoric acid 2%, Coomassie blue 0.05%) for 5 days. Finally,the gels were stored in a 5% acid acetic solution before scan-ning and spot picking after several days.
2.5 Image acquisition and spot detection
Stained gels were digitalized using the M141 image scannerand the LabScan software (Amersham Biosciences). First, acalibration with a grey scale was necessary to transform greylevels into OD values for each pixel of the gel picture. Thecalibration method used was the colloidal blue methoddescribed in the LabScan manual. All the gel pictures weresaved as tiff files. Image analysis was performed using theImage Master 2D-Elite software (IM2D; Amersham Bio-sciences). The 30 gel images were placed in one folder. Thewizard detection method proposed by the software was usedto detect the spots. Then, automatically detected spots weremanually checked, and some of them manually added orremoved. Following the detection procedure, the volume foreach spot corresponded to a gross value. In order to eliminatethe background from this gross value, the mode of non spotof IM2D was used. Finally, all the gels were matched in orderto attribute a common spot identity for the same spotsderived from different images. For this, we used the auto-matically matching options of IM2D. After visual checking ofthe matching, the IM2D software was used to construct amaster gel (reference gel, Fig. 2). For each sample, when aprotein was detected in all of the four replicates, this proteinwas automatically added to the master gel, thus creating areference map of wood forming tissue. Normalized volumeswere finally obtained using the total spot volume normal-ization procedure of IM2D.
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
3734 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751
Figure 2. Reference 2-DE map for maritime pine wood forming tissue (4–7 linear gradient). Proteins that were identified are marked witharrows and numbers following Table 1. Unknown function proteins are squared.
2.6 Characterization by MS
2.6.1 In-gel protein digestion
CBB-stained protein spots were manually excised from thegels and washed twice with ultra-pure water. Spots weresubsequently washed in H2O/MeOH/acetic acid (47.5:47.5:5)until destaining. The solvent mixture was removed and re-placed by ACN. After shrinking of the gel pieces, ACN wasremoved and gel pieces were dried in a vacuum centrifuge.Gel pieces were rehydrated in 10 ng/mL trypsin (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM NH4HCO3 andincubated overnight at 377C. The supernatant was removedand stored at 2207C, and the gel pieces were incubated15 min in 50 mM NH4HCO3 at room temperature underrotary shaking. This second supernatant was pooled with theprevious one, and a H2O/ACN/HCOOH (47.5:47.5:5) solu-tion was added to the gel pieces for 15 min. This step was
repeated once. Supernatants were pooled and concentratedin a vacuum centrifuge to a final volume of 30 mL. Digestswere finally acidified by addition of 1.8 mL of acetic acid andstored at 2207C.
2.6.2 On-line capillary HPLC nanospray ion trap
MS/MS analysis
Peptide mixtures were analyzed by on-line capillary HPLC(LC Packings, Amsterdam, The Netherlands) coupled to ananospray LCQ ion trap mass spectrometer (Thermo-Finnigan, San Jose, CA, USA). Peptides were separated on a75 mm id615 cm C18 PepMapTM column (LC Packings).The flow rate was set at 200 nL/min. Peptides were elutedusing a 5–50% linear gradient of solvent B in 30 min (sol-vent A was 0.1% formic acid in 5% ACN, and solvent B was0.1% formic acid in 80% ACN). The mass spectrometer wasoperated in positive ion mode at a 2.5 kV needle voltage and a
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
Proteomics 2005, 5, 3731–3751 Plant Proteomics 3735
44 V capillary voltage. Data acquisition was performed in adata-dependent mode consisting of alternatively in a singlerun, a full scan MS over the range m/z 50–2000 and a fullscan MS/MS in an exclusion dynamic mode. MS/MS datawere acquired using a 2 m/z units ion isolation window, a35% relative collision energy, and a 5 min dynamic exclusionduration. Peptides were identified with SEQUEST (Thermo-Finnigan, Torrence, CA, USA) using the 18 254 Pinus pinasterEST (http://cbi.labri.fr/outils/SAM/COMPLETE/index.php),and the 59 447 Pinus taeda xylem EST comprising 8046 con-tigs and 12 437 singletons (http://pinetree.ccgb.umn.edu/).The contig names in Table 1 correspond to the Novem-ber 2002 assembly [29]. The Swiss-Prot database (http://us.expasy.org/sprot/) was also used to evaluate the rate of proteinidentification using nonconiferous nucleotide sequences.
2.6.3 PMF by MALDI-TOF MS
For 57 spots (six of which being also sampled for LC ESI-MS/MS analysis), we used MALDI-TOF MS following the proto-col and analysis procedure described by Sarry et al. [30]. TheMASCOT search engine software (Matrix Science, London,UK) was used to search the NCBI nonredundant and specificpine EST databases on a local server.
2.7 Differentiating xylem cDNA library construction
and EST sequencing
A composite cDNA library was obtained using equal amountsof total RNA extracted from the same samples as describedfor the protein analysis, but for JW and MW. Total RNA weremixed and poly A(1) RNA isolated from this bulked sample.The cDNA library was made using the l-ZAP-cDNA synthe-sis kit (Stratagene, La Jolla, CA, USA). Approximately10 000 l clones were excised to generate plasmid clones. The10 000 plasmid clones were sequenced using the Templifi kit(Amersham Biosciences), by single pass from the 5’-end togenerate the EST collection. Only sequences longer than60 nucleotides were kept for further analysis. EST annotationwas based on a search for homology with public protein andnucleic acid sequence databases using the BLAST software[31]. Homologs were sequentially searched in Swiss-Prot(BLASTX), TrEMBL (BLASTX), EMBL (BLASTN), and lastlyin dbEST database (BLASTN). At each step, the process wasstopped if a gene with similar sequence was found (definedby an expected value lower than 1025 for BLASTX and 10210
for BLASTN searches). A total of 8429 EST were finally sub-mitted to dbEST (http://www.ncbi.nlm.nih.gov/dbEST/), andcan be retrieved using the search fields organism [Pinuspinaster] and tissue_type[xylem].
2.8 Statistical analyses
To appreciate the relatedness between the six types of wood(i.e., JW, MW, EW, LW, OW, CW), based on a proteomic dis-tance obtained from either the 1039 detected spots, or a
restricted dataset of 215 spots (the 175 known and 40 un-known function proteins), we used the hierarchical cluster-ing software EPCLUST available at URL: http://ep.ebi.ac.uk/EP/. The Euclidian distance and UPGMA algorithm wereused for the analysis. The same software and options wereused to cluster the 215 spots according their log2 trans-formed expression profiles along the six samples. Simplet-tests were performed for each pair-wise comparison,namely JW vs. MW, EW vs. LW, and OW vs. CW, to detectthose proteins showing significant (p-value , 0.01) onto-genic, seasonal, and gravitational effect.
3 Results and discussion
3.1 2-DE reference map of maritime pine wood
forming tissue
The 2-DE reference map of maritime pine wood formingtissue was established using proteins extracted from differ-entiating xylem associated to JW, MW, EW, LW, CW and OW,and separated by 2-DE. For each samples five replicated gelswere performed. After colloidal blue staining, they werescanned with the LabScan software and analyzed using theIM2D software. The image obtained for differentiating xylemassociated to OW after 2 years of bending (replicate #1) wasrandomly chosen to build the reference gel (master gel) onwhich spots specifically detected on other samples (through-out the four best replicates) were added. Overall, 445, 468,506, 581, 552, 570 spots were detected in MW, JW, EW, LW,OW, and CW, respectively. A total of 1039 spots were finallyplaced on the reference map (Fig. 2), among which 300 pro-teins (29%) were excised from the polyacrylamide gel andanalyzed by mass spectrometry or internal microsequencing.As shown in Fig. 2, spots were picked randomly to ensure agood representation in terms of pH, molecular weight andprotein abundance.
3.2 Protein identification success rate
LC ESI-MS/MS analysis of the 240 spots was used for proteinidentification using protein (Swiss-Prot) and nucleotide(Pinus pinaster and Pinus taeda EST and contigs) databases.The overall identification success rate was 67.9%, corre-sponding to 163 spots identified. It should be noted thatsearching both databases appears quite redundant, since inonly five cases protein identification was achieved usingSwiss-Prot. It should also be noted that for 71 of the identifiedspots, the same hit was obtained in both nucleotide and pro-tein databases. In other words, the overlap in the number ofproteins identified in both databases was 43.6%. Thus, thepine EST allowed the identification of an additional 87 spots.This result clearly indicates the utility of pine EST to achievea high rate of protein identification. As for the remainingspots, 40 (16.7%) presented no homology in the query data-bases. Given the amount of pine xylem EST in public data-
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
3736 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751Tab
le1.
IM2D
spot
IDMe
thod
Hiton
datab
asea)
(Matc
hesb) )
Cove
r-ag
ec)Co
nsen
suso
rsin
gleton
Id)Ac
cess
ionc)
Assig
nmen
tpI
/Mrf)
pI/M
rg)Ac
cess
ionnu
mber
Spec
iesMe
anno
rmali
zedv
olume
h)Me
anpro
tein
volum
ei)M
Jp-
value
EL
p-va
lueC
Op-
value
Amino
acid
metab
olism
166
MS/M
SPp
7(2)
19.1
RS09
E06(
root)
BX67
8462
2-Iso
propy
lmala
tesy
nthas
eA(EC
2.3.3.
13)
6.03/6
0687
5.66/6
4320
O049
73Ly
cope
rsico
nesc
ulentu
m–
––
722
0.011
36
90.0
231
11.21
184
MS/M
SPp
(3)15
.103
0C04
(xylem
)BX
2499
15Ald
ehyd
edeh
ydrog
enas
e(EC
1.2.1.
3)6.3
7/583
186.0
5/544
02P2
0000
Bost
aurus
134
520.1
462
148
182
0.454
815
218
40.2
301
166.4
423
5MS
/MS
Pp(2)
10CN
1576
(root)
AL75
1232
Amino
acyla
se-1
(EC3.5
.1.14
)5.4
6/531
015.7
7/458
55Q0
3154
Homo
sapie
ns–
––
029
0.001
519
130.4
333
15.04
241
MS/M
SPp
7(8)
46.3
CN15
77(ro
ot)AL
7512
33Am
inoac
ylase
-1(EC
3.5.1.
14)
5.44/5
4028
5.77/4
5856
Q031
54Ho
mosa
piens
3413
0.002
19
220.0
509
1113
0.384
313
.5523
MS/M
SPp
*(4)
23.8
087B
11(xy
lem)
BX25
3702
Amino
pepti
dase
N(EC
3.4.11
.2)5.4
9/104
359
5.14/9
8726
P048
25Es
cheri
chia
coli
2439
0.016
196
300.0
013
2313
<10-4
40.5
122
MS/M
SPp
*(3)
14.4
070G
05(xy
lem)
BX25
2790
D-3-p
hosp
hogly
cerat
edeh
ydrog
enas
e(EC
1.1.1.
95)
6.54/7
6786
5.22/6
0576
O041
30Ar
abido
psis
thalia
na–
––
––
–16
130.5
218
7.33
159
MS/M
SPp
*(3)
16.9
CN64
5(xy
lem)
BX25
2807
D-3-p
hosp
hogly
cerat
edeh
ydrog
enas
e(EC
1.1.1.
95)
5.82/6
2570
5.22/6
0576
O041
30Ar
abido
psis
thalia
na0
90.1
233
098
0.004
694
100
0.331
673
.0368
3MS
/MS
Pp7
(5)28
CN37
6(xy
lem)
BX25
0469
Glutat
hione
S-tra
nsfer
ase(
EC2.5
.1.18
)5.6
4/254
415.3
1/250
40P3
2111
Solan
umtub
erosu
m–
––
026
0.030
013
00.0
015
9.88
684
MS/M
SPp
7(8)
42.2
CN37
6(xy
lem)
BX25
0469
Glutat
hione
S-tra
nsfer
ase(
EC2.5
.1.18
)5.4
3/248
205.3
1/250
40P3
2111
Solan
umtub
erosu
m–
––
02
0.121
610
00.0
002
349
9MS
/MS
Pt7(3)
8.1co
ntig6
593_
3BG
0405
08Glu
tathio
neS-
trans
feras
e(EC
2.5.1.
18)
5.56/2
4431
5.78/2
5358
O498
21Ca
ricap
apay
a15
00.0
035
3717
90.0
002
8650
0.000
188
.1974
2MS
/MS
Pp*(
5)10
.3CN
167(
xylem
)BX
2556
17S-
Aden
osyl-
L-hom
ocys
teine
hydro
lase(
EC3.3
.1.1)
5.92/5
6287
5.51/5
3070
P502
48Ni
cotia
natab
acum
912
0.723
713
414
40.6
833
––
–69
.4619
7MS
/MS
Pp*(
9)26
.3CN
167(
xylem
)BX
2556
17S-
Aden
osyl-
L-hom
ocys
teine
hydro
lase(
EC3.3
.1.1)
6.00/5
5868
5.51/5
3070
P502
48Ni
cotia
natab
acum
093
0.003
797
890.7
938
211
215
0.922
615
2.86
260
MS/M
SPp
*(11
)36
.9CN
670(
xylem
)BX
2503
80S-
Aden
osylm
ethion
inesy
ntheta
se(EC
2.5.1.
6)6.1
7/497
455.5
3/431
41P5
0300
Pinus
bank
siana
––
–0
120.0
008
8053
0.015
536
.2126
3MS
/MS
Pp*(
9)21
.3CN
670(
xylem
)BX
2489
42S-
Aden
osylm
ethion
inesy
ntheta
se(EC
2.5.1.
6)5.8
9/490
905.5
3/431
41P5
0300
Pinus
bank
siana
––
–2
40.3
197
3822
0.001
016
.6827
3MS
/MS
Pp*(
14)
33.3
CN67
0(xy
lem)
BX24
8942
S-Ad
enos
ylmeth
ionine
synth
etase
(EC2.5
.1.6)
6.03/4
7389
5.53/4
3141
P503
00Pin
usba
nksia
na0
127
0.000
325
330.2
796
6449
0.223
242
.7927
5MS
/MS
Pp*(
10)
25.1
CN22
9(roo
t)BX
6660
55S-
Aden
osylm
ethion
inesy
ntheta
se(EC
2.5.1.
6)5.5
5/479
385.5
3/431
41P5
0300
Pinus
bank
siana
1627
0.162
617
120.3
169
2721
0.002
319
.2727
9MS
/MS
Pp*(
11)
31.6
CN22
9(roo
t)BX
6660
55S-
Aden
osylm
ethion
inesy
ntheta
se(EC
2.5.1.
6)5.4
5/475
365.5
3/431
41P5
0300
Pinus
bank
siana
145
301
0.003
223
616
00.0
038
220
179
0.014
619
8.73
280
MS/M
SPp
*(9)
30.2
CN22
7(roo
t)BX
6819
98S-
Aden
osylm
ethion
inesy
ntheta
se(EC
2.5.1.
6)6.1
9/466
305.7
4/431
93P4
6611
Orys
asati
va–
––
1667
0.003
223
317
00.0
397
121.6
128
2MS
/MS
Pp*(
3)20
.1CN
616(
xylem
)BX
2556
16S-
Aden
osylm
ethion
inesy
ntheta
se(EC
2.5.1.
6)5.6
7/471
355.7
6/426
25P4
3282
Lyco
persi
cone
scule
ntum
126
281
0,003
926
035
<10-4
100
540.0
002
112.2
328
3MS
/MS
Pp*(
9)50
CN61
5(xy
lem)
BX25
0974
S-Ad
enos
ylmeth
ionine
synth
etase
(EC2.5
.1.6)
5.93/4
5790
5.76/4
2625
P432
82Ly
cope
rsico
nesc
ulentu
m–
––
598
<10-4
262
78<1
0-411
0.730
3MS
/MS
Pp*(
10)
23.3
CN67
0(xy
lem)
BX24
8942
S-Ad
enos
ylmeth
ionine
synth
etase
(EC2.5
.1.6)
6.03/4
2731
5.53/4
3141
P503
00Pin
usba
nksia
na–
––
250
0.000
110
160.1
141
12.78
313
MS/M
SPp
(2)9
CN67
0(xy
lem)
BX25
0380
S-Ad
enos
ylmeth
ionine
synth
etase
(EC2.5
.1.6)
6.05/4
1844
5.53/4
3141
P503
00Pin
usba
nksia
na–
––
09
0.017
620
140.2
627
10.55
321
MS/M
SPp
*(2)
8.3CN
667(
xylem
)BX
2498
56S-
Aden
osylm
ethion
inesy
ntheta
se(EC
2.5.1.
6)5.9
5/424
245.7
4/431
93P4
6611
Orys
asati
va27
40,0
011
1421
0.083
415
140.7
202
15.69
668
MS/M
SSP
(2)10
.8P5
0303
S-Ad
enos
ylmeth
ionine
synth
etase
(EC2.5
.1.6)
5.94/4
1343
6.2/39
513
P503
03Ac
tinidi
achin
ensis
––
––
––
250
<10-4
6.27
782
MS/M
SPp
*(4)
9.8CN
667(
xylem
)BX
2498
56S-
Aden
osylm
ethion
inesy
ntheta
se(EC
2.5.1.
6)5.1
1/324
635.7
4/431
93P4
6611
Orys
asati
va12
00,0
351
30
0.132
7–
––
0.826
6Ed
man
SP(14
/15)
P503
00S-
Aden
osylm
ethion
inesy
ntheta
se(EC
2.5.1.
6)6.0
0/481
515.5
3/431
41P5
0300
Pinus
bank
siana
––
–20
622
80.3
547
618
455
0.016
337
6.67
Carbo
hydra
teme
taboli
sm13
4MS
/MS
Pt7(9)
27co
ntig7
733
CD02
7777
L-Asc
orbate
perox
idase
(EC1.1
1.1.11
)5.6
0/679
838.6
5/421
46Q3
9006
Arab
idops
istha
liana
817
0,029
144
880.0
052
3947
<10-4
54.54
431
MS/M
SPt
(4)13
.9co
ntig3
622_
1BG
0408
06L-A
scorb
atepe
roxida
se(EC
1.11.1
.11)
5.54/3
0528
8.65/4
2146
Q8LS
K6Ly
cope
rsico
nesc
ulentu
m–
––
20
0.143
710
150.0
561
6.73
476
MS/M
SPp
7(11
)30
.1CN
236(
xylem
)BX
2499
47L-A
scorb
atepe
roxida
se(EC
1.11.1
.11)
5.41/2
5533
5.52/2
7045
P485
34Pis
umsa
tivum
––
–28
530.1
233
2951
0.000
340
.3748
2Ed
man
SP(13
/15)
X800
36L-A
scorb
atepe
roxida
se(EC
1.11.1
.11)
5.55/2
4798
5.88/2
7928
Q390
06Ar
abido
psis
thalia
na31
930,0
108
110
377
0.001
725
724
30.3
1271
246.9
335
5MS
/MS
Pp7
(9)25
.8CN
515(
xylem
)BX
2488
14Fru
ctokin
ase(
EC2.7
.1.4)
4.75/3
8842
5.47/3
3743
P378
29So
lanum
tubero
sum
130
0,001
57
450.0
004
3125
0.363
027
.0336
0MS
/MS
Pp7
(9)26
.7CN
515(
xylem
)BX
2488
14Fru
ctokin
ase(
EC2.7
.1.4)
4.80/3
8680
5.47/3
3743
P378
29So
lanum
tubero
sum
180
260,0
010
127
205
0.002
519
215
90.0
310
170.9
232
2MS
/MS
Pt7(3)
24.1
conti
g625
1_2
BQ19
7338
NAD-
depe
nden
tsorb
itold
ehyd
rogen
ase
6.16/4
1315
6.75/4
0195
Q9ZR
22Ma
lusdo
mesti
ca3
40,8
182
2134
0.074
119
200.8
016
23.47
150
MS/M
SPp
*(9)
38.7
064E
07(xy
lem)
BX25
2436
Pyrop
hosp
hate
fructo
se6-p
hosp
hate
1-pho
spho
trans
feras
e(EC
2.7.1.
90)
6.31/6
6286
6.19/6
0076
Q411
41Ric
inusc
ommu
nis–
––
1376
0.007
292
112
0.064
373
.42
149
MS/M
SPt*
(2)8.6
conti
g683
6_1
BI64
3882
Pyrop
hosp
hate
fructo
se6-p
hosp
hate
1-pho
spho
trans
feras
e(EC
2.7.1.
90)
6.44/6
2870
6.19/6
0076
Q411
41Ric
inusc
ommu
nis0
00.3
559
2241
0.347
272
850.4
150
55.1
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
Proteomics 2005, 5, 3731–3751 Plant Proteomics 3737Tab
le1.C
on
tin
ued
IM2D
spot
IDMe
thod
Hiton
datab
asea)
(Matc
hesb) )
Cove
r-ag
ec)Co
nsen
suso
rsin
gleton
Id)Ac
cess
ionc)
Assig
nmen
tpI
/Mrf)
pI/M
rg)Ac
cess
ionnu
mber
Spec
iesMe
anno
rmali
zedv
olume
h)Me
anpro
tein
volum
ei)M
Jp-
value
EL
p-va
lueC
Op-
value
148
MS/M
SPp
7(1)
6.206
4E07
(xylem
)BX
2524
36Py
ropho
spha
tefru
ctose
6-pho
spha
te1-p
hosp
hotra
nsfer
ase(
EC2.7
.1.90
)6.1
7/627
626.1
9/600
76Q4
1141
Ricinu
scom
munis
--
-0
160.0
003
2535
0.179
019
221
MS/M
SPt7
(2)9.7
conti
g388
8_1
BM13
3421
Pyrop
hosp
hate
fructo
se6-p
hosp
hate
1-pho
spho
trans
feras
e(EC
2.7.1.
90)
5.93/5
4039
5.77/5
3781
Q9M3
94Ar
abido
psis
thalia
na0
70.0
451
1520
0.305
013
160.3
466
15.97
67MS
/MS
Pt7(4)
8.8co
ntig7
811_
1CD
0268
15Tra
nske
tolas
e(EC
2.2.1.
1)6.5
0/885
126.1
2/799
78Q9
FPB7
Oryza
sativ
a–
––
––
–7
90.4
008
4.06
69MS
/MS
Pt7(3)
6.1co
ntig7
811_
1CD
0268
15Tra
nske
tolas
e(EC
2.2.1.
1)6.3
1/848
346.1
2/799
78Q9
FPB6
Oryza
sativ
a0
00.3
559
1983
0.002
712
112
70.7
212
87.52
484
MS/M
SPp
*(4)
21.8
CN44
2(xy
lem)
BX25
5804
Trios
epho
spha
teiso
meras
e(EC
5.3.1.
1)5.7
5/246
845.3
8/270
33Q9
SKP6
Arab
idops
istha
liana
310
248
0.044
524
534
30.0
005
284
245
0.006
127
9.26
579
MS/M
SPp
*(3)
8CN
626(
xylem
)BX
2504
192,3
-Bisp
hosp
hogly
cerat
e-ind
epen
dent
phos
phog
lycera
temu
tase(
EC5.4
.2.1)
5.83/7
1263
5.52/6
0780
P354
93Ric
inusc
ommu
nis0
30.0
905
210
<10-4
90
0.006
17.4
1
104
MS/M
SPp
7(2)
4.8CN
626(
xylem
)BX
2504
192,3
-Bisp
hosp
hogly
cerat
e-ind
epen
dent
phos
phog
lycera
temu
tase(
EC5.4
.2.1)
5.80/7
1444
5.52/6
0780
P354
93Ric
inusc
ommu
nis5
110.2
665
230
0.000
16
60.8
272
8.81
108
MS/M
SPp
*(5)
13.7
CN62
6(xy
lem)
BX25
0419
2,3-B
ispho
spho
glyce
rate-i
ndep
ende
ntph
osph
oglyc
erate
mutas
e(EC
5.4.2.
1)5.9
8/712
695.5
2/607
80P3
5493
Ricinu
scom
munis
380
0.002
80
175
<10-4
4559
0.006
669
.98
305
MS/M
SPp
(3)14
.1CN
1715
(root)
BX68
1260
Alcoh
olde
hydro
gena
se(EC
1.1.1.
1).6.4
8/454
176.6
1/414
32P1
7648
Fraga
riaan
anas
sa3
00.1
534
1145
0.148
417
230.2
888
24.09
298
MS/M
SPp
*(2)
21.5
CN80
(xylem
)BX
2499
35Alc
ohol
dehy
droge
nase
(EC1
.1.1.1
)6.1
1/425
355.9
2/411
16P1
4675
Solan
umtub
erosu
m4
40.9
375
1250
0.000
548
540.3
862
41.17
246
MS/M
SPt
(3)22
.6co
ntig1
9317
_2BG
0398
98Dih
ydrol
ipoam
ideac
etyltra
nsfer
ase(
EC2.3
.1.12
)5.8
1/510
187.5
4/596
52Q9
LVK7
Arab
idops
istha
liana
2455
0.005
410
290
0.277
143
460.5
205
69.9
239
MS/M
SPp
*(6)
18.3
CN19
8(roo
t)BX
6660
27En
olase
(EC4.2
.1.11
)5.7
5/515
175.7
1/481
32P4
2895
Zeam
ays
151
215
0.004
018
917
50.3
129
9598
0.503
613
9.39
211
MS/M
SPp
*(7)
21.4
CN19
8(roo
t)BX
6660
27En
olase
(EC4.2
.1.11
)5.9
7/561
105.7
1/481
32P4
2895
Zeam
ays
––
–26
611
00.0
047
5252
0.927
911
9.837
5MS
/MS
Pp*(
2)5.3
CN52
1(xy
lem)
BX24
9304
Fructo
se-bi
spho
spha
teald
olase
(EC4.1
.2.13
)5.9
3/366
155.9
6/384
47P2
9356
Spina
ciaole
racea
177
0.019
30
210.0
010
2112
0.033
413
.5189
MS/M
SPp
*(2)
11.9
CN75
2(xy
lem)
BX25
2576
Phos
phog
lucom
utase
(EC5.4
.2.2)
5.33/7
4448
5.56/6
3442
Q9SG
C1Ar
abido
psis
thalia
na0
135
0.000
118
219
00.6
817
140
127
0.025
915
9.74
171
MS/M
SPp
(6)40
CN18
79(ro
ot)BX
6819
93Ph
osph
ogluc
omuta
se(EC
5.4.2.
2)5.3
5/614
975.7
9/615
81Q9
LF71
Arab
idops
istha
liana
––
–7
120.0
212
1717
0.772
613
.2984
MS/M
SPp
*(3)
15.8
CN75
2(xy
lem)
BX25
2576
Phos
phog
lucom
utase
(EC5.4
.2.2)
5.39/7
8618
5.56/6
3442
Q9SG
C1Ar
abido
psis
thalia
na12
00.0
098
1413
0.779
27
80.6
665
10.92
349
MS/M
SPp
*(11
)46
.1CN
784(
xylem
)BX
2508
05UD
P-glu
cose
protei
ntran
sgluc
osyla
se(EC
2.4.1.
15)
5.49/3
8689
5.71/4
1576
Q8RU
27So
lanum
tubero
sum
413
321
0.075
929
831
70.2
410
198
212
0.476
225
6.06
15MS
/MS
Pt7(2)
16.8
conti
g993
3_2
BF06
0543
Acon
itase
(EC4.2
.1.2)
6.25/1
0786
25.7
9/980
92Q9
SIB9
Arab
idops
istha
liana
––
––
––
1119
0.097
97.5
719
MS/M
SPp
7(4)
25.7
RS02
C10(
root)
AL75
0948
Acon
itase
(EC4.2
.1.3)
6.36/1
0383
15.8
8/980
08Q9
FVE9
Nico
tiana
tabac
um0
10.1
779
2035
0.300
133
390.4
820
31.8
Cellw
all31
0MS
/MS
Pp7
(10)
28.8
CN12
58(xy
lem)
BX25
1221
Arab
inoga
lactan
/prol
in-ric
hprot
ein4.0
0/410
003.8
/1217
9Q9
LLZ5
Pinus
taeda
227
950.0
098
840
0.001
118
210.5
129
30.87
586
MS/M
SPt
(5)22
.3co
ntig3
490_
3BE
1236
50Ar
abino
galac
tan/pr
oline
-rich
protei
n4.9
4/643
373.8
/1217
9Q9
LLZ5
Pinus
taeda
7274
0.849
039
00.0
044
––
–9.7
561
7MS
/MS
Pp(6)
34.3
CN73
7(xy
lem)
BX25
5373
Arab
inoga
lactan
/proli
ne-ri
chpro
tein
4.15/3
7074
3.8/12
179
Q9LL
Z5Pin
ustae
da30
378
0.000
275
190.0
561
70
0.000
825
.2262
3MS
/MS
Pp7
(4)25
.7CN
680(
xylem
)BX
2489
77Ar
abino
galac
tan/pr
oline
-rich
protei
n4.1
1/328
393.8
/1217
9Q9
LLZ5
Pinus
taeda
371
178
0.014
417
652
0.011
5–
––
57.04
618
MS/M
SPp
*(2)
001D
01(xy
lem)
BX24
8813
Caffe
icac
id3-O
methy
ltrans
feras
elike
protei
n(E
C2.1.
1.68)
5.40/3
6929
5.71/4
1904
Q8L8
L1Ar
abido
psis
thalia
na86
760.4
118
2687
<10-4
110
0.072
030
.97
315
MS/M
SPp
*(4)
34.8
RN72
G02(
root)
CR39
4041
Cinna
myl-a
lcoho
ldeh
ydrog
enas
e(EC
1.1.1.
195)
6.21/4
1493
5.6/38
821
P416
37Pin
ustae
da39
106
0.125
411
420
20.0
161
232
211
0.535
018
9.93
665
MS/M
SPp
7(5)
26CN
331(
root)
BX68
1393
Perox
idase
54pre
curso
r(EC1
.11.1.
7)5.5
0/488
754.5
4/341
59Q9
FG34
Arab
idops
istha
liana
260
0.000
519
130.2
852
60
0.007
69.4
724
7MS
/MS
Pt(2)
15.6
conti
g537
2_2
BF61
0181
Xylos
eiso
meras
e(EC
5.3.1.
5)5.4
0/528
245.3
1/536
14Q4
0082
Horde
umvu
lgare
922
0.109
70
76<1
0-426
210.0
188
30.63
439
Edma
nSP
(5/5)
Z829
82Ca
ffeoy
lCoA
O-me
thyltra
nsfer
ase(
EC2.1
.1.10
4)4.9
5/289
615.3
0/277
81Q4
2945
Nico
tiana
tabac
um51
837
60.0
232
339
365
0.343
637
930
20.0
035
346.2
348
9MS
/MS
Pt(2)
5.2co
ntig7
614_
2BF
5180
75Ca
ffeoy
lCoA
O-me
thyltra
nsfer
ase(
EC2.1
.1.10
4)6.1
2/253
865.4
1/295
78P2
2734
Rattu
snorv
egicu
s–
––
06
0.183
531
180.0
949
13.83
Cytos
kelet
on16
3MS
/MS
Pp7
(2)10
.1CN
869(
xylem
)BX
2493
35Ac
tin5.4
1/624
085.3
1/416
16P3
0171
Solan
umtub
erosu
m17
480.0
315
4631
0.004
923
210.4
936
30.2
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
3738 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751Tab
le1.C
on
tin
ued
IM2D
spot
IDMe
thod
Hiton
datab
asea)
(Matc
hesb) )
Cove
r-ag
ec)Co
nsen
suso
rsin
gleton
Id)Ac
cess
ionc)
Assig
nmen
tpI
/Mrf)
pI/M
rg)Ac
cess
ionnu
mber
Spec
iesMe
anno
rmali
zedv
olume
h)Me
anpro
tein
volum
ei)M
Jp-
value
EL
p-va
lueC
Op-
value
299
MS/M
SPp
*(5)
10.1
CN11
68(xy
lem)
BX25
5308
Actin
5.46/4
3549
5.31/4
1709
P534
92Ar
abido
psis
thalia
na52
332
60.0
228
515
207
0.002
612
812
80.9
785
244.6
530
0MS
/MS
Pp*(
3)7.2
CN54
9(xy
lem)
BX25
5342
Actin
5.59/4
3417
5.31/4
1709
P534
92Ar
abido
psis
thalia
na46
150.0
200
237
0.009
10
50.0
009
8.64
373
MS/M
SPp
*(10
)28
.4CN
1168
(xylem
)BX
2553
08Ac
tin5.6
1/372
265.3
1/417
09P5
3492
Arab
idops
istha
liana
1616
0.968
625
410.0
601
2044
0.000
832
.7729
7Ed
man
SP(5/
5)P2
4902
Actin
5.31/4
2734
5.31/4
1709
P534
92Ar
abido
psis
thalia
na43
7134
550.0
076
3305
2631
0.054
516
2115
670.7
122
2280
.8922
6MS
/MS
Pp*(
10)
22CN
179(
xylem
)BX
2488
16Tu
bulin
alpha
chain
5.07/5
4202
4.92/4
9621
P462
59Pis
umsa
tivum
338
286
0.409
734
324
10.0
060
158
127
0.019
921
7.38
242
MS/M
SPp
*(10
)40
.3CN
179(
xylem
)BX
2488
16Tu
bulin
alpha
chain
5.12/5
3290
4.92/4
9621
P462
59Pis
umsa
tivum
988
800
0.084
595
397
70.8
652
810
658
0.026
884
9.64
781
MS/M
SPp
*(2)
12.9
CN17
9(xy
lem)
BX25
5614
Tubu
linalp
hach
ain5.5
8/335
204.7
8/501
07Q9
ZPN6
Eleus
ineind
ica32
00.0
010
54
0.635
6–
––
2.21
212
MS/M
SPp
*(10
)19
.7CN
791(
xylem
)BX
2502
73Tu
bulin
beta
chain
4.88/5
7192
4.78/5
0107
Q9ZP
N7Ele
usine
indica
187
205
0.701
922
226
80.2
241
255
251
0.911
124
9.09
218
MS/M
SPp
*(18
)35
.6CN
791(
xylem
)BX
2502
73Tu
bulin
beta
chain
4.90/5
3820
4.78/5
0107
Q9ZP
N7Ele
usine
indica
141
160
0.433
012
219
90.1
101
138
191
0.003
916
2.521
7MS
/MS
Pp*(
8)14
.8CN
791(
xylem
)BX
2502
73Tu
bulin
beta
chain
4.92/5
4018
4.79/5
0011
Q436
97Ze
amay
s50
853
70.7
351
439
299
0.003
115
817
00.7
792
266.4
821
5MA
LDI-T
OFPp
7(11
1)BE
5821
28Tu
bulin
beta
chain
4.92/5
4542
4.82/4
9851
P180
26Ze
amay
s43
340
70.7
152
270
221
0.270
515
899
0.003
318
6.71
216
MALD
I-TOF
Pp7
(76)
BX25
5731
Tubu
linbe
tach
ain4.9
6/545
324.8
2/498
51P1
8026
Zeam
ays
450
266
0.030
129
411
90.0
071
5261
0.336
613
1.31
223
MALD
I-TOF
Pp7
(112)
BG03
9745
Tubu
linbe
tach
ain4.7
5/533
514.8
2/498
51P1
8026
Zeam
ays
970
<10-4
9323
00.0
016
251
231
0.412
820
1.27
Defen
se54
1MS
/MS
Pp*(
6)33
.8CN
1136
(xylem
)BX
2516
2317
.1kD
aclas
sIIh
eats
hock
protei
n5.4
7/197
716.3
2/170
59P1
9242
Pisum
sativ
um14
00.0
040
920
1<1
0-415
121
60.0
611
144.0
954
2MS
/MS
Pp7
(5)24
.9CN
523(
xylem
)BX
2502
1117
.1kD
aclas
sIIh
eats
hock
protei
n5.4
2/197
206.3
2/170
59P1
9242
Pisum
sativ
um–
––
081
0.000
525
560.0
157
40.36
733
MS/M
SPp
7(13
)43
.2CN
957(
xylem
)BX
2546
1217
.4kD
aclas
sIhe
atsh
ockp
rotein
4.90/2
0863
5.81/1
7365
P316
73Or
yzasa
tiva
––
–0
90.1
884
––
–2.1
653
3MS
/MS
Pp*(
9)31
.500
3D03
(xylem
)BX
2489
7017
.8kD
aclas
sIhe
atsh
ockp
rotein
6.25/2
0483
6.77/1
8123
P190
37Ar
abido
psis
thalia
na–
––
––
–5
80.0
651
3.24
836
MS/M
SPp
*(9)
28.1
CN10
82(xy
lem)
BX25
5777
18.0
kDac
lassI
heat
shoc
kprot
ein5.6
5/204
336.9
3/180
21P2
7397
Dauc
usca
rota
250
<10-4
014
80.0
331
––
–36
.9773
4MS
/MS
Pp*(
8)32
.9CN
1082
(xylem
)BX
2557
7718
.0kD
aclas
sIhe
atsh
ockp
rotein
5.62/2
0738
6.93/1
8021
P273
97Da
ucus
carot
a0
185
0.001
0–
––
––
–0
535
MS/M
SPp
7(3)
15.1
058F
10(xy
lem)
BX25
2024
22.7
kDac
lassI
Vhea
tsho
ckpro
tein
5.05/2
0381
6.17/1
9658
P192
44Pis
umsa
tivum
––
–6
650.0
220
3729
0.066
034
.0415
8MS
/MS
Pp7
(5)14
.2CN
748(
xylem
)BX
2514
9760
kDac
hape
ronin
5.75/6
2751
5.19/5
7611
P291
97Ar
abido
psis
thalia
na12
680.0
109
165
103
0.080
151
480.4
112
91.84
7MS
/MS
Pp7
(7)31
.7CN
1455
(root)
BX67
9313
70kD
ahea
tsho
ckpro
tein
5.42/1
1296
55.1
7/930
49Q9
AQZ5
Oryza
sativ
a–
––
––
–27
170.0
013
10.95
9MS
/MS
Pp*(
7)39
CN14
55(ro
ot)BX
6793
1370
kDah
eats
hock
protei
n5.3
4/112
791
5.17/9
3049
Q9AQ
Z5Or
yzasa
tiva
––
–0
370.0
032
4022
0.000
924
.7610
MS/M
SPp
7(4)
21.1
CN14
55(ro
ot)BX
6793
1370
kDah
eats
hock
protei
n5.3
8/112
791
5.17/9
3049
Q9AQ
Z5Or
yzasa
tiva
––
––
––
5328
<10-4
20.25
76MS
/MS
Pp*(
4)11
.5CN
769(
xylem
)BX
2491
7070
kDah
eats
hock
protei
n4.8
3/820
375.1
1/711
82P0
9189
Petun
iahy
brida
175
35<1
0-411
616
80.0
223
109
990.0
820
123.0
792
MS/M
SPp
*(2)
9.310
8H08
(xylem
)BX
2549
9270
kDah
eats
hock
protei
n5.4
3/753
865.2
7/669
53Q0
1899
Phas
eolus
vulga
ris0
10.0
402
445
0.000
323
380.0
001
27.13
80Ed
man
SP(12
/12)
Y170
53HS
P70k
Da5.0
0/758
684.9
7/711
03O6
5719
Arab
idops
istha
liana
030
0.162
00
284
<10-4
245
207
0.101
718
4.09
81Ed
man
SP(14
/14)
Y170
54HS
P70k
Da5.1
3/758
694.9
7/711
03O6
5719
Arab
idops
istha
liana
03
0.294
10
167
0.002
517
918
20.8
187
131.8
388
Edma
nSP
(15/15
)Y1
7055
HSP7
0kDa
5.07/7
5371
4.97/7
1103
O657
19Ar
abido
psis
thalia
na0
330.1
584
043
30.0
005
441
359
0.125
430
8.45
508
MS/M
SPp
(4)23
.6CN
1071
(xylem
)BX
2521
95Ab
scisi
cstre
ssrip
ening
protei
n15.9
8/241
616.8
1/131
22Q0
8655
Lyco
persi
cone
scule
ntum
––
–0
250.0
298
131
305
<10-4
115.2
790
7MS
/MS
Pp7
(2)10
.302
6F11
(xylem
)BX
2496
52Ab
scisi
cstre
ssrip
ening
protei
n15.1
1/269
336.8
1/131
22Q0
8655
Lyco
persi
cone
scule
ntum
––
–6
00.0
876
––
–1.5
639
0MS
/MS
Pp7
(5)18
.5CN
223(
root)
BX66
6054
Absc
isics
tress
ripen
ing-lik
eprot
ein4.8
7/357
425.6
8/207
47Q9
3WZ6
Prun
uspe
rsica
02
0.306
723
76<1
0-468
770.3
230
61.14
49MS
/MS
Pp*(
13)
44.8
CN94
2(xy
lem)
BX24
8869
Heat
shoc
kprot
ein4.9
7/938
164.9
6/800
86P3
6181
Lyco
persi
cone
scule
ntum
––
–0
140.0
134
142
129
0.287
371
.4197
MS/M
SPt
(3)10
.2co
ntig9
44_3
BE99
7207
Heat
shoc
kprot
ein5.1
6/732
475.0
3/713
13P2
2953
Arab
idops
istha
liana
00
0.355
93
90.1
433
2413
0.042
612
.3891
0MS
/MS
Pp7
(5)22
068B
04(xy
lem)
BX25
2628
Late
embry
ogen
esis
likep
rotein
5.41/2
0229
4.84/1
6412
P465
18Go
ssyp
iumhir
sutum
340
0.000
57
00.1
340
––
–1.6
538
4MS
/MS
Pt(3)
13co
ntig1
842_
3BG
3183
37La
teem
bryog
enes
is-lik
eprot
ein4.7
5/353
83NA
O813
66Pr
unus
armen
iaca
41
0.037
410
00.0
316
3450
0.023
123
.3571
9MS
/MS
Pt(2)
11.8
conti
g184
2_3
BG31
8337
Late
embry
ogen
esis-
likep
rotein
4.80/3
5110
NAO8
1366
Prun
usarm
eniac
a8
00.0
005
044
<10-4
––
–10
.9587
4MS
/MS
Pp7
(2)21
.9CN
1236
(xylem
)BX
2521
62Os
r40g3
protei
n(ab
scisi
cacid
ands
altstr
ess
respo
nsive
)5.3
7/259
787.6
6/228
07O2
4213
Oryza
sativ
a–
––
021
0.005
5–
––
5.28
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
Proteomics 2005, 5, 3731–3751 Plant Proteomics 3739Tab
le1.C
on
tin
ued
IM2D
spot
IDMe
thod
Hiton
datab
asea)
(Matc
hesb) )
Cove
r-ag
ec)Co
nsen
suso
rsin
gleton
Id)Ac
cess
ionc)
Assig
nmen
tpI
/Mrf)
pI/M
rg)Ac
cess
ionnu
mber
Spec
iesMe
anno
rmali
zedv
olume
h)Me
anpro
tein
volum
ei)M
Jp-
value
EL
p-va
lueC
Op-
value
183
MS/M
SPp
*(8)
58.1
039B
09(xy
lem)
BX25
0616
Putat
ivese
lenium
bindin
gprot
ein5.8
3/586
145.3
7/540
23O2
3264
Arab
idops
istha
liana
2215
0.107
127
120.0
023
76
0.608
613
.0768
2MS
/MS
Pp7
(4)15
.604
3B07
(xylem
)BX
2509
39Rip
ening
regula
tedpro
tein
4.35/2
4875
4.29/2
0248
Q9FR
34Ly
cope
rsico
nesc
ulentu
m58
10.0
001
125
0.249
512
00.0
033
7.49
524
MS/M
SPp
7(10
)25
.6CN
1167
(xylem
)BX
2499
53Sm
allhe
atsh
ockp
rotein
5.23/2
2024
4.97/1
7125
P118
90Ch
enop
odium
rubrum
––
–0
730.0
078
2125
0.269
329
.8452
5MS
/MS
Pp7
(10)
25.6
CN11
67(xy
lem)
BX24
9953
Small
heat
shoc
kprot
ein5.3
9/219
854.9
7/171
25P1
1890
Chen
opod
iumrub
rum–
––
60
0.135
421
210.8
339
11.93
516
MS/M
SPt
(4)14
.5co
ntig6
542_
2BE
0497
47Sm
allhe
atsh
ockp
rotein
5.35/2
2504
7.87/2
7451
Q9SE
11Fu
naria
hygro
metric
a–
––
046
0.033
832
360.6
422
28.31
86MS
/MS
Pt(2)
8.9co
ntig5
996_
3AW
8700
72Str
ess-i
nduc
edpro
tein,
sti1-l
ikepro
tein
6.09/7
8106
6.05/6
0408
Q8L7
24Ar
abido
psis
thalia
na–
––
013
0.014
625
310.1
555
17.15
94MS
/MS
Pt(2)
6.5co
ntig6
114_
3BF
6100
48Str
ess-i
nduc
edpro
tein,
sti1-l
ikepro
tein
6.28/7
5688
6/636
66Q9
STH1
Arab
idops
istha
liana
––
–0
220.0
137
5374
0.022
737
.3354
5MS
/MS
Pp*(
3)19
CN11
3(xy
lem)
BX24
9798
Supe
roxide
dismu
tase[
Cu-Zn
](EC1
.15.1.
1)5.9
5/195
715.7
5/153
67P2
4669
Pinus
sylve
stris
280
0.000
111
550.0
799
2530
0.214
530
.3548
5MS
/MS
Pt(2)
9.6co
ntig4
918_
4BF
1695
04De
hydro
asco
rbate
reduc
tase(
EC1.8
.5.1)
5.01/2
5420
7.59/2
8495
Q8LE
52Ar
abido
psis
thalia
na69
180.0
007
4065
0.056
646
430.7
849
48.37
Gene
sand
protei
nsex
press
ion25
2MS
/MS
SP(8)
25.2
Q9SE
I426
Sprot
ease
regula
torys
ubun
it6Bh
omolo
g5.9
3/500
425.4
2/457
51Q9
SEI4
Arab
idops
istha
liana
015
0.001
340
560.0
359
2735
0.009
339
.6512
6MS
/MS
Pt(6)
21.5
conti
g747
2_3
BM42
8171
40Sr
iboso
malp
rotein
5.21/6
7621
8.74/6
3290
Q8H2
L4Or
yzasa
tiva
––
–9
400.0
030
010
0.000
514
.955
2MS
/MS
Pp*(
4)21
.6CN
1785
(root)
BX68
1551
40Sr
iboso
malp
rotein
S12
5.06/1
8861
5.35/1
5285
Q9XH
S0Ho
rdeum
vulga
re38
0<1
0-412
110.9
261
03
0.292
46.3
938
7MS
/MS
Pp*(
4)24
.202
2B07
(xylem
)BX
2492
7760
Sacid
icrib
osom
alpro
tein
5.39/3
6015
5.15/3
4137
P503
46Gly
cinem
ax–
––
063
<10-4
3434
0.948
332
.9140
2MS
/MS
Pp7
(2)13
.502
2B07
(xylem
)BX
2492
7760
Sacid
icrib
osom
alpro
tein
5.36/3
4915
5.15/3
4137
P503
46Gly
cinem
ax51
350.0
312
4023
0.016
214
140.7
638
22.72
449
MS/M
SPt7
(2)11
.8co
ntig6
667_
3CD
0247
58Ch
apero
nin6.0
6/297
715.2
5/589
02Q9
4K05
Arab
idops
istha
liana
410
0.004
3–
––
1111
0.892
55.6
242
5MS
/MS
Pp7
(1)5.9
RN16
A02(
root)
BX67
6996
DNA-
dama
ge-re
pair/t
olerat
ionpro
teinD
RT10
25.1
8/316
785.2
7/252
41Q0
5212
Arab
idops
istha
liana
30
0.046
74
70.3
772
1212
0.992
08.8
439
1MS
/MS
Pp7
(8)27
.1CN
707(
root)
BX67
7176
Elong
ation
factor
1beta
4.45/3
6163
4.43/2
5117
P480
06Ar
abido
psis
thalia
na66
340.0
051
3413
0.011
622
190.7
335
22.05
432
MS/M
SPp
*(6)
33CN
1218
(xylem
)BX
2512
21Elo
ngati
onfac
tor1-b
eta4.2
8/303
384.4
1/243
51P9
3447
Pimpin
ellab
rachy
carpa
715
0.002
731
70.0
253
1112
0.701
215
.2943
3MS
/MS
Pp*(
8)24
.4CN
340(
root)
BX24
9583
Elong
ation
factor
1-beta
4.34/3
0267
4.41/2
4351
P934
47Pim
pinell
abrac
hyca
rpa83
620.0
032
2917
0.357
019
220.4
528
21.59
269
MS/M
SPp
*(6)
39.9
074E
06(xy
lem)
BX25
2896
Euka
ryotic
initia
tionf
actor
4A-11
5.24/4
7751
5.38/4
6872
Q404
65Ni
cotia
natab
acum
3849
0.258
036
140.0
028
2013
0.004
920
.6227
0MS
/MS
Pp*(
4)24
.507
4E06
(xylem
)BX
2528
96Eu
karyo
ticini
tiatio
nfac
tor4A
-115.1
9/473
305.3
8/468
72Q4
0465
Nico
tiana
tabac
um6
20.0
387
1235
0.002
59
120.0
974
16.7
271
MS/M
SPp
*(1)
4.9CN
918(
xylem
)BX
2492
25Eu
karyo
ticini
tiatio
nfac
tor4A
-145.3
1/472
175.3
8/468
46Q4
0467
Nico
tiana
tabac
um33
680.0
578
6171
0.298
560
600.9
837
62.77
551
MS/M
SPp
7(6)
56.2
CN13
29(ro
ot)BX
6783
86Gly
cine-r
ichRN
A-bin
dingp
rotein
5.62/1
8966
5.21/1
6006
P493
10Sin
apis
alba
381
238
0.001
014
618
0.004
60
32<1
0-448
.7547
0MS
/MS
Pp*(
5)16
.8CN
1726
(root)
BX68
1684
Prote
asom
esub
unita
lphat
ype3
(EC3.4
.25.1)
5.83/2
6313
6.11/2
7268
O243
62Sp
inacia
olerac
ea3
10.0
962
2232
0.467
729
270.4
121
27.45
477
MS/M
SPt
(4)13
.4co
ntig6
561_
1BQ
6548
71Pr
oteas
omes
ubun
italph
atyp
e4(EC
3.4.25
.1)5.3
6/254
836.1
7/274
48P5
2427
Spina
ciaole
racea
158
800.0
045
9214
50.0
647
042
<10-4
69.65
491
MS/M
SPp
*(8)
46.7
CN13
19(ro
ot)BX
6790
55Pr
oteas
omes
ubun
italph
atyp
e5-1
(EC3
.4.25
.1)4.5
5/242
574.7
/2596
4Q9
M4T8
Glycin
emax
320
0.000
39
90.9
368
910
0.672
99.1
754
8MS
/MS
Pp*(
8)40
CN12
81(ro
ot)CR
3929
19Pr
otein
kinas
eCinh
ibitor
5.58/1
9508
6.19/1
4292
P428
56Ze
amay
s72
80.0
005
827
0.005
410
170.1
668
29.15
229
MS/M
SPp
(13)
54.5
CN17
20(ro
ot)BX
6812
78Ra
bGDP
disso
ciatio
ninh
ibitor
alpha
5.52/5
3456
5/505
04P5
0398
Rattu
snorv
egicu
s–
––
068
0.000
532
290.6
855
32.25
257
MS/M
SPt
(3)7.3
conti
g772
4_3
BQ69
7516
RAD2
3prot
ein4.5
7/616
874.8
/4421
9Q9
M887
Arab
idops
istha
liana
123
580.0
053
7577
0.815
830
450.1
004
56.78
420
MS/M
SPp
(2)12
.910
2E08
(xylem
)BX
2546
80Ra
n-spe
cific
gtpas
e-acti
vatin
gprot
ein4.6
7/328
035.4
/2413
5Q0
9717
Schiz
osac
charo
myce
spom
be0
40.1
951
2510
90.0
001
8810
50.1
601
81.83
517
MS/M
SPp
(2)8.1
RS12
E06(
root)
BX67
8647
Ras-r
elated
protei
nARA
-34.8
1/222
167.6
5/238
20P2
8186
Arab
idops
istha
liana
160
0.001
912
130.9
214
1113
0.449
112
.3630
4MS
/MS
Pp7
(9)46
.4CN
327(
root)
BX67
8145
SRC2
onco
gene
4.94/4
2079
7.13/3
8989
Q9LN
V0Ar
abido
psis
thalia
na14
520.0
001
5476
0.186
676
910.1
420
74.17
Lipid
metab
olism
429
MS/M
SPp
7(3)
15.5
CN59
1(xy
lem)
BX25
0477
Isofla
vone
reduc
taseh
omolo
g(EC
1.3.1.
-)5.7
4/322
685.5
7/346
32P5
2579
Nico
tiana
tabac
um–
––
030
0.032
026
180.0
839
18.68
428
MS/M
SPp
(1)3.4
CN26
1(roo
t)BX
6675
58Iso
flavo
nered
uctas
ehom
olog(
EC1.3
.1.-)
6.24/3
0649
6.16/3
3831
P525
78So
lanum
tubero
sum
192
0.025
115
140
0.012
315
916
30.7
933
119.2
743
0Ed
man
SP(12
/15)
AF07
1477
Isofla
vone
reduc
tase(
EC1.3
.1.-)
5.94/3
1033
6.16/3
3831
P525
78So
lanum
tubero
sum
379
258
0.086
031
186
1<1
0-481
971
10.0
219
675.4
2
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
3740 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751Tab
le1.C
on
tin
ued
IM2D
spot
IDMe
thod
Hiton
datab
asea)
(Matc
hesb) )
Cove
r-ag
ec)Co
nsen
suso
rsin
gleton
Id)Ac
cess
ionc)
Assig
nmen
tpI
/Mrf)
pI/M
rg)Ac
cess
ionnu
mber
Spec
iesMe
anno
rmali
zedv
olume
h)Me
anpro
tein
volum
ei)M
Jp-
value
EL
p-va
lueC
Op-
value
Memb
ranea
ssoc
iated
364
MS/M
SPp
7(9)
28.2
CN86
5(roo
t)BX
6806
86Ste
roidm
embra
nebin
dingp
rotein
4.31/3
8375
4.56/2
1465
Q952
50Su
sscro
fa11
554
0.004
121
110.0
314
137
0.102
812
.89
Metab
olism
ofco
factor
sand
vitam
ins41
7MS
/MS
Pp7
(4)32
.9CN
627(
xylem
)BX
2525
28Cy
tochro
mec1
precu
rsor
4.00/3
2078
3.98/4
4864
P136
27Pa
racoc
cusd
enitri
fican
s19
358
0.001
894
00.0
030
2119
0.848
533
.54
Nucle
otide
metab
olism
503
MS/M
SPp
(1)3.7
CN11
84(ro
ot)BX
6777
86Ad
enine
phos
phori
bosy
ltrans
feras
e(EC
2.4.2.
7)5.0
2/239
905.3
5/197
27P3
1166
Arab
idops
istha
liana
110
0.019
018
70.0
392
109
0.672
111
.0855
4MS
/MS
Pp*(
2)15
.3CN
488(
root)
BX67
6865
Nucle
oside
dipho
spha
tekin
ase1
(EC2.7
.4.6)
6.08/1
8858
6.31/1
6341
O813
72Me
semb
ryanth
emum
crysta
llinum
109
5<1
0-453
105
0.269
60
410.0
960
49.76
638
MS/M
SPp
7(4)
16.5
CN10
44(xy
lem)
BX25
3409
UMP/C
MPkin
ase
5.06/2
6764
5.79/2
2468
O049
05Ar
abido
psis
thalia
na96
690.0
867
237
0.076
6–
––
7.57
Nucle
otide
suga
rsme
taboli
sm35
7MS
/MS
Pp*(
6)30
.1RS
16E0
9(roo
t)BX
6788
86UD
P-glu
cose
4-epim
erase
(EC5.1
.3.2)
6.09/3
8290
7.63/3
8966
Q430
70Pis
umsa
tivum
184
0.114
77
120.3
396
1713
0.257
912
.29
Photo
synth
esis
506
MS/M
SPp
7(2)
13CN
743(
xylem
)BX
2499
05Fla
vopro
teinw
rba6.3
9/246
735.7
9/206
93Q8
Z7N9
Salm
onell
atyp
hi25
00.0
437
013
0.218
034
430.5
624
22.4
Prim
aryme
taboli
sm(en
ergy)
202
MS/M
SPp
*(10
)31
.8CN
468(
xylem
)BX
2507
92AT
Psyn
thase
beta
chain
(EC3.6
.3.14
)5.3
1/574
275.1
3/540
97P1
7614
Nico
tiana
plumb
agini
folia
1167
897
0.002
571
767
90.4
964
404
443
0.378
656
0.79
532
MS/M
SPp
*(7)
17.8
CN46
8(xy
lem)
BX25
0792
ATPs
yntha
sebe
tach
ain(EC
3.6.3.
14)
6.00/5
7427
5.19/5
4029
P190
23Ze
amay
s–
––
541
60.0
006
149
207
0.001
619
4.29
945
MS/M
SPp
*(2)
10.5
CN21
23(ro
ot)AL
7507
20Ph
osph
oglyc
erate
kinas
e(EC
2.7.2.
3)5.7
3/365
215.2
9/426
01P2
9409
Spina
ciaole
racea
––
–0
40.0
006
––
–0.8
963
9MS
/MS
Pp7
(6)19
.6CN
406(
xylem
)BX
2498
48So
luble
inorga
nicpy
ropho
spha
tase(
EC3.6
.1.1)
6.25/2
6764
5.59/2
4246
Q431
87So
lanum
tubero
sum
71
0.014
85
30.5
636
80
0.000
83.9
283
MS/M
SPp
*(2)
24.6
RS16
G07(
root)
BX67
8905
Vacu
olarA
TPsy
nthas
e(EC
.3.6.
3.14)
5.47/7
8817
5.29/6
8835
P094
69Da
ucus
carot
a17
616
80.7
120
151
144
0.626
096
109
0.039
212
5.01
340
MS/M
SPp
(6)32
.600
9F03
(xylem
)BX
2557
74Va
cuola
rATP
synth
ase(
EC3.6
.3.14
)5.5
6/403
355.4
/4259
3Q9
SDS7
Arab
idops
istha
liana
70
0.355
911
90.6
634
58
0.256
47.9
210
9MS
/MS
SP(2)
4.2P3
4105
NADP
-depe
nden
tmali
cenz
yme(
EC1.1
.1.40
)5.7
6/703
576.5
/6522
3P3
4105
Popu
lustric
hoca
rpa9
00.0
003
45
0.271
17
100.1
249
6.63
Seco
ndary
metab
olism
519
MS/M
SPp
(6)29
.5CN
1427
(root)
BX67
8193
Glutat
hione
perox
idase
(EC1.1
1.1.9)
4.92/2
1599
5.92/1
9036
O238
14Sp
inacia
olerac
ea18
00.0
210
66
0.986
610
130.1
918
8.41
118
MS/M
SPt7
(2)13
.2co
ntig7
08_1
BE76
2017
beta
gluco
sidas
e5.3
2/691
575.3
9/560
42Q9
FIW4
Arab
idops
istha
liana
40
0.005
83
30.7
582
33
0.697
83.0
212
7MS
/MS
Pt7(2)
15.6
conti
g708
_1BE
7620
17be
taglu
cosid
ase
5.28/6
7570
5.39/5
6043
Q9FIW
4Ar
abido
psis
thalia
na12
00.0
068
2383
0.000
216
170.5
767
34.65
669
MS/M
SPp
7(3)
13.6
054F
12(xy
lem)
BX25
1740
Leuc
oanth
ocya
nidin
dioxy
gena
se(EC
1.14.1
1.19)
5.89/4
1222
5.13/4
8402
P510
92Pe
tunia
hybri
da–
––
––
–13
0<1
0-43.2
859
0MS
/MS
Pp7
(3)17
CN32
7(xy
lem)
BX25
0111
Limon
oidUD
P-glu
cosy
ltrans
feras
e(EC
2.4.1.
210)
5.55/6
1271
5.31/5
7441
Q9MB
73Cit
rusun
shiu
021
0.063
833
0<1
0-4–
––
8.18
168
MS/M
SPt
(8)11
.7co
ntig7
633_
1BE
5823
27Mi
tocho
ndria
lproc
essin
gpep
tidas
ebeta
subu
nit6.2
6/604
966.5
6/588
45Q9
AXQ2
Cucu
mism
elo0
60.3
352
3241
0.223
437
360.8
778
36.48
585
MS/M
SPp
*(9)
35.7
CN29
2(xy
lem)
BX25
1011
Prote
indis
ulfide
isome
rase(
EC5.3
.4.1)
4.67/6
6969
4.91/5
2998
Q431
16Ric
inusc
ommu
nis28
730.0
020
7910
0.000
17
00.0
131
24.15
940
MS/M
SPp
7(4)
25.1
RN62
D04(
root)
CR39
3604
Prote
indis
ulfide
isome
rase(
EC5.3
.4.1)
5.44/4
2052
5.29/3
7301
P386
61Me
dicag
osati
va–
––
026
0.000
2–
––
6.41
113
Edma
nSP
(9/10
)P5
2588
Prote
indis
ulfide
isome
rase(
EC5.3
.4.1)
4.63/6
7928
5.11/5
4655
P525
88Ze
amay
s44
690.0
463
117
130
0.401
928
300.8
511
76.3
Uncla
ssifie
d44
6MS
/MS
Pp*(
2)13
.5CN
1700
(root)
BX68
1197
14-3-
3-like
protei
n4.6
2/298
654.8
3/305
26Q9
C5W
6Ar
abido
psis
thalia
na71
70.0
004
5132
0.072
224
250.8
882
32.75
453
MS/M
SPp
*(11
)30
.8CN
1248
(xylem
)BX
2506
9914
-3-3-l
ikepro
tein
4.67/2
9089
4.79/2
9235
Q9SP
07Lil
iumlon
giflor
um11
557
0.140
815
415
20.9
405
8180
0.990
211
6.57
455
MS/M
SPp
*(8)
19.7
CN12
48(xy
lem)
BX25
0699
14-3-
3-like
protei
n4.7
4/281
454.7
9/292
35Q9
SP07
Lilium
longif
lorum
5653
0.795
171
730.8
508
4939
0.153
858
.143
7MS
/MS
Pp7
(3)14
.902
0A03
(xylem
)BX
2490
9514
-3-3-l
ikepro
tein
4.73/2
9839
4.79/2
9500
Q964
53Gly
cinem
ax44
180.1
842
8729
0.005
435
330.7
456
45.94
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
Proteomics 2005, 5, 3731–3751 Plant Proteomics 3741Tab
le1.C
on
tin
ued
IM2D
spot
IDMe
thod
Hiton
datab
asea)
(Matc
hesb) )
Cove
r-ag
ec)Co
nsen
suso
rsin
gleton
Id)Ac
cess
ionc)
Assig
nmen
tpI
/Mrf)
pI/M
rg)Ac
cess
ionnu
mber
Spec
iesMe
anno
rmali
zedv
olume
h)Me
anpro
tein
volum
ei)M
Jp-
value
EL
p-va
lueC
Op-
value
450
MS/M
SPp
*(5)
22CN
1087
(xylem
)BX
2494
0614
-3-3-l
ikepro
tein
4.84/2
9290
4.79/2
9500
Q964
53Gly
cinem
ax13
100.6
643
3573
0.002
339
470.2
892
48.43
57MS
/MS
Pt(4)
18.9
conti
g409
3_6
BF60
9020
acyl
CoAb
inding
protei
n5.5
1/838
035.8
9/709
65Q8
RWD9
Arab
idops
istha
liana
3423
0.050
319
50.0
158
106
0.116
19.9
524
MS/M
SSP
(5)6.4
P350
16En
dopla
smin
homo
logpre
curso
r(Grp9
4)4.7
5/106
433
4.86/9
3492
P350
16Ca
tharan
thusr
oseu
s–
––
032
0.000
411
711
20.6
773
65.22
496
MS/M
SPt
(2)2.8
conti
g586
8_1
BG03
9026
Ferri
tinsu
bunit
-relat
ed5.1
7/250
285.1
3/237
42Q9
SRL5
Arab
idops
istha
liana
3624
0.192
40
110.0
836
2222
0.896
513
.8682
1MS
/MS
Pp7
(6)33
.9CN
276(
xylem
)BX
2518
22GA
L4DN
A-bin
dinge
nhan
cerp
rotein
24.2
8/264
264.8
4/186
97P3
8879
Sacc
harom
yces
cerev
isiae
386
0.003
712
150.6
823
––
–6.6
849
7MS
/MS
Pp(5)
48.3
CN16
51(ro
ot)BX
6813
07Ge
rmin-
likep
rotein
5.35/2
4530
5.81/1
9362
P940
14Ar
abido
psis
thalia
na27
00.0
001
1543
0.026
863
250.0
000
36.54
730
MS/M
SPp
7(13
)42
.6CN
1875
(root)
BX68
1971
IN2-1
protei
n6.1
3/245
274.8
/2697
2P4
9248
Zeam
ays
657
0.003
60
50.1
447
––
–1.2
554
9MS
/MS
Pp*(
1)4.1
CN36
7(xy
lem)
BX24
9454
Profi
lin1
4.45/1
9058
4.88/1
4172
P492
31Ph
aseo
lusvu
lgaris
224
580.0
053
117
480.0
466
069
0.007
858
.6814
6MS
/MS
SP(5)
11.2
P287
69T-c
omple
xprot
ein1,
alpha
subu
nit6.1
4/631
085.9
3/592
29P2
8769
Arab
idops
istha
liana
––
–0
100.0
065
2129
0.171
515
.1
Nohit
201
MS/M
SNo
hit5.5
1/569
3758
00.0
008
––
–6
50.2
476
2.65
334
MS/M
SNo
hit4.7
7/418
339
00.0
096
89
0.875
16
90.1
180
7.85
649
MS/M
SNo
hit5.3
4/198
74–
––
06
0.147
9–
––
1.38
648
MS/M
SNo
hit5.9
3/204
28–
––
013
0.117
012
00.0
003
6.24
707
MS/M
SNo
hit5.5
7/504
810
10.3
559
02
0.004
2–
––
0.58
463
MS/M
SNo
hit5.7
2/289
380
00.0
805
521
0.132
714
170.1
249
14.26
540
MS/M
SNo
hit6.1
0/195
8537
140.1
984
7477
0.936
964
470.1
556
65.57
515
MS/M
SNo
hit5.5
0/228
1210
918
<10-4
4837
0.653
633
500.0
101
41.89
630
MS/M
SNo
hit5.7
5/301
43–
––
––
–6
0<1
0-41.5
651
0MS
/MS
Nohit
5.41/2
3219
509
0.001
977
138
0.015
059
600.9
212
83.57
467
MS/M
SNo
hit5.2
0/259
8641
10.0
002
2115
0.554
031
310.9
749
24.67
472
MS/M
SNo
hit4.9
8/266
8160
180.0
113
7958
0.075
034
590.1
354
57.39
720
MS/M
SNo
hit5.3
1/336
9817
00.0
061
––
––
––
025
3MS
/MS
Nohit
5.02/5
0485
920
0.141
944
260.0
418
2218
0.242
927
.690
5MS
/MS
Nohit
5.17/3
1656
30
0.026
42
00.1
247
––
–0.4
531
6MS
/MS
Nohit
5.29/4
1461
036
0.024
533
360.7
808
1018
0.003
924
.3631
4MS
/MS
Nohit
5.20/4
1426
711
0.228
816
110.2
350
07
0.007
08.3
612
1MS
/MS
Nohit
4.92/6
7765
200
0.012
921
280.3
898
1717
0.962
920
.5165
6MS
/MS
Nohit
6.29/8
5420
––
–4
00.0
642
180
0.003
65.4
247
5MS
/MS
Nohit
5.53/2
5582
2720
0.524
90
390.0
023
4662
0.060
136
.9844
0MS
/MS
Nohit
5.30/3
0588
80
0.001
26
50.7
998
68
0.340
16.5
452
3MS
/MS
Nohit
5.11/2
1951
30
0.003
10
240.0
921
1318
0.239
313
.9135
6MS
/MS
Nohit
6.13/3
7715
––
–6
00,0
015
131
101
0.092
159
.6589
7MS
/MS
Nohit
4.77/9
7180
––
––
––
100
<10-4
2.44
521
MS/M
SNo
hit5.9
5/216
988
00.0
003
60
0.168
37
70.9
375
5.27
302
MS/M
SNo
hit5.8
0/436
000
00.2
711
735
0.000
117
180.8
759
19.39
736
MS/M
SNo
hit5.8
5/203
71–
––
026
0.058
9–
––
6.58
95MS
/MS
Nohit
5.05/7
3399
67
0.844
54
110.2
109
1214
0.395
010
.0318
MS/M
SNo
hit6.6
1/104
293
––
––
––
2825
0.839
213
.37
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
3742 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751Tab
le1.C
on
tin
ued
IM2D
spot
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thod
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1120
0.088
013
150.7
775
14.96
47MS
/MS
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5.04/9
4480
––
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30.1
056
4142
0.781
921
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MS/M
SNo
hit5.0
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37–
––
––
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560.8
289
27.81
646
MS/M
SNo
hit5.1
1/224
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––
60
0.134
9–
––
1.43
262
MS/M
SNo
hit5.7
9/492
340
00.1
108
30
0.000
212
80.0
667
5.63
398
MS/M
SNo
hit5.8
6/348
6413
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0-42
20.9
883
05
0.001
92.3
383
MS/M
SNo
hit5.8
8/361
853
00.0
734
510
0.092
08
100.1
774
8.27
494
MS/M
SNo
hit6.5
0/254
61–
––
05
0.145
916
190.8
043
10.02
536
MS/M
SNo
hit5.3
6/203
918
00.0
011
69
0.732
917
120.3
703
10.92
289
MS/M
SNo
hit4.9
1/453
51–
––
09
0.002
86
80.2
634
5.67
877
MS/M
SNo
hit6.4
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63–
––
017
0.228
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––
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2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
Proteomics 2005, 5, 3731–3751 Plant Proteomics 3743
bases (,90 000), these proteins might correspond to very raretranscript that have not be sequenced in EST projects to date,and may indicate a specific role of these proteins in wood for-mation. Finally, 37 spots (15.4%) corresponded to mixtures ofproteins. Comparatively, only 9 (15.8%) of the 57 spots analyzedby MALDI-TOF MS were identified using the same databases,from which six were also analyzed and identified by MS/MS(#83, #212, #217, #226, #242, and #282), and three (#215, #216,and #223) corresponded to tubulin b chain isoforms. Suchdiscrepancy in identification rates between MS/MS and MSalone has recently been reported in Panax ginseng [32], and canlargely be attributed to the lack of genome database or extensiveEST datasets for most plant species, gymnosperms in particu-lar. To these 166 proteins identified by mass spectrometry, oneshould add another nine proteins corresponding to very abun-dant proteins in wood forming tissues, and whose function waspreviously determined by internal microsequencing [19].These proteins have been also localized on the reference gel(Fig. 2) and correspond to the following functions: #80, #81,and #88: HSP 70 kDa, #113: protein disulfide isomerase (EC5.3.4.1), #266: SAM-S (S-adenosylmethionine synthetase) (EC2.5.1.6), #297: actin, the most intense spot on our gels, #430:isoflavone reductase (EC 1.3.1.) probably the second mostintense spot not only in this experiment, but also reported asthe most highly expressed protein in poplar wood formingtissue [20], #439: caffeoyl-CoA-O-methyltransferase (EC2.1.1.104), and #482: ascorbate peroxidase (EC 1.11.1.11).Overall, 175 proteins were thus identified in this study. Theseproteins are marked with arrows and numbers in Fig. 2. Over-all, membrane proteins were under-represented, with theexception of a vacuolar ATPase subunit (#340). This observa-tion is not specific to the wood proteome, and can be attributedto the general poor solubilization of such proteins.
3.3 Protein identification of wood forming tissue and
functional classification
A summary of protein functions is given in Table 1. The func-tional distribution of the known function proteins is reportedin Fig. 3. Proteins were classified into 15 groups based on
functional categories using the DBGET system (http://www.genome.ad.jp/dbget/). Interestingly, 87% of the pro-teins fell into eight major groups, while 13% were classifiedin seven other minor groups. Major groups were for “defense”(34 spots), “carbohydrate metabolism” (29 spots), “amino acidmetabolism” (26 spots), “genes and proteins expression”(23 spots) (including signal transduction, transcription,translation, protein assembly, modification and degradation),“cytoskeleton” (14 spots), “cell wall” (10 spots), “secondarymetabolism” (9 spots), and “primary metabolism” (7 spots).Overall, 39.4% of the proteins appeared as multiple spots andaccounted for most of the proteins found in the groups. Sucha high number of spots attributed to one protein has recentlybeen reported in Medicago truncatula [33]. This observationmay reflect post-translational modification, allelic variation ofthe same protein (e.g., position shift variants), isozyme varia-tion (proteins encoded by paralogs), alternative splicingevents, but also protein degradation.
SAM-S for example was represented by 14 spots (3.36%of the 300 studied proteins), accounting for 53.8% of theproteins of the amino acid metabolism category. SAM servesas a universal methyl group donor in numerous trans-methylation reactions that involve many types of acceptormolecules (proteins, nucleic acids, polysaccharides, fattyacids). It is also the substrate of many reactions, such asvitamins, polyamines, the gaseous phytohormone ethylene,and nucleotide biosynthesis. SAM is believed to be next toATP for the number of reactions in which a biological com-pound is used [34]. The transcript of SAM-S was found at ahigh level in cDNA libraries constructed from pine andpoplar differentiating xylem [11, 12, 35]. Among others,SAM-S plays a role in the methylation of monolignol pre-cursors during lignin biosynthesis [36].
The carbohydrate metabolism category presented aredundancy of 51.7%, with most proteins present two to fourtimes, e.g., pyrophosphate fructose 6-phosphate 1-phospho-transferase and ascorbate peroxidase (four spots), 2,3-bisphos-phoglycerate-independent phosphoglycerate mutase andphosphoglucomutase (three spots), fructokinase, transketo-lase, alcohol dehydrogenase, enolase, aconitase (two spots).
Figure 3. Functional distributionof the major proteins in mar-itime pine wood forming tissue,as separated by 2-DE.
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
3744 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751
Carbohydrates (cellulose, hemicelluloses) are essential com-ponents of the cell wall. The abundance of these proteinsclearly shows the importance of carbohydrate biosyntheticpathways during xylogenesis [37].
Arabinogalactan proteins (AGP) represented 40% of thecell wall category. EST sequencing in trees [12, 35, 38] hasrevealed a high level of transcript accumulation of these pro-teins in wood forming tissue. In the maritime pine xylemEST database, this protein ranked first among the mosthighly expressed genes. Moreover, Loopstra and Sederoff [39]reported that some AGP were preferentially expressed indifferentiating xylem compared to other tissue, suggestingan important role of these proteins in vascular development.Enzymes involved in the second most abundant compoundof the cell wall, lignins, were also well represented in the setof studied proteins, with four proteins identified, corre-sponding to C-CoA-OMT (caffeoyl-CoA-O-methyltransfer-ase), COMT (caffeic acid 3-O methyltransferase), CAD (cin-namyl-alcohol dehydrogenase) and a peroxidase.
The 10 spots classified in the cytoskeleton category cor-responded to only two proteins, namely tubulin (a and bsubunits) and actin. These proteins were also found to behighly expressed at the transcriptome level in loblolly pine[14] and poplar [35] wood forming tissues. Actin and tubulinconstitutes essential component of the cytoskeleton. Corticalmicrotubules are mainly composed of a and b tubulin. Cor-tical microtubules could determine the cell wall pattern bydefining the position and the orientation of cellulose micro-fibrils during the differentiation of tracheid elements [40],probably by guiding the movement of the cellulose-synthe-sizing complex in the plasma membrane.
The defense category mainly comprised heat shock pro-teins (HSP): 20 spots in total of low and high molecularweight, representing 11.4% of the identified proteins in thisstudy. Canton et al. [16] showed that HSP were much moreexpressed in differentiating xylem of maritime pine com-pared to other tissues (pollen, bud, phloem, cambium, nee-dles, and root). HSP are well known to be produced in re-sponse to various stresses [41–43]. Synthesis also occursduring developmental processes such as pollen or seedmaturation [44], and early seedling growth [45]. Recently, LeProvost et al. [46] hypothesized that some HSPs could havespecific role during wood formation, showing that theseproteins are important proteins for the normal developmentof secondary wood. The presence of multiple spots corre-sponding to LEA (late embryogenesis abundant) like pro-teins, abscisic stress ripening-like protein and stress inducedproteins is also worth noting and could be related to thepresence of drought stressed tissues, namely LW, OW andCW, sampled in summer.
The gene and protein expression category was repre-sented by 23 spots involved in signal transduction, tran-scription, translation, protein assembly, modification anddegradation. Most of these proteins (11 spots) correspondedeither to ribosomal protein or initiation and elongation fac-tors. It has been reported that the expression of initiation
factor could be related to GTP-binding protein. Moreover,Schultheiss et al. [47] have shown that GTP-binding proteinsare potentially involved in cellular shape and cell wall for-mation.
Subunits of the ATP-synthase complex were the mostabundant proteins of the primary metabolism category. Thisobservation is certainly related to the high energy demandfor tracheids elongation and growth.
The secondary metabolism was represented by eightproteins, two of which being similar to protein disulfideisomerase (PDI). In endoplasmic reticulum of eukaryotes,PDI catalyzes the formation, isomerization and reduction ofdisulfide bonds to ensure the correct folding of secretoryproteins prior to their further modification and transport[48]. High expression of PDI during wood formation isprobably related to the high metabolic activity existing invascular cambium.
3.4 Correlation between protein and mRNA
abundance
The relationship between mRNA and protein abundances isneeded to elucidate the processes and regulation of tran-scription and translation. For this comparison, mRNAabundance was estimated for unique functional annotationsby counting the number of ESTs corresponding to thesefunctions among the 8429 xylem ESTs. Protein amount wasestimated by determining the volume of each spot averagedacross the four samples used to generate the cDNA library(i.e., OW, CW, EW, and LW). Average values for a given func-tional annotation (e.g., SAM-S) were then summed to obtainthe global volume of that function. Raw data for proteinamount and number of EST are provided in Table 2. Thecorrelation between mRNA and protein level is shown inFig. 4. There was a general trend of increased protein level,resulting from increase in mRNA level. The Pearson corre-lation coefficient for the whole dataset was 0.46. However,when highly expressed genes at the transcript (AGP) andprotein (SAM-S, actin, a and b tubulin, ATP synthase bchain, isoflavone reductase and HSP70 kDa) levels wereremoved from the dataset, the correlation coefficient was stillpositive but dropped down to 0.31. Although our analysiswas restricted to a limited number of highly abundant pro-teins (i.e., revealed by 2-DE), this result indicates a weaklypositive correlation between mRNA and protein abundance.A similar result has also been reported in yeast by Gygi et al.[49] and Washburn et al. [50]. In a recent report on the pro-teome of Medicago truncatula, Watson et al. [51] reported on amoderate level (r = 0.50) of correlation between mRNAabundance (estimated by EST counting) and protein amount(estimated by 2-DE). Given the biased representation of thepresent proteome (poor representation of membrane, highmolecular weight and basic proteins), we believe that 0.31may represent a lower limit of the wood proteome.
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
Proteomics 2005, 5, 3731–3751 Plant Proteomics 3745
Table 2. Correspondence between protein [Q (protein), normalized volume] and mRNA [Q (transcript), number of EST] abundance
Spot ID Function Q(protein) Q(transcript)
297 Actin 10389 37223 Tubulin beta chain 4789 36266 S-Adenosylmethionine synthetase (EC 2.5.1.6) 4324 83781 Tubulin alpha chain 4277 7888 HSP70 kDa 3322 18
430 Isoflavone reductase (EC 1.3.1.-) 3254 20532 ATP synthase beta chain (EC 3.6.3.14) 3020 6489 Caffeoyl-CoA O-methyltransferase 1440 19482 L-ascorbate peroxidase (EC 1.11.1.11) 1394 21450 14-3-3-like protein 1207 13484 Triosephosphate isomerase (EC 5.3.1.1) 1117 6211 Enolase (EC 4.2.1.11) 1037 8349 UDP-glucose protein transglucosylase (EC 2.4.1.15) 1024 23197 S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) 889 0360 Fructokinase (EC 2.7.1.4) 792 11315 Cinnamyl-alcohol dehydrogenase (EC 1.1.1.195) 760 6542 17.1 kDa class II heat shock protein 738 084 Phosphoglucomutase (EC 5.4.2.2) 736 5
390 Abscisic stress ripening-like protein 712 19221 Pyrophosphate fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) 654 12340 Vacuolar ATP synthase (EC 3.6.3.14) 532 16623 Arabinogalactan/proline-rich protein 492 127113 Protein disulfide isomerase (EC 5.3.4.1) 427 0499 Glutathione S-transferase (EC:2.5.1.18) 404 4158 60 kDa chaperonin 389 969 Transketolase (EC 2.2.1.1) 366 1
108 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (EC 5.4.2.1) 345 497 Heat shock protein 335 20
420 Ran-specific gtpase-activating protein 327 2159 D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) 321 3304 SRC2 oncogene 297 1246 Dihydrolipoamide acetyltransferase (EC 2.3.1.12) 280 0516 Small heat shock protein 280 3477 Proteasome subunit alpha type 4 (EC 3.4.25.1) 279 0298 Alcohol dehydrogenase (EC 1.1.1.1) 261 624 Endoplasmin homolog precursor (Grp94) 261 1
271 Eukaryotic initiation factor 4A-14 251 0433 Elongation factor 1-beta 236 14463 60S acidic ribosomal protein 233 0257 RAD 23 protein 227 294 Stress-induced protein, sti1-like protein 218 0
554 Nucleoside diphosphate kinase 1 (EC 2.7.4.6) 199 3551 Glycine-rich RNA-binding protein 195 44485 Dehydroascorbate reductase (EC 1.8.5.1) 193 0734 18.0 kDa class I heat shock protein 169 923 Aminopeptidase N (EC 3.4.11.2) 162 0
252 26S protease regulatory subunit 6B homolog 159 219 Aconitase EC:4.2.1.3 157 0
127 Latex cyanogenic beta glucosidase 151 0270 Eukaryotic initiation factor 4A-11 149 1168 Mitochondrial processing peptidase beta subunit 146 0497 Germin-like protein 146 5719 Late embryogenesis-like protein 143 15535 22.7 kDa class IV heat shock protein 136 0417 Cytochrome c1 precursor 134 18229 Rab GDP dissociation inhibitor alpha 129 6618 Caffeic acid 3-O methyltransferase like protein (EC 2.1.1.68 ) 124 0
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
3746 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751
Table 2. Continued
Spot ID Function Q(protein) Q(transcript)
247 Xylose isomerase (EC 5.3.1.5). 123 0545 Superoxide dismutase [Cu-Zn] (EC 1.15.1.1). 121 10548 Protein kinase C inhibitor 117 1241 Aminoacylase-1 (EC 3.5.1.14) 114 0470 Proteasome subunit alpha type 3 (EC 3.4.25.1) 110 1322 NAD-dependent sorbitol dehydrogenase 94 0506 Flavoprotein wrba 90 30552 40S ribosomal protein S12 85 0146 T-complex protein 1, alpha subunit 60 0496 Ferritin subunit-related 55 1375 Fructose-bisphosphate aldolase (EC 4.1.2.13) 54 5183 Putative selenium binding protein 52 2364 Steroid membrane binding protein 52 0517 Ras-related protein ARA-3 49 9357 UDP-glucose 4-epimerase (EC 5.1.3.2) 49 17166 2-isopropylmalate synthase A (EC 2.3.3.13) 45 157 Acyl CoA binding protein 40 6
665 Peroxidase 54 precursor (EC 1.11.1.7) 38 16491 Proteasome subunit alpha type 5–1 ( EC 3.4.25.1) 37 2425 DNA-damage-repair/toleration protein DRT102 35 1519 Glutathione peroxidase (EC 1.11.1.9) 34 1590 Limonoid UDP-glucosyltransferase (EC 2.4.1.210) 33 15682 Ripening regulated protein 30 1638 UMP/CMP kinase 30 0109 NADP-dependent malic enzyme (EC 1.1.1.40) 27 2821 GAL4 DNA- binding enhancer protein 2 27 0874 Osr40g3 protein (abscisic acid and salt stress responsive) 21 9639 Soluble inorganic pyrophosphatase (EC 3.6.1.1) 16 6669 Leucoanthocyanidin dioxygenase (EC 1.14.11.19) 13 5730 IN2–1 protein 5 1945 Phosphoglycerate kinase (EC 2.7.2.3) 4 3
3.5 Protein expression in the six types of wood
The clustering of the 215 proteins (175 known and40 unknown function proteins) analyzed in this study andquantified over the six types of wood, clearly showed thatseasonal effect was the main factor controlling protein accu-mulation in wood forming tissue. Indeed, the six samplesclustered together into two distinct sub-trees (Fig. 5), withthe three samples collected in summer (LW, CW and OW)forming a first branch, and the three samples collected inspring (EW, JW and MW) forming a second branch. Then,ontogenic (JW vs. MW) and gravitational (OW vs. CW) effectsranked second and third, respectively. The same conclusionscould be drawn from the analysis of the whole dataset(1039 spots, data not shown). A simple pair-wise comparison(t-test), confirmed that the seasonal factor presented themost important effect on protein accumulation in secondaryxylem during wood formation. Indeed, 39.5, 30.7, and 20% ofthe identified proteins exhibited distinctive expression pat-terns between EW vs. LW, JW vs. MW, and OW vs. CW,respectively (Table 1). Examples of differentially expressed
proteins are given in Fig. 6. Given that the quality of woodand derived products largely depends on the physical andchemical properties of xylem secondary cell wall, and giventhe phenotypic differences in terms of wood quality betweenthese six samples, it can be hypothesized that some of theseproteins could be related to the changes in secondary cellwall structure and composition, and therefore representinteresting targets potentially controlling wood and end-useproperties.
To cluster the proteins showing similar expressionprofiles in the six types of wood, hierarchical clusteringwas applied to the 215 proteins (Fig. 7A). Interestingly,while among the most abundant proteins, actin, tubulins,AGP, 14-3-3 tended to be clustered and expressed con-stitutively across the six types of wood, SAM-S spots werefound to be distributed throughout the dendrogram,showing that members of this multigene family are eitherexpressed constitutively (#279, #282, #275, #321), or speci-fically regulated by environmental and/or ontogenic factors(Fig. 7B, cluster C1). In the following paragraphs we willonly discuss three clusters characteristic of protein over-
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Proteomics 2005, 5, 3731–3751 Plant Proteomics 3747
Figure 4. Correlation betweenprotein (x axis, normalized vol-ume) and mRNA (y axis, num-ber of EST) abundance. Plainline: correlation for the wholedataset (88 functions listed inTable 2, r = 0.46). Inset: correla-tion for a restricted datasetwhere AGP, SAM-S, actin, a andb tubulin, ATP synthase b chain,isoflavone reductase andHSP70 kDa were removed (r =0.31).
Figure 5. Clustering result between JW, MW, EW, LW, OW, andCW based on the proteins listed in Tab. 1.
expressed in either LW, MW, or CW, as well as one clusterand one protein overexpressed in normal wood and EW,respectively.
The LW cluster contained 21 proteins (Fig. 7B, clus-ter C2), most of which already reported as drought respon-sive in plants, such as: (i) the abscisic stress ripening protein,a plant gene with unknown biological role that becomesoverexpressed under water- and salt-stress conditions [52];(ii) low and high molecular weight HSP, including theendoplasmin precursor 94 kDa glucose-regulated protein (aholomog of HSP90) and stress-induced proteins (sti1-likeprotein) (reviewed by [53]); and (iii) isoflavone reductase [54].As for the two SAM-S accumulating in LW tissue (#260,#213), it should be reminded that besides their central role inplant growth and development [55], these proteins have alsobeen reported as drought stress regulated [56, 57]. Con-versely, one spot (#586) corresponding to an arabinoga-lactan/proline-rich protein (AGP) was identified as an EWprotein. AGP are abundant in the plant cell wall. They havebeen reported as among the most expressed genes in differ-entiating xylem of poplar and pine stems, undergoing radialexpansion by secondary growth [11, 12, 14, 16, 18]. Althoughtheir exact functions are unclear, they are implicated indiverse processes associated with plant growth and develop-ment, including cell proliferation (reviewed in [58]). Its over-expression in EW could be related to the higher rate of celldivision occurring in spring time.
The MW cluster contained 23 co-regulated proteins(Fig. 7B, cluster C3), most of which were also significantlydifferentially expressed between JW and MW as revealed byt-tests. As opposed to differentiating xylem formed by young
cambium, MW differentiating xylem is characterized bylarge cells with thick cell wall and lower microfibril angle,high cellulose content, and lower lignin content [4]. MW isalso characterized by higher LW and lower CW content per-centage. We hypothesized that the molecular mechanisms,as revealed by the protein overexpressed in MW, contributedto the delay of programmed cell death (PCD), therefore pro-longating cell wall deposition, resulting in the higher wooddensity characteristic of MW. Our results suggest that fourmechanisms, DNA reparation, cell detoxication, proteolysisregulation, and reduction of cytoplasm acidosis contribute toprolonged cell life.
In plants, PCD is involved in the terminal differentiationof xylem vessels [59]. DNA degradation has been reported asone of the key events associated with tracheary element dif-ferentiation [60]. In this study, a low abundant protein(spot #425) corresponding to a DNA-damage repair/tolera-tion protein DTR 102 was found to be overexpressed in MW,supporting our hypothesis.
In addition, several proteins of the MWcluster belonged tothe defense category, particularly involved in detoxicationmechanisms, also likely to contribute to the delay of PCD.These proteins included a superoxide dismutase [Cu-Zn](#545), one glutahione S-transferase (#499), and one glutathi-one peroxidase (#519). One germin-like protein (GLP, #497)categorized in the “unclassified” category was also present inthis cluster. Germins/GLP have been reported as proteinsinvolved in defense mechanisms (with either oxalate oxidaseor extracellular [Mn] superoxide dismutase activities [61, 62]).Interestingly, spot #497 was tightly linked with spot #545,reinforcing the putative role of this GLP in oxidative stressdefense. Kim and Tripplett [63] recently reported that a cottonfiber germin-like protein (GhGLP1) exhibited a maximalexpression with stages of maximal cotton fiber elongation,suggesting that some GLP may be important for cell wallexpansion. All together, these results suggest an importantrole of GLP in MW differentiation. The accumulation of ade-nine phosphoribosyl transferase (APT) (spot #503), a proteinimplicated in salvage of adenine to AMP, in MW differentiat-ing cells could also be interpreted as a defense mechanismagainst adenine, a toxic compound for the cell.
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3748 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751
Figure 6. Cuttings from 2-D gels showing different types of behavior in protein accumulation between JW vs. MW, EW vs. LW, and OW vs.CW.
Ubiquitin-dependent proteolysis plays a crucial role dur-ing the development in all organisms. Especially in plants, ithas been shown that phytohormone action depended on theubiquitin-proteasome pathway [64]. Recently, Paux et al. [17]showed the importance of auxin signaling through ubiqui-tin-dependent proteolysis, during wood formation. Ourfindings, i.e., up-regulation of a GTP-binding protein (Ras-related protein ARA-3, #517) and a proteasome protein(#491) in MW forming tissue, highlight the importance ofprotein degradation in the regulation of MW differentiation.
According to Drew [65], metabolic changes under anoxiahelp maintain cell survival by generating ATP anaerobicallyand minimizing the cytoplasmic acidosis associated with celldeath. In anaerobic treatment of maize seedlings, 20 anaero-bic proteins (ANP), which accounted for more then 70% oftotal translation, were selectively synthesized [66]. Most ofthese ANP corresponded to enzymes of glycolysis or sugar-phosphate metabolism [67]. In this study, proteins of thecarbohydrate and primary metabolisms category were alsofound to be up-regulated in MW forming tissue, namely afructokinase (#355), an alcohol dehydrogenase (#305), aphosphoglucomutase (#84), and NADP-dependent malicenzyme (spot #109). Besides these mechanisms, the accu-mulation of a vacuolar ATP synthase (spot #340; H1-ATPase(V-ATPase)) in MW corroborates the hypothesis of cyto-plasmic acidosis reduction during MW formation.
The CW cluster contained four proteins (Fig. 7B, clus-ter C4), including a structural enzyme of the flavonoid bio-synthetic pathway, leucoanthocyanidin dioxygenase (antho-cyanidin synthase). This enzyme catalyzes the reaction from
the colorless leucoanthocyanidin compound to the antho-cyanidin pigment responsible of the dark red or purple colorin plant tissues [68]. CW is also characterized by a reddishcolor (Fig. 1D). Although the molecular basis for this colorhas not been established, it has been suggested that it couldbe attributed to the polymerization of coniferaldehyde [69].Our finding suggests that the typical color of CW formingtissue also results from the biosynthesis of flavonoids.
The OW or “normal” wood (NW) cluster contained eightproteins (Fig. 7B, cluster C5) down-regulated in CW. CW ishighly lignified with more p-hydroxyphenyl subunits, andcontains less cellulose than NW. Microfibril angle of the cel-lulose fibers in the S2 layer of the cell wall is high, tracheidlength is reduced, the cross-sectional profile becomes iso-diametric, and intercellular spaces become larger comparedto NW [5]. We hypothesized that the molecular mechanismsdetermining cell shape and cell size [70], as revealed by someof the proteins underexpressed in CW (actin, profilin,nucleoside diphosphate kinase), are disturbed in gravi-stimulated tissue, leading to the typical cell phenotypeobserved in CW. Actin (spot #300) filaments are responsiblefor many aspects of cell behavior, including cell division,movement, and expansion. Profilin (PFN, #549) is a ubiqui-tous actin monomer-binding protein involved in the organi-zation of the cytoskeleton of eukaryotes, including higherplants. It is thought to regulate actin polymerization in re-sponse to extracellular signals. In cotton, it was observed thatone PFN-like protein was activated during the fiber elonga-tion period [71]. In Arabidopsis, it was observed that PFNplays an important role in cell elongation, cell shape main-
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Proteomics 2005, 5, 3731–3751 Plant Proteomics 3749
Figure 7. Hierarchical clustering analysis of the 175 identified and the 40 unknown proteins and example of clus-ters. Samples correspond to mature (M), juvenile (J), early (E), late (L), compression (C) and opposite (O) wood.Left panel (A): clustering of the whole dataset, right panels (B): C1/SAM-S cluster, C2/LW cluster, C3/MW cluster,C4/CW cluster, C5/normal wood cluster.
tenance, and polarized growth of root hair [72]. Cells of Ara-bidopsis plants expressing antisense PFN were shorter, andmore isodiametric, compared to wild-type. These resultsstrongly suggest that the specific shape of CW tracheids canresult (at least in part) from the down-regulation of actin andPFN in wood forming tissue. By comparing coleoptilelengths, nucleoside diphosphate kinase (NDK, #554) en-zyme activities, and cell size in non-transformants and anti-NDP kinase rice plants, Pan et al. [73] found that the cellelongation process was predominantly inhibited in epi-dermal cells of coleoptiles in antisense plants. This resultsuggests that the reduction of NDK accumulation couldcontribute to the shorter cells characteristic of CW. The threeother known function proteins of the normal wood cluster(glycine-rich RNA-binding protein #551, proteasome sub-
unit alpha type 4 #477, 40S ribosomal protein S12, #552)indicate that proteins of the genes and proteins expressioncategory were underexpressed in CW forming tissue.
3.6 A database to store and query the maritime pine
wood proteome
We recently described a complete web-based application“PROTICdb” (http://moulon.inra.fr/,bioinfo/PROTICdb,[74]), dedicated to the storage, query, and analysis of plantproteomic data. Maritime pine proteomes of differentiatingxylem, corresponding to different developmental stages andtreatments, have been stored with this application. We havedeveloped a new website to make these data publicly avail-able (http://cbib1.cbib.u-bordeaux2.fr/Protic/Protic/home/
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3750 J.-M. Gion et al. Proteomics 2005, 5, 3731–3751
index.php). As a first level of information, the ‘Plants’, ‘Pro-tocols’, and ‘Protein identification’ hyperlinks providerespectively details about (i) the plant material and theexperimental conditions used, the organs sampled, the pro-tein quantity loaded on IPG strips; (ii) the protocols used forprotein extraction, first and second dimension electrophore-sis, staining, and image digitalization; and (iii) identifiedspots, including the query databases, number of matchingpeptides, protein coverage by the matching peptides, acces-sion number, matching species, assignment, theoretical andobserved pI and Mr (Da), MS techniques, and the MS plat-form where the spots have been processed.
A Java applet is used to access diverse information di-rectly on 2-DE images. 2-DE images can be downloaded byselecting their own ID and press the ‘load’ button. Up to fourimages can be visualized at the same time. By selecting the‘detected’ or ‘identification’ mode, all detected spots or allidentified spots are displayed with blue or red crosses,respectively. By Moving the mouse over a cross, a first level ofinformation is displayed, i.e., spot ID for any gel or referencespot ID for the master gel, Mr, pI and annotation (as inTable 1). More information (spot relationships, identificationand quantification) can be retrieved by left clicking on themouse. In respect to the identified spots, SEQUEST data(including peptide sequences) and links to nucleotide orprotein databases are also provided.
4 Concluding remarks
In this report, we have identified for the first time a highnumber (175) of known function proteins expressed in thewood forming tissue in a forest tree species. Identificationsuccess rate of MS/MS was high, over 70%, resulting mainlyfrom the use of pine EST. This is to be compared to the 16%success rate of MADLI-TOF MS. It is concluded that thecombined analysis of MS/MS spectra and EST sequences,provides an efficient and accurate protein identificationmethod for pine proteome analysis. A comparison betweenprotein and mRNA levels showed that at least 30% of theproteins were correlated with their corresponding mRNAlevels. This also means that for the majority of the proteinstheir level could not be predicted from transcript accumula-tion. This result demonstrates that a proteomic approach iscertainly a relevant approach for tracking genes involved inwood formation and determining wood quality.
The reference map represents the most comprehensivewood proteome projects to date and provides a good basis forfuture proteomic comparisons. Approximately 20 samples ofdifferentiating xylem taken along, (1) a gradient of gravi-stimulated xylem tissue, with samples collected on treesbended for few hours to few months, (2) a seasonal gradient,with samples taken every 15 days during the growing season(i.e., from April to August), and (3) a cambial age gradientwith samples taken every 4 m along the bole of an adult tree,have been collected, and are being analyzed by 2-DE com-
bined with LC ESI-MS/MS. This new dataset should shednew light onto the protein machinery involved in wood for-mation.
We thank anonymous reviewers for helpful comments on themanuscript. This research was supported by grants from the Eu-ropean Union (GEMINI project no. QLK-5-CT-1999-00942)and France (DERF no. 01.40.40/99; Région Aquitaineno. 20000307007, and INRA “Lignome”). JP was supported byfellowship SFRH/BD/3129/2000 from FCT/MCT Portugal.
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