plant cell physiol. doi:10.1093/pcp/pci089, available …...proteomes for cell wall regeneration in...

Plant Cell Physiol. 46(6): 843–857 (2005)

doi:10.1093/pcp/pci089, available online at www.pcp.oupjournals.org

JSPP © 2005

A Proteomic Approach to Apoplastic Proteins Involved in Cell Wall Regeneration in Protoplasts of Arabidopsis Suspension-cultured Cells

Hye-Kyoung Kwon, Ryusuke Yokoyama and Kazuhiko Nishitani 1

Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, 980-8578 Japan

;

To clarify the mechanisms of cell wall construction, we

used a proteomic approach to investigate the proteins

secreted into cell wall spaces during cell wall regeneration

from the protoplasts of Arabidopsis suspension-cultured

cells. We focused on cell wall proteins loosely bound to the

cell wall architecture and extractable with 1 M KCl solu-

tions from: (i) native suspension cultured cells; (ii) proto-

plasts that had been allowed to regenerate their cell walls

for 1 h; and (iii) protoplasts allowed to regenerate their cell

walls for 3 h. We adopted a non-destructive extraction pro-

cedure without disrupting cellular integrity, thereby avoid-

ing contamination from cytoplasmic proteins. Using two-

dimensional polyacrylamide gel electrophoresis (2-D PAGE)

and matrix-assisted laser desorption ionization-time-of-

flight/mass spectrometry (MALDI-TOF/MS), we separated,

mapped and identified 71 proteins derived from the native

cell wall, and 175 and 212 proteins derived from the 1 and

3 h regenerated protoplasts, respectively. Quite different

sets of proteins with differing status of their post-transla-

tional modifications, including phosphorylation and glyco-

sylation, were identified in the three protein fractions. This

indicated dynamic in muro changes in the cell wall proteins

during cell wall regeneration in the protoplasts. The analy-

sis revealed a set of enzymes specifically involved in cell

wall expansion and construction in suspension-cultured

cells. This approach has also determined a set of cell wall

proteins that had not been predicted to be localized in cell

wall spaces.

Keywords: Arabidopsis — Cell wall regeneration — Extra-

cellular proteins — Post-translational processing — Proteomics

— Suspension cultured cells.

Abbreviations: CBB, Coomassie brilliant blue; 2-D PAGE, two-dimensional polyacrylamide gel electrophoresis; DTT, dithiothreitol;FDA, fluorescein diacetate; G6PDH, glucose-6-phosphate de-hydrogenase; IEF, isoelectric focusing; MALDI-TOF/MS, matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry;SEM, scanning electron microscope; xyloglucan endotransglucosylase/hydrolase, XTH.

Introduction

The plant cell wall is a dynamic structure that plays a crit-

ical role not only in determining cell shape and formation of

the plant body, but also in interactions with environmental

factors including those required for nutrition, response to abi-

otic stress and biological attack by other organisms (Carpita

and McCann 2000). It is made of various types of macromole-

cules, and undergoes extensive construction and remodeling

during cell division and differentiation. Cell wall dynamics are

achieved via two distinct steps: (i) the biosynthesis of cell wall

components by the actions of membrane-bound enzymes at the

plasma membrane and endoplasmic reticulum (ER)–Golgi

apparatus; and (ii) the assembly and rearrangement of cell wall

structures in muro via the actions of extracellular proteins.

Significant progress has been made recently in the identifica-

tion of membrane-bound enzymes involved in the syntheses

of cellulose and matrix polymers (Arioli et al. 1998, Perrin et

al. 1999, Hong et al. 2001, Faik et al. 2002, Iwai et al. 2002,

Li et al. 2003), as well as with certain cell wall proteins such

as expansin and the XTH family of genes (Cosgrove 1997,

Nishitani 1997). However, only a limited number of extra-

cellular proteins compared with those predicted based on the

genomic database have thus far been characterized, par-

ticularly in terms of cell wall dynamics (Girke et al. 2004,

Yokoyama and Nishitani 2004).

The functional and structural diversity of the plant cell

wall largely mirrors the abundance and diversity of extra-

cellular proteins, which make up approximately 10% of the cell

wall mass, and are typically encoded by large gene families

(Chivasa et al. 2002, Yokoyama and Nishitani 2004). More

than 2,000 putative cell wall-related genes are estimated to

occur in the genome database of Arabidopsis thaliana

(Arabidopsis Genome Initiative 2000). These genes are consid-

ered to encode proteins in categories with differing biological

functions (Cassab 1998, Darley et al. 2001, Nishitani 2002, Qin

et al. 2003). Reverse genetic approaches based on oligo DNA

microarray technology coupled with phenotypic analyses of

T-DNA insertion lines have paved the way for understanding

the functions of this large number of gene sets involved

with the cell wall (Bonetta et al. 2002, Li et al. 2004, Manfield

et al. 2004). Despite these genomic approaches, a complete

picture of the concerted action of these cell wall proteins in

the cell wall construction and remodeling processes is largely

unknown.

A proteomic approach has been adopted to gain insight

into the function and localization of individual cell wall pro-

teins in muro. This promising technology allows the large-scale

1 Corresponding author: E-mail, [email protected]; Fax, +81-22-795-6700.

843

Proteomes for cell wall regeneration in protoplasts844

analysis of protein expression profiles, including their post-

translational modifications, and protein–protein interactions

(Pandey and Mann 2000). This is particularly the case for pro-

teins found in specific organs and cellular compartments, or for

those expressed under specific biological conditions (Kuster et

al. 2001, Martin and Nelson 2001). Using this proteomic

approach, lists of proteins expressed in the cell wall of A.

thaliana have been determined recently (Rose et al. 2004a,

Rose et al. 2004b).

However, even with current proteomic approaches,

dynamic aspects of cell wall proteins, particularly those during

cell wall construction, remain elusive. To better understand cell

wall proteins, we considered the process of cell wall regenera-

tion in the present study. We used protoplasts generated from

suspension-cultured Alex cells of Arabidopsis, which is not

only a model plant with a complete genome database and

abundant transcriptomes, but also provides us with intact cells

ready to extract cell wall proteins without damaging cell

integrity. Using this ‘intact model cell’ system, we extracted

extracellular proteins that were secreted from the protoplasts

and bound loosely to its surface during the cell wall regenera-

tion process with 1 M KCl solutions. We then analyzed them

comprehensively with the aid of two-dimensional polyacryla-

mide gel electrophoresis (2-D PAGE) and matrix-assisted

laser desorption ionization-time-of-flight/mass spectrometry

(MALDI-TOF/MS) technologies. This approach has revealed

dynamics of protein profiles that are distinct from those found

by conventional cell wall proteome analyses. In addition, this

approach also disclosed the presence of a certain set of pro-

teins that had not been identified as being secreted.

Results

Purification of cell wall proteins secreted from suspension-cul-

tured cells of Arabidopsis during cell wall regeneration on

protoplasts

To gain insight into dynamic aspects of cell wall construc-

tion, we focused on a set of proteins secreted onto the surface

of protoplasts during cell wall regeneration. Fig. 1 shows dif-

ferential interference contrast images (Fig. 1A–D) and epifluo-

rescent images (Fig. 1E–L) of native cells and protoplasts

stained with Calcofluor White M2R (Fig. 1E–H) and aniline

blue (Fig. 1I–L), which preferentially stain cellulose and β-1,3

glucan, respectively. Fresh protoplasts were not stained with

either Calcofluor White M2R (Fig. 1F) or aniline blue (Fig.

1J). The deposition of glucans, as determined by staining with

the two fluorescent compounds, appeared within 1 h of cell

wall regeneration, and the extent of deposition increased and

spread over the surface during 3 h of regeneration (Fig. 1G, H,

K, L).

A close-up examination of the cell surface using a scan-

ning electron microscope (SEM, Fig. 1M–P) showed that the

native cell surface was covered with a tangled network of fine

fibrils (Fig. 1M), whereas the surface of freshly prepared proto-

plasts was smooth, and some dumbbell-shaped structures were

sparsely deposited onto the surface (Fig. 1N). As cell wall

regeneration proceeded, the dumbbell-shaped threads elon-

gated. On the surface of the protoplasts that had been allowed

to regenerate cell walls for 3 h, a network of fine threads simi-

lar to those found on the native cell surface was observed (Fig.

1M, P). These results indicate that the secretion of cell wall

material begins within 1 h of the incubation of protoplasts, and

that the formation of β-glucan-containing cell wall structures

similar to those found in native cells begins within 3 h of cell

wall regeneration. Therefore, we decided to examine proteins

on the surface of protoplasts that had been incubated for 1 and

3 h. We also extracted loosely bound cell wall proteins using a

1 M KCl solution according to the procedure examined previ-

ously (Robertson et al. 1997, Borderies et al. 2003).

To validate whether cytosolic proteins leak out of the

protoplasts during protein extraction processes, we tested for

the activity of glucose-6-phosphate dehydrogenase (G6PDH), a

typical marker enzyme used to detect contamination by cyto-

plasmic proteins (Li et al. 1989). Table 1 shows that specific

activities of G6PDH in the cell wall protein fraction from the

native cells contained 1.6% of the activity of those from the

cytosolic extracts. Furthermore, a much lower amount of

enzyme activity was detected in the cell wall protein fraction

derived from protoplasts compared with those in native cells.

From these results, we consider that contamination of intra-

cellular proteins is minimized in the present study.

We also examined the viability of cells and protoplasts

that had been extracted with 1 M KCl by staining with fluores-

cein diacetate (FDA). We counted the viable cells under a fluo-

rescence microscope. This showed that >90% of protoplasts

remained viable after the 1 M KCl extraction (data not shown).

Judging from the results obtained from the two different tests,

we conclude that the cell membrane is not severely damaged

during protein extraction, and that contamination of the extra-

cellular protein fraction by cytosolic proteins is negligible.

Characterization of cell wall proteomes

Cell wall proteins were separated by 2-D PAGE using dif-

ferently immobilized isoelectric point (pI) gradient (IPG) strips

for the first-dimension separation (pI 3–10 and pI 6–11) and

12% SDS–PAGE for the second separation. These were visual-

ized with Coomassie brilliant blue (CBB) staining (Fig. 2).

Using Image Master 2D-Elite software, we mapped 71 pro-

teins in the 2-D gel derived from the native cultured cells, and

175 and 212 protein spots in those derived from the 1 and 3 h

regenerated protoplasts, respectively (Fig. 2, 3). By matching

individual spots in different gels derived from the three protein

fractions, we identified 327 protein spots (Tables 2–4). This

spot matching revealed that 26 of the 71 spots found in native

cells are not in the protoplasts, while 75 of the 175 spots, and

104 of the 212 spots are specific to the 1 and 3 h regenerated

protoplasts, respectively, as summarized in Fig 3. Conse-

quently, 256 of the 327 spots detected in the present study were

Proteomes for cell wall regeneration in protoplasts 845

Fig. 1 Time course of changes in the cell surface during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells.

Cells and protoplasts stained with Calcofluor White M2R (E–H) and aniline blue (I-L) were observed under a differential interference contrast

microscope (A–D) and an epifluorescent microscope (E–L). For scanning electron microscopic observation (M–P), cells and protoplasts were

fixed in glutaraldehyde and ruthenium tetroxide, and then coated with silver. Native suspension-cultured cells (A, E, I and M), fresh protoplasts

(B, F, J and N), protoplasts regenerated for 1 h (C, G, K and O) and those regenerated for 3 h (D, H, L and P) are shown. Bar: 25 µm (A–L);

1.5 µm (M–P)

Table 1 Examination of cytosolic contamination in the cell wall protein fraction as assessed by the

cytosolic protein marker, glucose-6-phosphate dehydrogenase (G6PDH)

Cytosolic proteins were extracted with 1 M KCl after cells or protoplasts were disrupted by homogenization, while cell

wall proteins were extracted with 1 M KCl without disrupting cellular integrity. A unit of G6PDH activity is given as

1 µmol of substrate turnover per minute.

FractionTotal protein (mg flask–1)

Specific activity (mU mg–1 protein)

Total activity (mU flask–1)

Cytosolic protein from native cells 4.28 375.70 1607.4

Cell wall protein from native cells 0.74 5.68 4.2

Cytosolic protein from nascent protoplasts 6.09 371.58 2264.7

Cell wall protein from 1 h regenerated protoplasts 0.58 1.03 0.6

Cell wall protein from 3 h regenerated protoplasts 0.51 4.04 2.0


specifically expressed during cell wall regeneration on the cell

wall surface.

After individual protein spots were picked and in-gel

digested with trypsin, protein sequences and genes encoding

the proteins were identified with a MALDI-TOF/MS analysis

coupled with a database search using the Mascot sequence

database search program. The protein spots with their genes

predicted were numbered on each of the gels as shown in Fig. 2

and listed in Tables 2–4, respectively. Protein spots with the

same numbers are identical. The data in Tables 2–4 indicate

that two or more spots are predicted to be encoded by the same

gene, indicating the occurrence of post-translational modifica-

tions. Consequently, 55 protein spots derived from the native

cells were identified as being encoded by a total of 39 inde-

pendent genes (Table 2). Similarly, 108 protein spots derived

from the 1 h regenerated protoplasts were identified as being

encoded by 61 independent genes (Table 3), and 116 protein

spots from the 3 h regenerated protoplasts were identified as

being encoded by 67 independent genes (Table 4). Other spots

that were recognized on the gel by staining, but were not iden-

tified in terms of genes, were not numbered in Fig. 2.

This analysis has identified several carbohydrate-related

enzymes as being localized in the cell wall of intact cells and/or

in the protoplasts during cell wall regeneration. These enzymes

include expansin, chitinase, xyloglucan endotransglycosylase/

hydrolase, β-glucosidase, α-glucosidase, α-xylosidase, β-

xylosidase, α-galactosidase, β-galactosidase, β-fructosidase,

α-mannosidase, polygalacturonases, fructokinase and β-

hexosaminidases (Tables 2–4). Given that protein families have

dozens of family members (Yokoyama and Nishitani 2004), it

is worth noting that only a single, or at most two, members of

each of these protein families is identified in each protein

fraction. This implies specific roles for individual proteins in

specific aspects of cell wall dynamics in suspension-cultured

cells. In addition to these carbohydrate-related enzymes, the

present study identified several types of proteases, esterases,

kinases and oxidoreductases as cell wall resident proteins.

Furthermore, we have identified a few proteins whose

molecular functions have not yet been predicted. For these

unknown proteins, we predicted their functions by examining

their structural features using domain and motif analyses.

These analyses identified a group of proteins that are classified

into lectin families with potential domains for interaction with

certain polysaccharides or proteins present in the cell wall

(Tables 2–4).

Post-translational modifications

As denoted in Tables 2–4, identification using the

MALDI-TOF/MS analysis showed that some spots are attrib-

uted to a single protein encoded by a single gene, suggesting

post-translational modifications. A comparison of the relative

positions in the 2-D gel among the protein spots derived from

the same gene indicates that modifications would have affected

the pI and/or the molecular weight. To characterize these modi-

fications, we assayed for phosphorylated and glycosylated pro-

teins in the 2-D polyacrylamide gel by staining with Pro-Q

Diamond dye (Molecular Probes, Eugene, OR, USA) and Pro-

Q Emerald dye (Molecular Probe), respectively. Data indicated

that of 39 independent proteins found in native cells, 18 pro-

teins (denoted by asterisks in Table 2) were phosphorylated,

and 28 proteins (denoted by underlines in Table 2) were

glycosylated. Similarly, 22 and 43 proteins of 61 proteins

derived from the 1 h regenerated protoplasts were phosphoryl-

ated and glycosylated, respectively (Tables 2, 4), while 31 and

45 proteins derived from the 3 h regenerated protoplasts were

phosphorylated and glycosylated, respectively. For example,

spots 150, 151 and 152 in the native cells (Fig. 2A) are the

same α-xylosidase (At1g68560). Whereas they have the same

molecular weight, they have a different pI. Spots 95, 96 and

118 from 1 h regenerated extracts (Fig. 2C, D) with different

molecular weights and pI represent the same β-galactosidase

(At5g63810). α-Mannosidase (At3g26720) was also identified

as different spots with different molecular weights and pI in

individual steps of cell wall regeneration: native (spots 159 and

160), 1 h (spot 119) and 3 h (spot 33). These results suggest

that cell wall proteins undergo various types of post-transla-

tional modifications during cell wall regeneration.

Fig. 3 Summary of the number of protein spots identified in the apo-

plastic fraction derived from suspension-cultured native cells (native),

protoplast cell walls that had been regenerated for 1 h (1 hour), and

those regenerated for 3 h (3 hours). Numbers in parentheses indicate

the total number of protein spots found in each fraction.

Fig. 2 2-D PAGE patterns of cell wall proteins extracted with 1 M KCl from native cell walls of suspension-cultured cells (A and B), from

protoplast cell walls that were regenerated for 1 h (C and D) and those regenerated for 3 h (E and F). An isoelectric point (pI) gradient strip with

pI 3–10 (A, C and E) or pI 6–11 (B, D and F) was loaded with 180 µg of total cell wall proteins. Protein spots disclosed by CBB staining were

mapped, identified, and summarized in Tables 2–4, respectively.


Table 2 Summary of the proteins identified as 1M KCl extractable from the native cell walls of Arabidopsis suspension cultured

cells

a Protein spot numbers correspond to the numbers on gel images (Fig. 2A, B). An asterisk indicates that the protein spot is phosphorylated, while

underlined spots are glycosylated.b Accession numbers are based on the NCBI database.c The number of transmembrane domains (TMDs) as predicted by TMHMM is shown.d The potential signal peptide as predicted by iPSORT, Signal P and Target P is represented by +.e † in the 1 h and 3 h columns indicates that proteins encoded by the same gene are also identified in the 1 and 3 h regenerated protoplasts, respec-

tively.

Protein spot number a

Accession no. b

Protein nameTheoretical mol. wt/pI

TMD cSignal peptide d

1 h e 3 h

146*,174 At3g45970 Expansin 29253/8.28 2 +

147 At5g24090 Chitinase 33501/9.22 0 +

59 At3g48580 Xyloglucan endotransglucosylase/hydrolase 31062/8.62 0 + † †

148 At1g71380 β-Glucanase 53638/8.81 0 +

149 At2g44450 β-Glucosidase 57211/7.56 0 +

150*,151*,152 At1g68560 α-Xylosidase 102961/6.31 0 + † †

153,154*,155*, 156 102210/6.32 0 + † †

175* At5g20950 β-Xylosidase 67910/9.4 0 + † †

157*,158 At5g08380 α-Galactosidase 46079/6.19 0 + †

95* At5g63810 β-Galactosidase 83619/8.58 1 + † †

54* At3g13790 β-Fructosidase 66255/9.19 1 + † †

159*,160 At3g26720 α-Mannosidase 115547/6.27 1 + † †

176 At5g41870 Polygalacturonase 49013/6.89 0 +

161,177 At5g06860 Polygalacturonase 37293/8.24 0 +

178,179* At3g54400 Aspartyl protease 46259/9.31 0 +

162 At5g67360 Serine protease 80024/5.91 0 +

163* At3g02750 Phosphatase-2C 54332/5.04 0 –

184* At1g13750 Phosphoesterase 68826/6.29 0 + †

49* At4g34870 Peptidylprolyl isomerase 18594/8.90 0 – † †

57 At1g30730 FAD-linked oxidoreductase 59161/9.23 0 + † †

56* At5g44380 FAD-linked oxidoreductase 60920/9.51 1 + † †

164 At5g18860 Nucleoside hydrolase 99580/7.59 0 + †

165 At2g02990 Ribonuclease 26007/5.08 1 +

166 At3g47800 Aldose 1-epimerase 39861/9.12 1 +

48* At4g25900 Aldose 1-epimerase 36229/9.65 1 + † †

167,168 At1g03220 Glycoprotein 46373/8.98 0 +

77 At1g03230 Glycoprotein 46803/9.33 0 + †

99*,180* At1g53070 Legume lectin 30416/8.49 1 + † †

110 At3g16530 Legume lectin 30547/6.97 0 + †

85 At3g16420 Jacalin lectin 32096/5.46 0 – † †

87* At3g16450 Jacalin lectin 32003/5.06 0 – † †

35*,62*,71 At1g78850 Curculin-like lectin 49818/7.82 0 + † †

182,183* At1g33590 Leucine-rich repeat protein 561123/9.10 0 +

169 At1g21670 Unknown 77468/9.33 0 +

170 At1g51630 Unknown 47454/6.44 1 +

171 At2g32330 Unknown 35134/8.61 0 –

172 At2g41800 Unknown 40630/9.22 0 +

181,173 At3g08030 Unknown 39326/7.17 1 +

50 At5g26260 Unknown 839802/9.21 0 + † †


Table 3 Summary of the proteins identified as 1 M KCl extractable from the 1 h regenerated protoplasts


Accession no. b



3 h e Native

111 At2g43610 Chitinase 30892/9.54 1 +

59 At3g48580 Xyloglucan endotransglucosylase/hydrolase 32204/8.76 0 + † †

32 At1g66280 β-Glucosidase 60142/6.74 0 +

2*, 3*, 104*, 114* 115*, 116*

At1g68560 α-Xylosidase 102210/6.32 0 + † †

52* At5g20950 β-Xylosidase 67910/9.49 0 + † †

117 At5g08380 α-Galactosidase 46079/6.19 0 + † †

95*, 96, 118* At5g63810 β-Galactosidase 83619/8.58 1 + † †

51, 54*, 73, 100 At3g13790 β-Fructosidase 66581/9.14 1 + †

119* At3g26720 α-Mannosidase 115547/6.27 1 + † †

63 At5g19440 Cinnamyl-alcohol dehydrogenase 35914/6.67 0 – †

106 At2g04160 Serine protease 81364/9.34 0 +

120* At5g10540 Oligopeptidase 79223/5.45 0 –

121 At1g63770 Aminopeptidase 99495/5.43 0 –

185, 186* At1g13750 Phosphoesterase 68826/6.29 0 + †

42* At4g24340 Phosphorylase 36024/8.24 1 + †

101 At3g27950 GDSL-motif lipase/hydrolase 40120/8.89 0 + †

49 At4g34870 Peptidylprolyl isomerase 18594/8.90 0 – † †

69 At4g38740 Peptidylprolyl isomerase 18589/7.68 0 – †

57 At1g30730 FAD-linked oxidoreductase 59161/9.23 0 + † †


17, 122, 123* At3g55440 Triosephosphate isomerase 27380/5.39 0 – †

102 At1g74000 Strictosidine synthase 34819/9.66 0 +

76 At5g04590 Sulfite reductase 72391/8.51 0 – †

124 At4g25100 Iron superoxide dismutase 23776/6.06 0 –

98, 125 At5g43940 Alcohol dehydrogenase 41528/6.51 0 – †

89* At3g48000 Aldehyde dehydrogenase 58951/7.11 0 – †

126 At2g41530 Esterase 30759/5.91 0 –

10* At2g31390 Fructokinase 35424/5.31 0 – †

48, 127 At4g25900 Aldose 1-epimerase 36229/9.65 1 + † †

11, 21 At5g07440 Glutamate dehydrogenase 45013/6.07 0 – †

41, 12 At1g78380 Glutathione transferase 25691/5.80 0 – †

58, 105 At1g65590 β-Hexosaminidase 60375/8.30 1 + †

22, 30, 60*, 103 At1g13440 Glyceraldehyde-3-phosphate dehydrogenase 37004/6.67 0 + †

31 At3g04120 Glyceraldehyde-3-phosphate dehydrogenase 36897/7.15 0 +

75 At3g02360 6-Phosphogluconate dehydrogenase 53829/7.02 0 + †

55, 74, 128* At4g37870 Phosphoenolpyruvate carboxykinase 73929/6.61 0 – †

129 At1g79550 Phosphoglycerate kinase 42162/5.49 0 –

130 At1g80050 Adenine phosphoribosyltransferase 21241/5.69 0 –

1*, 28, 113, 131*, 132*, 133

At5g17920 Methionine synthase 84646/6.09 0 – †

134 At3g52880 Monodehydroascorbate reductase 46629/6.41 0 – †

135* At5g03630 Monodehydroascorbate reductase 47408/5.18 0 – †

64*, 109 At3g22110 20S proteasome α-subunit C 27475/7.14 0 – †

66, 65 At5g66140 20S proteasome α-subunit D2 27306/8.96 0 – †

20, 72* At1g56450 20S proteasome β-subunit G1 27691/6.09 0 – †

67, 99, 136, 137 At1g53070 Legume lectin 30416/8.49 1 + † †

110 At3g16530 Legume lectin 30547/6.97 0 + †

85*, 138, 139* At3g16420 Jacalin lectin 32138/5.46 0 – † †


Discussion

Proteins potentially involved in cell wall expansion

In this work, we carefully extracted cell wall proteins that

were ionically bound to the cell surface using a non-destruc-

tive procedure without disrupting cellular integrity, thereby

avoiding contamination from cytoplasmic proteins, and ana-

lyzed the protein profiles comprehensively. This non-destruc-

tive cell wall proteome approach disclosed a set of proteins that

are expressed specifically during cell wall regeneration, and are

loosely attached to the cell surface during the cell wall regener-

ation process in Arabidopsis suspension-cultured cells. In the

cell wall protein fraction of the native cells, we identified sev-

eral key proteins implicated in the remodeling of the cellulose/

xyloglucan network, in which xyloglucan functions as major

load-bearing cross-links between cellulose microfibrils. These

proteins include expansin, XTH, α-xylosidase and chitinase.

Expansins are a class of proteins capable of causing

cell wall expansion under acidic conditions (Cosgrove 2000).

Whereas the mechanism of expansin action still remains elu-

sive, it is implicated in a reversible disruption of hydrogen

bonding between cellulose microfibrils and xyloglucan. This

results in a loosening of the cell wall and allows turgor-driven

deformation of the cellulose/xyloglucan framework (McQueen-

Mason and Cosgrove 1994, Cosgrove 2000, Whitney et al.

2000). These proteins are encoded by a large multigene family

(McQueen-Mason and Cosgrove 1994), which can be divided

into four subfamilies: two expansin subfamilies, EXPA and

EXPB; and two expansin-like subfamilies, EXLA and EXLB

(Kende et al. 2004). The EXLA1 (At3g45970) identified in this

study is a member of the EXLA subfamily. Unlike EXPA and

EXPB (Cosgrove 1997), no biological or biochemical function

has yet been established for any member of the EXLA or

EXLB family (Kende et al. 2004), despite the fact that mem-

bers of EXLA and EXLB possess both expansin domain I and

II. Thus, the present result might imply a possible role for

EXLA1 protein in the cell wall construction, particularly in

suspension-cultured cells.

The chitinase family in Arabidopsis consists of two

distinct subfamilies. One, acidic endochitinase, encodes pro-

teins highly homologous to ‘yieldin’, which is implicated in

the modulation of mechanical properties of the cell wall in

cowpea plants (Okamoto-Nakazato et al. 2000, Yokoyama and

Nishitani 2004). A phylogenetic analysis reveals that the chiti-

nase isoform (At5g24090), which we have identified in the

present study, is the one and only yieldin-like protein in

Arabidopsis (Table 2; Yokoyama and Nishitani 2004). It is

likely that this Arabidopsis yieldin ortholog is involved in the

regulation of mechanical properties in the plant cell wall, thereby

modulating cell expansion in the suspension-cultured cells.

Two proteins encoded by AtXTH11 (At3g48580) and

AtXYL1 (At1g68560), which were found to be present in the

apoplastic fraction in both the native cells and protoplasts

(Tables 2–4), are implicated in the modification of these

Table 3 Continued

a Protein spot numbers correspond to the numbers on gel images (Fig. 2C, D). An asterisk indicates that the protein spot is phosphorylated, while

underlined spots are glycosylated.b Accession numbers are based on the NCBI database.c The number of transmembrane domains (TMDs) as predicted by TMHMM is shown.d The potential signal peptide as predicted by iPSORT, Signal P and Target P is represented by +.e † in the 3 h and Native columns indicates that proteins encoded by the same gene are also identified in the 3 h regenerated protoplasts and native

cell walls, respectively.


Accession no. b



3 h e Native

26, 86, 140 At3g16430 Jacalin lectin 32138/5.86 0 – †

87* At3g16450 Jacalin lectin 32003/5.06 0 – † †

38 At3g16460 Jacalin lectin 72430/5.31 0 – †

35*, 62*, 71, 80*, 112, 140*, 141*, 142, 143*

At1g78850 Curculin-like lectin 49818/7.82 0 + † †

29, 82 At1g54000 Myrosinase-associated protein 43498/6.65 1 + †

144 At1g54010 Myrosinase-associated protein 43572/8.33 1 + †

107 At5g12940 Leucine-rich repeat protein 40497/9.52 1 +

61 At1g18080 WD-40 repeat protein 36124/9.62 0 – †

53 At1g07930 Elongation factor 1-α 49781/9.15 0 – †

68 At3g53430 60S ribosomal protein L12 18073/9.05 0 – †

145 At1g21350 Unknown 28413/9.14 0 +

70 At4g09320 Unknown 15797/6.84 0 –

50 At5g26260 Unknown 39802/9.21 0 + † †

108 At5g26280 Unknown 39421/8.46 1 +


Table 4 Summary of the proteins identified as 1M KCl extractable from the 3h-regenerated protoplasts


Accession no. b



1 h e Native

59 At3g48580 Xyloglucan endotransglycosylase/hydrolase 31922/8.78 0 + † †

32 At1g66280 β-Gucosidase 60142/6.74 0 + †

82* At3g09260 β-Glucosidase 60196/6.45 0 +

2*, 3*, 114*, 115*

116*, 187*At1g68560 α-Xylosidase 102210/6.32 0 + † †

52* At5g20950 β-Xylosidase 67910/9.49 0 + † †

117 At5g08380 α-Galactosidase 46079/6.19 0 + † †

95*, 96 At5g63810 β-Galactosidase 83619/8.58 1 + † †

13*, 51*, 54*, 73, 100

At3g13790 β-Fructosidase 66581/9.14 1 + †

33* At3g26720 α-Mannosidase 115203/6.7 1 + † †

63* At5g19440 Cinnamyl-alcohol dehydrogenase 35576/7.15 0 – †

42* At4g24340 Phosphorylase 36876/9.04 1 + †

8* At4g23590 Aminotransferase 47919/5.76 0 –

88 At3g29636 Transferase 22757/8.33 0 –

9 At5g65020 Annexin 36358/5.76 0 –

81 At2g05710 Aconitate hydratase 98720/5.79 0 –

83 At3g14220 GDSL-motif lipase/hydrolase 40565/6.37 1 +

101 At3g27950 GDSL-motif lipase/hydrolase 40120/8.89 0 + †

49 At4g34870 Peptidylprolyl isomerase 18594/8.90 0 – † †

69 At4g38740 Peptidylprolyl isomerase 18355/7.65 0 – †



36*, 94* At5g18860 Nucleoside hydrolase 99580/7.59 0 + †

18 At1g76680 12-Oxophytodienoate reductase 41526/6.51 0 –

17* At3g55440 Triosephosphate isomerase 27380/5.39 0 – †

102 At1g74000 Strictosidine synthase 34819/9.66 0 + †

76 At5g04590 Sulfite reductase 71933/8.47 0 – †

43 At3g10920 Manganese superoxide dismutase 25426/8.83 0 –

98, 125 At5g43940 Alcohol dehydrogenase 41528/6.51 0 – †

89* At3g48000 Aldehyde dehydrogenase 58951/7.11 0 – †

10* At2g31390 Fructokinase 35424/5.31 0 – †

48*, 93 At4g25900 Aldose 1-epimerase 36234/10.24 1 + † †

11, 21 At5g07440 Glutamate dehydrogenase 45013/6.07 0 – †

12, 41 At1g78380 Glutathione transferase 25691/5.80 0 – †

78* At1g04410 Malate dehydrogenase 35890/6.11 0 +

79* At1g53240 Malate dehydrogenase 36010/8.54 0 –

58 At1g65590 β-Hexosaminidase 59996/8.26 1 + †

22*, 23*, 30, 31, 60*, 103

At1g13440 Glyceraldehyde-3-phosphate dehydrogenase 37004/6.67 0 + †

75 At3g02360 6-Phosphogluconate dehydrogenase 53560/7.49 0 + †

27*, 55*, 74* At4g37870 Phosphoenolpyruvate carboxykinase 73929/6.61 0 – †

1*, 28, 131*, 132*, 133

At5g17920 Methionine synthase 84646/6.09 0 – †

134 At3g52880 Monodehydroascorbate reductase 46629/6.41 0 – †

90 At3g53880 Monodehydroascorbate reductase 46629/6.41 0 –

25* At5g03630 Monodehydroascorbate reductase 47507/5.24 0 + †

34 At1g16470 20S proteasome α-subunit B 25685/5.53 0 –

64* At3g22110 20S proteasome α-subunit C 27572/6.60 0 – †


xyloglucan molecules. AtXTH11 is a member of the endotrans-

glucosylase/hydrolase (XTH) family of proteins, which can

mediate splitting and/or reconnection of the xyloglucan cross-

links in the cell wall (Nishitani and Tominaga 1991, Nishitani

and Tominaga 1992, Okazawa et al. 1993, Campbell and

Braam 1999, Yokoyama and Nishitani 2001, Rose et al. 2002).

As such, it is considered essential for cell wall dynamics. In

Arabidopsis, 33 genes encoding this family of proteins have

been identified in the genome. The present study disclosed that

only one of the 33 XTH members is specifically expressed both

in the native cell wall and during the cell wall regeneration

processes in the protoplasts. This AtXTH11 protein belongs to

the group I/II subfamily of the XTH family of proteins, and is

predicted to exhibit xyloglucan endotransglucosylase activity

exclusively, suggesting its role in the integration of newly

secreted cellulose/xyloglucan complexes into the cell wall

components during cell wall construction (Ito and Nishitani

1999, Yokoyama and Nishitani 2001).

The AtXYL1 protein, which shows a signature region of

family 31 glycosyl hydrolases, is implicated in the degradation

of an unsubstituted α-xylosyl residue situated nearest to the

non-reducing end of xyloglucan oligosaccharides. Higher lev-

els of the AtXYL1 transcript as well as α-xylosidase activity

were observed in younger rosette leaves of Arabidopsis, indi-

cating developmental regulation of their expression (Sampedro

et al. 2001). It should be noted that several protein spots for the

AtXYL1 protein with differing molecular sizes and pI have

been identified. This indicates that this protein undergoes

extensive post-translational modifications. It is also interesting

that some spots are phosphorylated, while others are not. This

implies that kinase-mediated regulation in muro may be

involved in the regulation of this enzyme. Thus, the data imply

that AtXTH11 and AtXYL1 play roles in the modification of

the cellulose xyloglucan network during cell wall regenera-

tion, as well as in the native cell walls. In the present study, we

did not examine incorporation of phosphate into individual pro-

Table 4 Continued

a Protein spot numbers correspond to the numbers on gel images (Fig. 2E, F). An asterisk indicates that the protein spot is phosphorylated, while

underlined spots are glycosylated.b Accession numbers are based on the NCBI database.c The number of transmembrane domains (TMDs) as predicted by TMHMM is shown.d The potential signal peptide as predicted by iPSORT, Signal P and Target P is represented by +.e † in the 1 h and native columns indicates that proteins encoded by the same gene are also identified in the 1 h regenerated protoplasts and native

cell walls, respectively.


Accession no. b



1 h e Native

65*, 66* At5g66140 20S proteasome α-subunit D2 27306/8.96 0 –

7 At4g31300 20S proteasome β-subunit A 25193/5.31 0 –

19, 44 At4g14800 20S proteasome β-subunit D2 22084/6.21 0 + †

91 At3g60820 20S proteasome β-subunit F1 24856/6.95 0 –

20*, 72* At1g56450 20S proteasome β-subunit G1 27691/6.09 0 – †

77 At1g03230 Glycoprotein 46803/9.33 0 + †

67*, 99* At1g53070 Lgume lectin 30361/8.84 1 + † †

84 At3g15356 LEgume lectin 29788/8.91 0 +

24 At2g39310 Jacalin lectin 50602/5.15 0 –

45 At3g16420 Jacalin lectin 32138/5.46 0 –

16, 40, 85*, 97 At3g16420 Jacalin lectin 32138/5.46 0 – † †

26, 86 At3g16430 Jacalin lectin 32213/5.86 0 – †

46*, 47, 87 At3g16450 Jacalin lectin 32003/5.06 0 – † †

4*, 5*, 6, 14*, 15*, 37*, 38*, 39*

At3g16460 Jacalin lectin 72430/5.31 0 – †

35*, 80*, 62*, 71, 142, 141*

At1g78850 Curculin-like lectin 49818/7.82 0 + † †



61 At1g18080 WD-40 repeat protein 35730/7.81 0 – †

53 At1g07930 Elongation factor 1-α 49485/9.6 0 – †

68 At3g53430 60S ribosomal protein L12 17952/9.57 0 – †

70, 92 At4g09320 Unknown 16500/6.79 0 –

50 At5g26260 Unknown 39802/9.21 0 + † †


teins or kinase activities responsible for the putative

phosphorylation reactions. Further investigation is required to

demonstrate the in muro phosphorylation of these proteins.

A polygalacturonase (At5g41870), which was also found

in the native cells, hydrolyses the α-1,4-D-galacturonan back-

bone of pectic polysaccharides (Brummell and Harpster 2001).

Enzyme activity of the polygalacturonase reportedly increases

throughout the expansion period of ripening fruit in many plant

species (Hobson 1962, Brummell and Labavitch 1997). A

polygalacturonase present in liverwort cell cultures increased

in parallel with cell growth until the cessation of the exponen-

tial growth phase, at which time the activity declined (Konno et

al. 1983). In addition, polygalacturonases extracted from cul-

tured carrot cells degraded pectic polymers from these cultures

(Konno et al. 1984). Based on these developmental correla-

tions, it is possible that polygalacturonases act coordinately

with other cell wall-modifying enzymes to increase cell wall

extensibility (Konno et al. 1983). We also identified other pro-

teins involved in the exo- or endo-type hydrolysis of poly-

saccharides in the native cells (Table 2). These enzymes may

also act coordinately with cell wall-loosening proteins to

increase cell wall extensibility, thus permitting cell expansion.

Proteins possibly involved in cell wall regeneration

Whereas expansin, yieldin and polygalacturonase were

not detected in the cell wall fraction derived from the proto-

plasts, several protein spots encoded by AtXTH11 and AtXYL1

were found to be expressed in the protoplasts as well as in

native cells. However, given the incomplete construction of the

cellulose/xyloglucan network during the first hours of cell wall

regeneration on the protoplasts (Fig. 1), it is unlikely that

AtXTH11 participates in the remodeling of the pre-existing cel-

lulose/xyloglucan framework as in native cell walls. It is more

probable that AtXTH11 plays a role in the integration of newly

secreted xyloglucan material into the pre-secreted xyloglucan

molecules, thereby assembling the precursor for cellulose/

xyloglucan construction (Ito and Nishitani 1999, Nishitani

1997). Accordingly, AtXTH11 may fill dual roles, wall assem-

bly and restructuring, during the cell wall regeneration and

expansion processes (Nishitani 1997).

XTH can mediate the depolymerization of xyloglucan

molecules by transferring a xyloglucan fragment generated by

splitting a xyloglucan donor substrate to a small xyloglucan

acceptor substrate. This depolymerization activity of XTH

directly depends on the concentration of xyloglucan oligomers

in muro. Since AtXYL1 is demonstrated to remove the xylosyl

residue at the non-reducing terminus of xyloglucan oligomers

(Sampedro et al. 2001), and since the terminal xylosyl substitu-

tion is a prerequisite of the acceptor substrate activity of

xyloglucan for XTH (Nishitani 1997), it is quite likely that

AtXYL1 may suppress the depolymerization of xyloglucan by

degrading or inactivating the acceptor substrate (Lorences and

Fry 1993). The AtXYL1-mediated suppression of xyloglucan

depolymerization would result in the generation of higher

molecular weight xyloglucans outside the plasma membrane.

As we have mentioned before the AtXYL1 protein under-

goes a variety of post-translational modifications throughout

the cell wall assembly and remodeling processes (Tables 2–4,

Fig. 2). These modifications in the AtXYL1 protein raise the

possibility that differently modified AtXYL1 proteins exhibit

differing enzymatic activities on xyloglucan oligomers, and

thereby on the regulation of AtXTH11 activity.

Proteins involved in defense and extracellular signaling

The present study revealed that several defense-related

proteins, including chitinase (At2g43610) and β-glucanase

(At1g66280 and At3g09260), are secreted into the extra-

cellular matrix of the protoplasts. Some defense-related pro-

teins are directly involved in the structural organization of cell

walls and take part in an active defense system. These defense

proteins may be induced by various stresses that would be gen-

erated during protoplast preparation. Alternatively, proteins

required for cell wall dynamics during cell wall regeneration in

the protoplasts may be mediated by the same molecular com-

ponents that are also recruited as defense proteins upon defense

response. It is worth noting that the β-glucanase (At3g09260),

which was found in the protoplast cell wall in the present study,

has recently been identified as being a major component of

the ER body, a subcellular compartment discovered recently

(Matsushima et al. 2003).

The presence of phosphorylated proteins in plant cell

walls has recently been reported (Chivasa et al. 2002, Baluska

et al. 2003). These proteins may participate in extracellular

signaling events. An acid phosphatase (At3g02750), which was

identified in the present study, could be involved in such signal

mediation. The phosphorylation of cell wall proteins in muro

would play an important role in regulating their localizations

and enzyme activities, thereby allowing multiple locations

and/or functions of the proteins encoded by a single gene. It

might be possible that a post-translational modification may

participate in the extracellular localization of proteins without

any conventional signal peptide via an unknown trafficking

mechanism.

Glycolytic enzymes and other unexpected proteins found in

extracellular spaces during cell wall regeneration processes

In cell wall-regenerating protoplasts, we identified sev-

eral glycolytic enzymes that are known to catalyze the initial

steps of the glycolytic pathway. These enzymes have never

been considered to be secreted outside of the protoplasm.

However, some glycolytic enzymes have already been classi-

fied as cell wall components in some organisms (Chaffin et al.

1998, Pardo et al. 2000, Chivasa et al. 2002). For example,

glyceraldehyde-3-phosphate dehydrogenase (At1g13440 and

At3g04120) has been immunologically localized in yeast cell

walls (Gozalbo et al. 1998). Although how these glycolytic

enzymes actually cross the plasma membrane has not been fully


elucidated, some previous substantial data are in support of

the idea that some glycolytic enzymes may be secreted out

of the protoplasts during the cell wall regeneration process in

A. thaliana. It should be noted that in the present study

glycolytic enzymes were identified in the apoplasts around the

protoplasts only, not in native cells. This means that these

glycolytic enzymes may fulfill certain special functions that are

unknown, but might be required for the cell wall regeneration

of protoplasts.

In addition to glycolytic enzymes, we identified in the

extracellular protein fractions a few proteins that have been

considered present exclusively in the cytosol or in some intra-

cellular compartments. Although we used a non-destructive

method for protein extraction to minimize cross-contamination

of cytosolic marker proteins (Table 1), we cannot exclude the

possibility of minor contamination from cytoplasmic proteins

that cannot be detected by the activity assay of G6PDH or by a

simple viability test using fluorescein diacetate. The presence

of 60S ribosomal protein L12 in the 1 M KCl extract might

imply possible leakage of some cytoplasmic protein from the

cortical region of cytoplasm during extraction of the protoplast

with 1 M KCl solution. Thus, we should be careful in interpret-

ing localization of proteins that lack a conventional secretory

signal peptide. Nevertheless, apart from the 60S ribosomal pro-

tein, there is reason to believe that most proteins without

known secretory signal peptides are actually secreted. Whereas

the conventional theory for protein trafficking cannot explain

the mechanism by which proteins without any secretory signal

peptide are exported into the extracellular spaces, several pro-

teins that lack signal peptides are reportedly localized in the

extracellular matrix (Cleves 1997). For example, an ortholog of

the elongation factor 1-α protein that we have identified in the

present study was localized in tobacco cell walls by immuno-

gold labeling (Zhu et al. 1994), despite the lack of a secretory

signal peptide. A cell wall-bound malate dehydrogenase (Li et

al. 1989, Karkonen et al. 2002), which is implicated in the

regeneration of NAD(P)H required for cell wall peroxidase-

mediated free radical generation associated with lignin poly-

merization, is another example of this interesting class of extra-

cellular proteins without a signal peptide. Judging from this

line of evidence, we conclude that most of the proteins identi-

fied in the 1 M KCl fractions have been localized in the apo-

plastic spaces. However, the real functions of these proteins

remain unknown at present, and require further investigation.

Taken together, this proteomic approach using the cell

wall regeneration system on protoplasts derived from

Arabidopsis suspension-cultured cells has disclosed a set of

proteins required for cell wall expansion as well as wall con-

struction processes. Most of these proteins show a differing sta-

tus of post-translational modifications such as phosphorylation

and glycosylation among the three protein fractions. This indi-

cates dynamic changes in muro of these cell wall proteins dur-

ing the cell wall regeneration of protoplasts. More interestingly,

the use of a non-destructive extraction procedure for cell wall

proteins allowed us to avoid contamination from cytosolic frac-

tions, thus making it possible to identify a new class of cell

wall proteins that were predicted not to be localized in the cell

wall spaces. These results strongly vindicate the usefulness of a

proteomic approach for cell wall dynamics. Functional analy-

ses for each of these newly identified cell wall proteins,

together with their biochemical characterization and expres-

sion analyses, should extend our understanding of the whole

picture of cell wall dynamism from a genomic point of view,

and give an invaluable molecular basis for metabolomic

approaches of this complex supermolecule.

Materials and Methods

Plant material

Suspension-cultured Alex cells of A. thaliana L. (Heynh.), eco-

type Columbia, established by Mathur et al. (1998), were kindly pro-

vided by Dr. Masaaki Umeda from The University of Tokyo. The cell

line was maintained in a modified Murashige and Skoog medium

(Murashige and Skoog 1962) containing 3% sucrose, B5 vitamins

(Gamborg et al. 1976) and 0.5 mg l–1 2,4-dichlorophenoxyacetic acid

(2,4-D), adjusted to pH 5.8, at 23°C with constant shaking (120 rpm, 2

inches) in the dark. The suspension-culture was subcultured every 7 d

by transfer of 15 ml of culture into 35 ml of fresh medium (Mathur et

al. 1998).

Cell wall regeneration from protoplasts

Five-day-old suspension-cultured cells were collected by centrif-

ugation at 190×g for 10 min and incubated for 1 h at 37°C in an

enzyme mixture containing 1% Cellulase Onozuka RS (Yakult Phar-

maceutical Ind. Co. Ltd., Tokyo, Japan) and 0.1% Pectolyase Y-23

(Kikkoman Shoyu Co. Ltd., Tokyo, Japan). This was supplemented

with 120 g l–1 sorbitol and adjusted to pH 5.8 with KOH. After com-

pletion of incubation, the protoplasts were collected by centrifugation

at 70×g for 10 min and washed twice with 40 ml of 120 g l–1 sorbitol.

The purified protoplasts were gently resuspended in a modified cell

wall regeneration medium as described by Schirawski et al. (2000) at a

final density of about 105 protoplasts ml–1, and incubated for 1 or 3 h at

25°C to regenerate cell walls.

Microscopic observation

For light microscope observation, native cells, fresh protoplasts

and protoplasts that had been allowed to regenerate cell walls for 1 and

3 h were fixed in 1% glutaraldehyde and stained with 0.001% Cal-

cofluor White M2R (Sigma, St Louis, MO, USA) or aniline blue in

120 g l–1 sorbitol at 25°C (Smith and McCully 1978). Both epifluores-

cent images and differential interference contrast images for individ-

ual specimens were observed under a fluorescence microscope

equipped with a UV fluorescence filter set (excitation filter, 350 nm;

barrier filter, 430 nm) and differential interference contrast optics

(DMRXP, Leica, Heerbrugg, Switzerland). The images were then pho-

tographed with a digital camera (Color Chilled 3CCD CAMERA C-

5810, Hamamatsu Photonics K.K., Hamamatsu, Japan).

For electron microscopic observation, native cells and proto-

plasts were fixed in 2% glutaraldehyde with 120 g l–1 sorbitol and

adjusted to pH 6.0 for 16 h. This was followed by an additional 30 min

of fixation in 0.2% (v/v) ruthenium tetroxide (RuO4) at room tempera-

ture. After completion of fixation, specimens were dehydrated through

an ethanol series and n-butyl alcohol, lyophilized (ES-2030, Hitachi

Science Systems Ltd., Tokyo, Japan), coated with silver (E-1030,

Hitachi Science Systems Ltd.), and observed with an SEM (S-4100,


Hitachi Science Systems Ltd.) at 3 kV according to a similar procedure

described by Konomi et al. (2000).

G6PDH activity assay

The activity of G6PDH (EC 1.1.1.49) was examined according to

the procedure described by Weimar and Rothe (1986), with some mod-

ifications. Briefly, protein extracts were mixed with a substrate solu-

tion containing 1 mM glucose-6-phosphate, 0.2 mM NADP and 1 mM

MgCl2. In 0.1 M Tris, the extracts were adjusted to pH 8.0 and incu-

bated at 25°C for 60 min. During the incubation period, the reduction

of NADP in the reaction mixture was measured by its absorbance at

340 nm using a spectrometer (UV-160A, Shimadzu Co., Kyoto, Japan).

Viability assay

The viability of native cells and protoplasts that had been

extracted with 1 M KCl was examined by staining with FDA

(Ishikawa et al. 1995, Amano et al. 2003). Briefly, cells or protoplasts

were mixed with 12 µM FDA in a 120 g l–1 sorbitol solution with gen-

tle shaking and kept for 5 min in the dark at 25°C. Viable cells and

protoplasts were counted under a fluorescence microscope equipped

with a UV fluorescence filter set (excitation filter, 490 nm; barrier fil-

ter, 514 nm).

Protein extraction and separation by two-dimensional gel electro-

phoresis

To extract extracellular proteins ionically bound to the cell wall,

5-day-old suspension-cultured cells were pelleted by centrifugation at

60×g for 10 min, resuspended in an equal volume of 1 M KCl and kept

under a reduced pressure at –70 kP at 25°C for 10 min (Vanacker et al.

1998, Ito and Nishitani 1999, Souza and MacAdam 2001). This was

followed by centrifugation to obtain the supernatant. Extracellular pro-

teins on the surface of the protoplasts were also extracted with essen-

tially the same procedure. An exception was that extraction with 1 M

KCl was conducted under atmospheric pressure by gently stirring the

protoplasts. This extraction procedure was repeated twice. The extracts

were combined and reduced to 1/100 volume by ultrafiltration

(Amicon Ultra-4 10000MWCO, Millipore, Milford, MA, USA).

Proteins were precipitated with the addition of 9 vol of 80% acetone

containing 10% trichloroacetic acid and 200 mM dithiothreitol (DTT),

pelleted by centrifugation at 20,400×g for 30 min, washed three times

with 80% acetone, and resuspended in 2D-PAGE sample buffer (5 M

urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-

propanesulfonate (CHAPS), 2% SB3-10, 2 mM tributyl phosphate

(TBP), 40 mM Tris base and 0.5% Pharmalyte; pH 3–10 and 6–11).

After the protein contents were determined using a modification of the

method of Bradford (1976), a 180 µg protein portion was loaded into

13 cm isoelectric focusing (IEF) gel strips (Amersham Pharmacia

Biotech, Uppsala, Sweden) with pH ranges of 3–10 or 6–11. These were

rehydrated according to the procedure provided by the manufacturer

(Rabilloud et al. 1994). Electrofocusing was performed at 17 kV h–1

using a Multiphor II system (Amersham Pharmacia Biotech), equili-

brated for 15 min in a buffer (6 M urea, 30% glycerol, 1% SDS, and

0.5 M Tris–HCl, pH 6.8) containing 1% DTT, and for another 10 min

in the same buffer containing 30 mM iodoacetamide. After equilibra-

tion, the IEF gel strips were loaded on top of 12% polyacrylamide gels

(160×160×1 mm) for SDS–PAGE, which was performed using the 2-D

gel system (Nihon Eido Co., Ltd, Tokyo, Japan; Laemmli 1970). The

gels were stained with CBB.

Protein identification by MALDI-TOF/MS

Proteins separated by the 2D-PAGE were mapped using Image

Master 2D-Elite software (Amersham Pharmacia Biotech). All protein

spots were picked from the 2D-polyacrylamide gels, washed in 25 mM

bicarbonate buffer, destained, and desiccated in the presence of

acetonitrile. Each gel piece was then rehydrated in 25 µl of 50 mM

bicarbonate buffer containing 200 ng of modified trypsin (Promega,

Madison, WI, USA), followed by incubation for 16 h at 37°C. Peptides

released by trypsin digestion were extracted from the gel piece with

75 µl of 50% acetonitrile solution containing 5% trifluoroacetic acid

followed by evaporation under a reduced pressure using Speed Vac

(SC110A, Savant Instruments, Inc., NY, USA) to reduce the concentra-

tion to about 20 µl. After the concentrated samples were treated with

ZipTip C18 (Milipore, Milford, MA, USA) and mixed with 1 µl of an

α-cyano-4-hydroxycinnamic acid (α-CHCA) matrix, they were sub-

jected to MALDI-TOF/MS analysis (Applied Biosystems, Foster City,

CA, USA). The mass spectrum of the peptide derived from each pro-

tein spot was recorded using MALDI-TOF/MS (Applied Biosystems,

Voyager DE-STR). Each peptide mass was matched to the theoretical

tryptic digests of proteins predicted based on the complete non-redun-

dant Arabidopsis database using the Mascot sequence database search

program (Matrix Science, Boston, MA, USA). We selected sequence

results that matched all conditions as follows: (i) the sequence cover-

age was >30%; (ii) the mass error margin was within 50 ppm; and (iii)

the molecular weight search (MOWSE) score was significantly (P <

0.05) greater than 76.

Data analysis

The presence of putative cleavable signal peptides or non-cleava-

ble signal anchors was predicted using PSORT (http://psort.nibb.ac.jp/

form.html), Target P (http://www.cbs.dtu.dk/services/TargetP/) and the

Signal-P V2.0.b2 program (http://www.cbs.dtu.dk/services/SignalP/;

Nielsen et al. 1997). The amino acid sequences of hypothetical proteins

were also analyzed using the TMHMM prediction method (http://www.

cbs.dtu.dk/services/TMHMM/) for transmembrane helices (Sonnhammer

et al. 1998). Homologies to other proteins were searched using BLAST

programs (http://www.ncbi.nlm.nih.gov/BLAST/) and TAIR (http://

www.arabidopsis.org). The search of protein domains was performed

using Pfam (http://www.sanger.ac.uk/software/Pfam) and InterPro

(http://www.ebi.au.uk/iterpro).

Characterization of post-translational modifications

Phosphorylated proteins on 2-D polyacrylamide gels were deter-

mined using the Pro-Q Diamond phosphoprotein gel stain kit (Molecu-

lar Probes; Schulenberg et al. 2003, Hart et al. 2003). Briefly, after the

gel was fixed in a solution containing 50% methanol and 10% tri-

chloroacetic acid overnight, it was stained in a Pro-Q Diamond phos-

phoprotein gel stain solution for 3 h in the dark, followed by

destaining with successive washes of 2% acetonitrile in 25 mM

sodium acetate, pH 4.0, for 1 h. Protein spots on the Pro-Q-stained gels

were determined by staining with CBB. Fluorescent images of the Pro-

Q-stained gels were acquired using a Fuji FLA 3000 laser scanner

(Fuji Photo Film Co., Ltd., Tokyo, Japan) with a 532 nm excitation

and 580 nm emission filter for Pro-Q Diamond dye detection and total

protein detection.

Glycosylated proteins on 2-D polyacrylamide gels were deter-

mined using the Pro-Q Emerald 488 glycoprotein gel staining kit

(Molecular Probes; Hart et al. 2003). Briefly, after 2-D polyacryl-

amide gels were fixed in a solution containing 50% methanol and 5%

acetic acid, and extensively washed in 3% acetic acid, glycans were

oxidized by incubation in a solution containing 1% periodic acid and

3% acetic acid for 1 h. After the gels were washed in 3% acetic acid,

they were incubated in a Pro-Q Emerald 488 dye solution for 2.5 h in

the dark, followed by incubation in 3% acetic acid. The gels were then

stained for total protein with a CBB stain. Fluorescence images were

acquired on a Fuji FLA 3000 laser scanner (Fuji Photo Film Co., Ltd.)

http://psort.nibb.ac.jp/form.html

http://www.cbs.dtu.dk/services/TargetP/

http://www.cbs.dtu.dk/services/SignalP/

http://www.cbs.dtu.dk/services/TMHMM/

http://www.ncbi.nlm.nih.gov/BLAST/

http://www.arabidopsis.org

http://www.sanger.ac.uk/software/Pfam

http://www.ebi.au.uk/iterpro

http://psort.nibb.ac.jp/form.html

http://www.cbs.dtu.dk/services/TMHMM/


with 473 nm excitation and a 520 nm emission filter for Pro-Q Emer-

ald 488 dye detection and total protein detection.

Acknowledgments

The authors thank Dr. Csaba Koncz (Max-Planck-Institut fur

Zuchtungsforschung) and Dr. Masaaki Umeda (The University of

Tokyo) for kindly providing the Arabidopsis thaliana suspension-cul-

tured Alex cell line. This work was supported by a Grant-in-Aid for

Scientific Research on Priority Areas (15031202) and Scientific

Research (B) (15370016) from the Ministry of Education, Culture,

Sports, Science, and Technology of Japan, and by the Program for

‘Development of Fundamental Technologies for Controlling the Proc-

ess of Material Production of Plants’ from the New Energy and Indus-

trial Technology Development Organization, Japan.

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(Received on February 9, 2005; Accepted on March 11, 2005)

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