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DOI 10.1002/pmic.200600953
RESEARCH ARTICLE
A large proportion of the apoplast proteome resides in the intercellular fluid (IF) or is ionically
bound (IB) to the wall matrix. A combined analysis of IF and IB proteins of the Medicago truncatula leaf apoplast was performed. 2-DE analyses demonstrated the reproducible presence of 220
IF and 84 IB proteins in the apoplast. These two protein populations were largely distinct; 22
proteins could be spatially matched, but MALDI-TOF/TOF analyses suggested a considerably
smaller number had common identities. MALDI-TOF/TOF characterisation identified 81 distinct proteins. Analyses of selected IF proteins (45) indicated 17 distinct proteins with mainly
defence-related functions, whereas analyses of IB proteins (70) identified 63 distinct proteins of
diverse natures, including proteins of non-canonical natures. The presence of non-canonical
proteins in IB extracts is discussed in the light of evidence supporting a low level of contamination of purified walls from symplastic proteins. This work indicates that IB and IF proteins are
functionally distinct fractions of the apoplast. The data obtained complements earlier studies of
the Medicago proteome and therefore will be useful in future studies investigating the role of
apoplastic proteins in plant processes.
Keywords:
Apoplast / Intercellular fluid / Ionically bound wall proteins / MALDI-TOF/TOF / Medicago truncatula
Introduction
ments [10], partly due to the difficulty in isolating a representative fraction of apoplastic proteins demonstrably free
from intracellular contamination.
Many studies have used non-disruptive methods as a
convenient means to isolate secreted proteins from cell cultures free from intracellular contaminants [11, 12], but for
more complex tissues, vacuum-infiltration techniques are
required to extract apoplastic proteins without incurring cellular disruption [9, 13, 14]. A major problem associated with
this technique is that the protein yield is generally low [7, 10,
14, 15]. In addition, infiltrates prepared with single buffers
fail to provide information describing possible differences in
protein solubility in the apoplast, which can provide insight
into protein function. Furthermore, potentially large differences between the quantities of soluble and more tightly
bound forms could lead to difficulty in spot detection which
could be avoided by the use of fractionated samples.
* These authors contributed equally to this work.
www.proteomics-journal.com
In some cases, sequential vacuum-infiltration with different salts and chelating agents could result in isolation of
specific fractions of wall proteins. However, in Arabidopsis,
sequential extractions have been shown to result in increased
wall disruption and a greater risk of contamination from the
symplastic space [11]. Thus, in order to isolate more tightly
bound proteins from the wall, it is often necessary to employ
disruptive techniques to obtain purified walls for sequential
extraction as suggested by Chivasa et al. (2002) [16]. On the
other hand, cell-disruptive methods could result in wall contamination through the electrostatic binding of intracellular
proteins with the extracellular matrix (ECM), which is largely
negatively charged [17]. Several studies using disruptive
techniques have in fact not only identified several classical
cell wall proteins but also unexpected proteins that generally
have been considered to be non-secretory or traditionally
associated with organelles other than the wall [8, 16, 1820].
The presence of these proteins in the wall is a current subject
of controversial discussion. Nonetheless, while the possibility of intracellular contamination has not been excluded in
many cases, proteins of unexpected nature have also been
isolated from the cell wall using undisruptive techniques
[1114], suggesting they are targeted to the ECM in vivo. In
this study, we provide corroborative evidence supporting
these novel proteins as true cell wall proteins.
The intercellular fluid (IF) is important for the transport
of essential solutes in extracellular spaces. As the outermost
cellular compartment, it also serves to release IF, or guttation
fluid at sites of damage, leading to the transport of, e.g.
pathogen-related (PR) proteins to the wounded surface to
help impede pathogen ingress. In agreement, many studies
have found several PR-proteins present in the IF [2123], and
PR-proteins represent the major quantitative changes in soluble proteins during defensive responses [24].
The intercellular spaces are also likely to contain signalling proteins which can interact with specific receptors at the
ECM to initiate signal cascades in cell-cell communication
and other events. In Arabidopsis, it has been demonstrated
that the stem cell-specific protein, CLAVATA3 (CLV3), is soluble in the extracellular space where it is required for the
activation of the CLV1/CLV2 receptor complex at the plasma
membrane of the underlying cells [4]. In maize, the small
extracellular and hydrophilic protein, embryo surrounding
region, shares a conserved region with CLV3, and is also
thought to participate in protein-protein interactions [25].
It might also be expected that the protein population
more tightly bound to the ECM to be more enriched in proteins functionally dedicated to this compartment. Matrixmodifying proteins such as, e.g. expansin [26], xyloglucan
endotransglycosidase/ hydrolase [1] or extensin [27], and
those thought to be involved in wall/cytoplasm communication (e.g. Arabinogalactan-rich proteins [28]), might be
expected to be largely restricted to the ECM where they are
functionally relevant.
Nevertheless, a few examples have been documented of
apoplastic proteins which can be found both bound to cell
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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N. C. Soares et al.
2.3 2-DE
Protein concentration in the extracts was determined with a
BioRad protein assay kit (BioRad, Germany), using a modified Bradford assay [43] as suggested by Ramagli et al. (1999)
[44].
For IEF, the IPGphor system was used (Amersham Biosciences) with 310 non-linear (NL) pH gradient strips (IPG
strips, Amersham Biosciences; Sweden). Proteins were
solubilised in 8 M urea, 2% w/v CHAPS, 40 mM DTT and
0.5% v/v IPG buffer (310 NL (Amersham Biosciences). IEF
was carried out at 30 V for 12 h, followed by 250 V for 1 h,
500 V for 1.5 h, 1000 V for 1.5 h, a gradient to 8000 V over
1.5 h and 8000 V for a further 4 h, all at 207C. Prior to the
second dimension SDS-PAGE, the focused IPGstrips were
equilibrated for 2615 min in 50 mM Tris-HCl (pH 8.8) buffer containing 6 M urea, 30% v/v glycerol, 2% w/v SDS and a
trace of Bromophenol Blue. DTT at 1% w/v was added to the
first equilibration step and 2.5% w/v iodoacetamide to the
second. SDS-PAGE was performed on 12 or 15% polyacrylamide gels [45]. For analytical 2-DE gels, silver-staining was
performed according to Blum et al. (1987) [46] and gels were
loaded with 40 mg of total protein. In all cases the experiments used at least three replicate protein samples prepared
from separate biological samples. For preparative gels, MScompatible silver staining [47] was utilised and the gels
loaded with 150 mg of total protein. Gels were scanned using
the ImageQuant v3.3 densitometer (Molecular Dynamics)
and were analysed by the Image Master Platinum software
v.5.0 (Amersham Biosciences).
2.4 Trypsin digestion of proteins and characterisation
by MALDI-TOF/TOF
Selected spots were excised from the gels and destained
with a solution containing 20% w/v sodium thiosulphate
and 1% w/v potassium ferricyanide for 5 min. The supernatant was removed and the gel spots were washed twice
with 25 mM ammonium bicarbonate in 50% v/v ACN for
20 min. The gel spots were then washed in ACN, dried in a
speed-vac (Savant, USA) and digested overnight with 20 mg/
mL of trypsin in 25 mM ammonium bicarbonate at 377C.
Tryptic peptides were passed through C18 Zip-Tips and
mixed with 5 mg/mL of an CHCA as matrix and subject to
MALDI-TOF/TOF analysis (4700 Proteomics Analyzer,
Applied Biosystems).
2.5 Database queries and protein identification
Databases (Est-others, NCBInr, NRDB, ) were queried with
either MASCOT data files obtained from MALDI -TOF/TOF
mass spectral data or the compiled partial de novo sequences
obtained per spot, using MASCOT software available at
www.matrixscience.com, or MS-Blast [48] at http://
dove.embl-heidelberg.de/Blast2, respectively. All the protein
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Results
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N. C. Soares et al.
Table 1. MALDI-TOF/TOF characterisation of leaf IF proteins. Databases were queried with either MASCOT data files or the compiled partial
de novo sequences. In all cases, peptide charges were 11
Spot
No.
kDa
69
69SIe)
70
71
71SI
72
73
81
83
83SI
85
87
93
94
97
97SI
100
103
105
106
109
112
114
34.9
34.9
31.6
33.3
33
34.7
29.0
39.5
39.2
39.2
32.6
28.8
37.1
37.1
28.6
28.3
26.4
26.2
23.5
23.0
21.1
15.5
17.2
pI
4.0
4.0
4.0
4.3
4.3
4.8
4.6
5.9
5.95
5.95
6.4
6.2
7.8
8.4
7.45
7.45
7.4
6.0
6.2
6.6
7.3
6.7
4.3
Homologue
b21,3 Glucanase
b21,3 Glucanase
b21,3 Glucanase
Thaumatin-like protein
Thaumatin-like protein
b21,3 Glucanase
Class II chitinase
Class II chitinase
Class Ib chitinase
Class Ib chitinase
Endochitinase
Chitinase
b21,3 Glucanase
b21,3 Glucanase
NtPRp27-like protein
NtPRp27-like protein
Thaumatin-like protein
Germin-like protein
Class Ib chitinase
Thaumatin-like protein
Class Ib chitinase
b21,3 Glucanase
Putative glycine-rich
Accession
No.a)
Classb)
Q8GT15
Q8GT15
Q6S9W0
AY035168
AY035168
Q9ZP12
P29024
Q9SDY6
Q7X9F6
Q7X9F6
AAD34596
CAA64868
AF435088
O23473
AY185207
AY185207
O04364
CAC34417
Q42428
O04364
Q42428
AF239617
Q6Z498
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
OR
D
D
D
D
S
MALDI-TOF/TOFc)
de novo Sequencingd)
Score
Matched
peptides
Score
Matched
peptides
2
2
2
2
2
2
2
2
65
2
2
2
2
154
2
2
2
2
2
81
114
2
129
2
2
2
2
1
2
2
2
2
1
2
2
2
2
2
1
1
2
1
2
2
264
350
256
161
161
224
2
532
369
492
365
2
124
310
241
459
102
2
2
102
2
355
208
3
4
4
2
3
4
2
5
6
7
5
2
2
5
4
9
2
2
2
2
2
4
5
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Table 2. MALDI-TOF/TOF characterisation of leaf IB proteins. The databases were queried with either MASCOT data files or the compiled
partial de novo sequences. In all cases peptide charges were 11
Spot
No.
kDa
pI
1
2
3
24.1
21.5
18.8
4.0
4.2
4.2
4
4SIe)
5
6
7
8
9
16.3
16.1
14.7
18.0
20.6
20.3
22.8
4.3
4.3
4.3
4.6
4.8
5.1
5.4
10
11
12
21.2
23.1
23.1
5.5
5.2
5.6
13
14
14.0
16.5
5.0
5.3
15
16
18.8
20.6
5.5
5.6
17
18
18.3
15.6
5.7
5.6
19
20
21
22
16.0
15.6
15.6
21.5
5.8
6.0
6.5
8.5
23
20.3
8.8
24
18.8
9.5
25
26
30.1
31.0
4.9
5.0
27
32.8
4.9
28
28.8
5.3
29
29.2
5.5
30
26.8
5.6
31
28.8
5.6
33
33SI
34
35
36
37
29.2
29.2
26.0
35.3
36.7
33.3
6.0
6.0
6.0
4.9
4.8
5.3
Homologue
Accession
No.a)
Classb)
NP_910497
BAD04724
AAD03022
MALDI-TOF/TOFc)
de novo Sequencingd)
Score
Matched
peptides
Score
Matched
peptides
D
T/B
GA
2
2
2
2
2
2
62
70
111
1
1
2
CAA26709
CAA26709
P10496
CAA67375
AAB41811
AAP54051
CAC81066
EPC
EPC
S
D
OR
S
T/B
2
2
2
114
2
2
2
2
2
1
2
2
216
229
510
2
332
475
285
3
3
5
2
4
9
7
AAC14127
AAB05888
XP_465138
OR
OR
S
2
127
2
2
2
2
194
2
167
3
2
4
CAA53900
AB043960
OR
EPC
72
2
196
2
3
Q8ZDQ7
CAC49966
T/B
T/B
2
2
2
2
64
106
1
2
AAD10219
AAG05168
Misc
ID
225
2
4
2
2
64
2
1
BG449895
AW775706
AW775375
AAL14244
N/A
OR
N/A
Misc
322
84
147
2
5
1
4
2
2
2
2
2
118
BAC46013
Misc
66
AJ498300
EPC
422
AY389763*
CAD76101
S
OR
2
2
2
2
130
65
3
1
AAR34625
ID
68
AW559699
EPC
CAA78043
EPC
Q6L1G8
88
191
Misc
161
BAC08163
GA
65
CAC34417
CAC34417
BAC74347
AW696867
AAK72818
AAB41811
OR
OR
U
Misc
EPC
OR
284
217
2
2
174
2
2
2
2
2
2
2
173
2
173
324
2
2
4
2
3
5
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N. C. Soares et al.
Table 2. Continued
Spot
No.
kDa
pI
38
39
31.0
35.1
5.4
5.8
40
41
34.6
30.1
5.7
5.9
42
43
44
44SI
45
46
32.8
33.8
32.4
32
33.6
40.3
6.3
6.8
7.4
7.4
9.5
5.4
47
48
49
36.4
36.7
34.8
5.5
5.6
5.6
50
42.8
6.0
51
44.7
6.5
52
47.4
6.8
53
54
55
56
57
58
45.3
51.0
50.2
50.2
53.3
51.0
9.5
4.8
5.3
5.5
5.5
5.6
59
60
61
46.7
48.8
46.7
5.8
5.9
6.0
62
63
55.7
54.1
6.2
6.5
64
54.8
6.7
65
66
67
52.1
63.5
40.3
7.8
5.5
5.3
68
40.2
5.3
Homologue
Ferritin
S-Adesonylmethionine
synthase
Triophosphate isomerase
Epithelial cell adhesion
molecule
Unknown protein
Germin-like protein
Germin-like protein
Germin-like protein
Germin-like protein
Oxygen evolving
enhancer protein 1
Harpin binding protein 1
Type I anti-freeze protein
Vacuolar sorting-associated protein
ATP synthase (beta
subunit)
ATP synthase (beta
subunit)
Arabinogalactan protein
AGP2
Peroxidase
Threonine dehydratase
Hypothetical protein
Phosphoribulosekinase
Hypothetical protein
Putative ABC transporter
ATP-binding
Sensor histidine kinase
Unknown protein
Ferredoxin-NADP(H)
oxidoreductase
Unknown protein
D-3-phosphoglycerate
dehydrogenase
ABC transporter
ATP-binding
Peroxidase
ATP synthase
Oxygen evolving
enhancer protein 1
Oxygen evolving
enhancer protein 1
Accession
No.a)
Classb)
CAA65771
Q5P2V5
MALDI-TOF/TOFc)
de novo Sequencingd)
Score
Matched
peptides
Score
Matched
peptides
T/B
Misc
92
2
2
2
2
60
2
1
AAT46998
AB161197
EPC
ID
311
2
4
2
2
63
2
1
AACY01181595
P94040
AW776317
AW776317
P94040
P12359
U
OR
OR
OR
OR
EPC
2
2
166
325
2
2
2
2
2
2
2
2
67
75
2
2
75
311
1
1
2
2
1
5
BF006528
AAR22529
CAA81876
D
D
Misc
184
2
2
3
2
2
2
113
134
2
3
3
AAD46914
EPC
519
AAD46914
EPC
358
AAB35284
143
BI272831
Q63BA8
AAG39973
CAA72118
AE013598
Q5Z2A9
OR
Misc
U
EPC
U
T/B
456
2
2
88
2
2
6
2
2
4
2
2
2
72
63
2
62
70
2
1
1
2
1
1
Q6HC69
AACY01426312
CV283317
ID
U
OR
2
2
170
2
2
3
102
406
2
2
3
2
AAB41813
BAC07877
U
EPC
2
2
2
2
84
67
1
1
Q9A7G4
T/B
67
CAA62226
AAD46914
AW317313
OR
EPC
EPC
2
380
227
2
3
2
197
2
2
4
2
2
AW776405
EPC
388
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Figure 2. Assaying the relative abundance of IF and IB proteins in the leaf apoplast. (A) Spot volumes of four proteins in the IF prepared by
vacuum-infiltration with aqueous buffer (grey bars) and vacuum-infiltrates prepared with 1 M KCl (white bars). (B) Spot volumes of three IB
proteins extracted from purified leaf cell walls (grey bars) or through vacuum infiltration with 1 M KCl (white bars). Error bars represent the
SD based on three biological replicates. In all cases, 40 mg of proteins were loaded onto gels and the normalised spot volumes expressed as
a % of total spot volume.
Table 3. Activities of the cytosolic marker enzyme, malate dehydrogenase, in soluble (cytosolic) and apoplastic extracts
of callus material and leaves. Specific activity is given as
units activity mg-1 protein6102 (1 U = 1 mmol/s)
Material
Leaf
Callus
Fraction
Cytosol (soluble)
Intercellular fluid (aq.)
Intercellular fluid
(1 M KCl)
Purified cell walls
Cytosol (soluble)
Saline eluate
Purified cell walls
Specific activity
malate dehydrogenase
24.30 6 3.00
0.01 6 0.00
0.13 6 0.01
0.20 6 0.02
25.0 6 1.00
0.58 6 0.04
0.43 6 0.09
2078
N. C. Soares et al.
Figure 3. An assay for contamination by electrostatically charged proteins of the symplast during callus cell wall purification. (A) 2-DE of
proteins extracted from intact cells eluates with 1 M KCl and (B) from purified cell walls. Arrowheads or circular heads indicate the spots
present exclusively in gel (A) and (B), respectively. The number of proteins considered as potential contaminants was estimated from the
number of spots exclusively present in (B). Gels were loaded with 40 mg of total protein.
Table 4. MALDI-TOF/TOF characterisation of spatially matched proteins in gels of saline eluates of intact callus and of purified callus cell
walls. The databases were queried with either MASCOT data files or the compiled de novo sequences. In all cases peptide charges
were 11
MASCOTa)
MS-Blastb)
Spot
No.
kDa
pI
Homologue
Accession
No.c)
Score
Matched
peptides
Score
Matched
peptides
122Ed)
122He)
123E
123H
124E
124H
125E
125H
126E
126H
127E
127H
16.2
16.2
32.4
32.6
39.2
39.2
46.6
46.8
57.3
57.5
53.8
53.8
3.8
3.8
5.6
5.6
6.5
6.5
5.8
5.8
5.5
5.5
5.7
5.7
PR-10
PR-10
Glycine rich protein
Glycine rich protein
Secretory peroxidase
Secretory peroxidase
Malate dehydrogenase
Malate dehydrogenase
Secretory peroxidase
Secretory peroxidase
Hypothetical protein
Hypothetical protein
BE239885
BE239885
Q39682
Q39682
BF644273
BF644273
BF006134
BF006134
AW559660
AW559660
CAE60064
CAE60064
266
93
2
2
241
218
140
178
156
99
2
2
3
1
2
2
3
3
2
2
1
1
2
2
2
2
416
272
2
2
2
2
2
2
117
244
2
2
4
3
2
2
2
2
2
2
3
1
a)
b)
c)
d)
e)
molecular weight and/or pI (e.g. spots 105 and 109, spots 100
and 106; spots 43 and 45, spots 50 and 51; see also Tables 1
and 2). These observations are a common feature in 2-DE
gels [7, 20], possibly arising from heterogeneity in PTMs.
Other spots shared homology to the same protein families,
but with different corresponding gene sequences, indicating
the presence of multi-gene families in the apoplast.
3.4.1 IF proteins
Of the 45 IF protein spots (see Fig. 1A) selected for MALDITOF/TOF analysis, 19 showed homology with known proteins. For the remaining proteins, although it was possible to
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Discussion
3.4.2 IB proteins
MALDI-TOF/TOF analyses of IB proteins revealed that 59
proteins demonstrate homology with known proteins,
whereas six have homology with unknown or hypothetical
proteins (Table 2). In addition, two spots had no homology
with any protein in the current data-base.
The results demonstrate that IB cell wall proteins of
Medicago are diverse in nature, and can be conveniently
divided into nine groups (see Fig. 4B). The most representative group is related with energy production and conversion (15). Energy production-related proteins included
those with homology with chloroplastic oxygen-evolving
enhancer proteins 1 and 3 (7). Proteins associated with
energy conversion include ATP synthase-like proteins (4),
triosephosphate isomerase (1) and D-3-phosphoglycerate
dehydrogenase (1). The second most abundant group
revealed homology with oxidoreductases (14), including
germin-like proteins (4), peroxidase (4), superoxide dis 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2080
N. C. Soares et al.
hypersensitive response in many species [62]. Proteins homologous to a type I anti-freeze, PR-10 and a stress-inducible
protein were also present. Several oxidoreductases were
identified, including SOD, peroxidase and germin-like proteins, which could contribute to the regulation and/or generation of ROS during the oxidative burst, thereby contributing to the mobilisation of defence genes [63].
4.4 Concluding remarks
In summary, we have demonstrated that purified cell walls
can be a convenient source of IB proteins which can be isolated with minimal contamination, yet which contain noncanonical proteins. Our comparison of IF and IB proteins
indicated them to be largely distinct protein populations.
With the exception of defence-related proteins which are
present in both IF and IB proteins, these two protein fractions appear to represent distinct functional compartments
of the apoplast.
This study therefore suggests that the combined study of
IF and IB proteins will enable a more comprehensive overview of the role of apoplastic proteins in plant processes.
Plant Proteomics
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[12] Kwon, H.-K., Yokoyama, R., Nishitani, K., Plant Cell Physiol.
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Physiol. 2006, 140, 311325.
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U., Ebermann, R., Penel, C., Greppins, H. (Eds.), Plant peroxidases, Biochemistry and Physiology, Universit de Genve press, Vienna 1996, pp. 255258.
[16] Chivasa, S., Ndimba, B. K., Simon, W. J., Robertson, D. et al.,
Electrophoresis 2002, 23, 17541765.
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