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Journal of Experimental Botany, Vol. 58, No. 1, pp. 103–112, 2007

Intracellular  Compartmentation: Biogenesis and Function Special Issue

doi:10.1093/jxb/erj209 Advance Access publication 27 June, 2006

SPECIAL ISSUE PAPER 

Proteomics selaput sel [pabrik/tumbuhan]

The  proteomics of  plant cell membranes

Setsuko Komatsu*, Hirosato Konishi and Makoto Hashimoto

 National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan

Received 11 December 2005; Accepted 21 March 2006

Abstract

Membrane proteins are involved in many different functions depending on their  location in the cell.

Characterization of the membrane proteome can  bring new insights to the function of  different plant

membrane systems and the subcellular compartments where the  proteins are found. Plant membrane pro-teomics can also provide valuable information about  plant-specific biological  processes. Despite recent ad-

vances in the separation and techniques for the anal- ysis of  plant membrane proteins, characterization of these

 proteins, especially the hydrophobic ones, is still challenging. In this review, plant membrane proteo- mics data,

compiled from the literature on Arabidop- sis thaliana, are described. In addition, initial attempts towards

determining the  physiological significance of  some proteins identified from membrane proteomics in rice are

also described.

Key words: Arabidopsis, cell membrane, plant, proteomics, rice.

Introduction

Plant cells contain many membrane systems specialized to particular functions. Their lipid component provides a barrier to solute movement, whilst membrane-associated proteins undertake their unique biological roles. For ex- ample,the plasma membrane is an organized system that serves both a structural role and acts as a communication interfacewith the extracellular environment for the ex- change of information and substances. Environmental stresses causesignificant intracellular restructuring in plants (Buchanan et al., 2000), and the processing of signals involved inresponses to biotic and abiotic stresses occurs in the plasma membrane. Therefore, a better knowl- edge of the plasmamembrane proteome would help in developing strategies to increase plants’ natural defences.

In plant cells, as well as in animal cells, the plasma mem- brane controls many primary cellular functions, such as

metabolite and ion transport, endocytosis, and cell differ- entiation and proliferation. All these processes involve a

large number of proteins with highly diverse structures and functions.

The degree of association of proteins with a membrane varies. Some proteins are embedded in the membrane lipid

core, while others are more peripheral, and associated by reversible interactions with either lipids or other membrane

 proteins (Marmagne et al., 2004). Plant membrane proteo- mics can give valuable information on plant-specific pro-

cesses; however, the challenge for proteomics is to find

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Plasma membrane proteomics of Arabidopsis thaliana

Many plasma membrane proteomics studies have been conducted (Prime et al., 2000; Santoni et al., 2000; Kawamuraand Uemura, 2003; Alexandersson et al.,2004; Marmagne et al., 2004). Marmagne et al. (2004) reported that about 100 putative plasma membrane proteinswere identified in plasma membrane fractions, obtained from an Arabidopsis cell suspension culture, enrichedin hydrophobic proteins. Ninety-five per cent of these pro-

teins represent newly identified  plasma membrane  proteins, and 50 of these proteins had predicted transmembrane do-

mains. Marmagne et al. (2004) used one-dimensional gels. To the authors’ knowledge, no non-ionic or zwitterionicdetergent has been found to resolve plasma membrane proteins quantitatively from eukaryotic cells in IEF for the

first dimension of 2D-PAGE. Thus, so far, it has not been possible to use 2D-PAGE to resolve integral proteinsof eukaryotic plasma membranes.

Alexandersson et al. (2004) analysed highly purified Arabidopsis plasma membranes from leaves and petioles by

mass spectrometry to identify integral and peripheral proteins associated with the plasma membrane. In total,238 putative plasma membrane proteins were identified, of which 114 are predicted to have trans-membrane do-mains or to be glycosyl phosphatidylinositol anchored.

About two-thirds of the integral proteins identified have not previously been shown to be plasma membrane pro- teins.

Of the 238 proteins identified, 76% could be classi- fied according to their function. Major classes represented aretransport (17%), signal transduction (16%), membrane trafficking (9%), and stress responses (9%). Almost a quarter of the proteins identified in this study have noknown function and more than half of these are predicted to be integral membrane proteins (Alexandersson et al.,2004). Functional characterization of these unknown pro- teins should provide clues that may lead to the identifi- cation

of novel functions for plant plasma membranes.

Proteomics of other  membrane systems of 

Arabidopsis thaliana

The interest of combining several complementary extrac- tion procedures to take into account specific features of 

membrane proteins will be discussed in the light of recent proteomics data, notably for the chloroplast envelope,mitochondrial membranes and the plasma membrane from Arabidopsis. Chloroplasts perform vital biosynthetic func-tions, and many processes are located exclusively within these unique organelles including the light and dark reac- tionsof photosynthesis, and the biosynthetic pathways for fatty acids, vitamins, and amino acids. The envelope, a two-

membrane system surrounding all plastid types, is involved in the synthesis of very specific components likeglycerolipids, pigments, and prenylquinones (Joyard et al.,1998). In recent years, a number of comprehensive pro- teomic studies have focused on the chloroplast envelope(Froehlich et al., 2003; Rolland et al., 2003; Eichacker et al., 2004) and the thylakoid membrane and lumen(Peltier et al., 2002, 2004; Schubert et al., 2002; White- legge, 2003; Friso et al., 2004). Peltier et al. (2004)reported a simple, fast, and scalable off-line procedure based on three-phase partitioning with butanol to frac-tionate membrane proteomes in combination with both in- gel and in-solution digestions and mass spectrometry. Theyanalysed the salt-stripped thylakoid membrane of chloroplasts of Arabidopsis, identifying 242 proteins of which at least40% were integral membrane proteins. The functions of 86 proteins remain unknown. This analysis showed strongdifferentiation in cellular functions be- tween the two membrane systems and elucidated the suborganellar localizationof many chloroplast membrane proteins (Peltier et al., 2004).

Mitochondria play a central role in eukaryotic cells by providing ATP from oxidative phosphorylation. Mitochon- driaare also involved in many other cellular functions including numerous catabolic or anabolic reactions and apoptotic cell

death (Balk et al., 2003; Newmeyer and Ferguson-Miller, 2003). Recent developments in proteo- mics also open the

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 path toward a deeper exploration of mitochondrial function using 2D-PAGE, SDS–PAGE, or blue native PAGE (Millar et al., 2001; Giege et al., 2003; Millar and Heazlewood, 2003; Brugiere et al., 2004; Heazlewood et al., 2004). Thus far,the Arabidopsis mitochondrial membrane proteome is one of the best characterized in plants. Brugiere et al.(2004) reported that highly purified mitochondrial membrane proteins, prepared from Arabidopsis cultured cells, provided the most exhaustive view of the protein repertoire of these membranes. Various extraction procedures wereapplied, and LC-MS/MS analyses were then performed on each membrane subfraction, leading to the identification of 114 proteins. About 40% of these proteins had not been identified during previous proteomic studies performed onmitochondria (Brugiere et al., 2004). Despite the large number of previous proteomic studies of plant mitochon- dria,the strategy that was shown to be most efficient for identifying new membrane proteins from chloroplast envelopemembranes was also efficient for mitochondrial

Proteomics of plant cell membranes 105

membranes. Comparative proteomics of mitochondrial and chloroplast envelope/thylakoid membranes provides a

framework to which additional membrane proteins can be added as their functions are experimentally determined.

Proteomics of rice membranes

Materials and methods for rice membrane proteomics

Isolation of vacuolar membranes: Rice seedling leaf sheaths and roots were utilized for isolation of vacuolar mem- branes.

All procedures were carried out at 4 8C. Fresh tissues of rice seedlings were chopped and ground in ahomogenization medium consisting of 0.25 M sorbitol,50 mM TRIS-acetate (pH 7.5), 1 mM EGTA, 1% polyvinyl- pyrrolidone (PVP), 10 lM phenylmethylsulphonyl fluo-

ride, and 2 mM dithiothreitol (DTT) using a mortar and pestle. The homogenate was filtered through a layer of Miracloth (Calbiochem, La Jolla, CA, USA). The extract was centrifuged at 3600 g for 10 min. The supernatant was

collected and centrifuged at 120 000 g for 25 min. The precipitate was suspended in TRIS-sucrose buffer consist- ing of 0.5 M sucrose, 20 mM TRIS-acetate (pH 7.5), 1 mM

EGTA, 2 mM MgCl2, and 2 mM DTT, and the suspension was overlaid with an equal volume of TRIS-sorbitol buffer 

consisting of 0.25 M sorbitol, 20 mM TRIS-acetate (pH7.5), 1 mM EGTA, 2 mM MgCl2, and 2 mM DTT. After centrifugation at 120 000 g for 45 min, vacuolar mem- branes

that formed a band at the interface between the two solutions were collected, diluted with the TRIS-sorbitol buffer, andcentrifuged at 130 000 g for 25 min. The resulting pellet was suspended in the TRIS-sorbitol buffer and used as thevacuolar membrane fraction.

Isolation of plasma membranes: Rice seedling plasma membranes were extracted from leaf blades, leaf sheaths, and roots.A portion (70 mg) of fresh tissues was chopped and ground in 210 ml of extraction buffer containing 0.4 M sucrose, 75mM MOPS/KOH pH 7.6, 5 mM EDTA/KOH pH 7.5, 5 mM EGTA/KOH pH 8.2, and 10 mM KF containing 1

mM DTT with 2% PVP using a mortar and pestle on ice. These homogenates were filtered through four layers of Miracloth. The filtrates were centrifuged at 9000 g for 15 min and 42 000 g for 30 min at 4 8C, plasma membrane-enrichedfractions were obtained using a two- phase partitioning method (Kawamura and Uemura, 2003). Two-phase partitioningwas repeated three times for the root samples, and four times for leaf blade and leaf sheath samples for increased purity.

Isolation of Golgi membrane:  Nucleoside diphosphate and (NDPase)-associated Golgi membrane were prepared fromcultured suspension cells as described by Mikami et al. (2001). The NDPase-associated Golgi membranes were further 

 purified by a second centrifugation in a discontinu- ous density gradient consisting of 28%, 30%, and 34%

106 Komatsu et al.

sucrose in 50 mM glycylglycine–NaOH (pH 7.0) and 5 mM MgCl2 at 100 000 g for 2 h. The Golgi membranes weretrapped on the 34% sucrose cushion. Enzymes charac- teristic of the endoplasmic reticulum, mitochondrion, microbody,vacuolar membrane, and plasma membrane were assayed as described previously (Mitsui et al.,

1990; Mikami et al., 2001).

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Purity of membrane fractions: KNO3, Na3VO4, and  NaN3 are specific inhibitors of V-, P-, and F-type H+-ATPases that

are specifically associated with the vacuolar  membrane, the plasma membrane, and the mitochondrial membrane,

respectively (Sze, 1985). The quality of isolated mem- branes was determined by assaying these specific ATPaseactivities. ATPase activity was assayed in a reaction solution containing 30 mM MES-TRIS (pH 6.5), 50 mM

KCl, 10 mM MgSO4, and 10 mM ATP with or without inhibitors of ATPase, 100 mM Na3VO4 and 50 mM KNO3 and 2mM NaN3. The solutions were incubated at 30 8C for 30 min. The reactions were stopped by the addition of 

a solution containing 0.5% ammonium molybdate, 1% SDS, and 1.96% H2SO4, to which ascorbate was added to a

final concentration of 10%. The reaction solution was incubated at room temperature for 30 min, and the absorbance at750 nm was measured. Products of ATPase activity were calculated from a standard curve generated with K 2HPO4.

Gel electrophoresis: Proteins (50 lg, 100 ll) of vacuolar membranes solubilized with lysis buffer (O’Farrell, 1975) wereseparated in the first dimension  by isoelectric focusing (IEF) or linear immobilized pH gradient (IPG) tube gels (DaiichiPure Chemicals, Tokyo, Japan) and in the second dimension by SDS-PAGE. The IEF tube gel solution consisted of 8 M urea, 3.5% acrylamide, 2% NP-40, 2% ampholytes (pH 3.5–10 and pH 5.0–8.0, Amersham Biosciences, Piscataway, NJ, USA), ammonium persulphate, and TEMED. Electrophoresis was carried out at 200 V for 30 min, followed by 400V for 16 h and 600 V for 1 h. For IPG electrophoresis, samples were applied to the acidic side of gels and electrophoresisusing IPG tube gels (pH6.0–10) was carried out at 400 V for 1 h, followed by 1000

V for 16 h and 2000 V for 1 h. After IEF or IPG, SDS- PAGE in the second dimension was performed using 15%

 polyacrylamide gels. Proteins (20 lg) from plasma mem- branes were extracted by SDS-sample buffer, and subjected toSDS-PAGE. The gels were stained with silver or Coomassie brilliant blue (CBB), and image analysis was

 performed. Images of two 2D-PAGE gels; one using IEF in

the first dimension and the other using IPG, were synthe- sized and the positions of individual proteins on the gels wereevaluated automatically using ImageMaster 2D Elite software (Amersham Biosciences). The pI and M r  of each proteinwere determined using 2-D markers (Bio-Rad, Hercules, CA, USA).

Mass spectrometry analysis: The stained protein spots were excised from gels, washed with 25% methanol and 7% aceticacid for 12 h, and destained with 50 mM  NH4HCO3 in 50% methanol for 1 h at 40 8C. Proteins were reduced with 10

mM DTT in 100 mM NH4HCO3 for 1 h at 60 8C and incubated with 40 mM iodoacetamide in 100 mM NH4HCO3 for 

30 min. The gel pieces were minced and allowed to dry and then rehydrated in 100 mM  NH4HCO3 with 1 pmol trypsin

(Sigma, St Louis, MO, USA) at 37 8C overnight. The digested peptides were extracted from the gel slices with 0.1% TFAin 50% acetonitrile three times. The peptide solution thus obtained was dried and recon- stituted with 30 ll of 0.1% TFAin 5% acetonitrile and then desalted with ZipTip C18 pipette tips (Millipore, Bedford, MA, USA). The above peptide solution was mixed with a-cyano-4-hydroxycinnamic acid and analysed using MALDI-TOF MS (Voyager,

Applied Biosystems, Framingham, MA, USA). In another experiment, each lane of the SDS-gel was cut out andsectioned into 20 pieces. Each section was further cut into smaller pieces and digested as described above. These peptidesolutions were concentrated down to 10 ll by vacuum centrifugation and reconstituted with 10 ll of 0.1% formic acid inwater and analysed using ESI-MS/MS (Nano-Frontier L, Hitachi High-Technologies Co., Tokyo, Japan and/or Q-TOFmicro, Micromass Co., Manchester, UK). The mass spectra were subjected to sequence database searches using Mascotsoftware (Matrix Science Ltd, London, UK).

For MALDI-TOF analysis, three criteria were used to assign a positive match with a known protein. (i) The deviation between the experimental and theoretical peptide masses had to be less than 50 ppm. (ii) At least four dif- ferent predicted peptide masses needed to match the ob- served masses for an identification to be considered valid. (iii) Thecoverage of protein sequences by the matching peptides had to reach a minimum of 10%. Furthermore, the score that wasobtained from the analysis with Mascot software had to indicate the probability of a true positive identification and thevalue had to be at least 50.

Plasma, vacuolar and Golgi membrane proteomics of rice

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Rice is not only a very important agricultural resource; it is also a model plant for biological research because itsgenome is smaller than those of other cereals (Devos and Gale, 2000). Publication of the draft genome sequences for Oryza sativa L. ssp. indica (Yu et al., 2002) and for Oryza sativa L. ssp. japonica (Goff et al., 2002), and a completemap-based sequence of chromosome 1 (Sasaki et al., 2002) and chromosome 4 (Feng et al., 2002) for Oryza sativa L. cv. Nipponbare provide rich resources for understanding the biological processes in rice. Recently, the International RiceGenome Sequencing Project (2005) presented a map-based, finished-quality sequence that covers 95% of 

the 389 Mb genome of rice, including virtually all of the euchromatin and two complete centromeres. Once the ricegenome is completely sequenced, the challenge ahead for the plant research community will be to identify the func- tion,

regulation, and type of  post-translational modification of each encoded protein. Also, whereas the genome is static; the proteome is highly dynamic in its response to external and internal cellular events. The responses of the proteome caninclude changes not only to the relative abundance but also to the post-translational modification of each protein.

In addition to tissue-specific analyses (Komatsu et al.,2003; Komatsu and Tanaka, 2004; Komatsu, 2005), the Rice Proteomics Project has analysed biological samples that are

specific to a subcellular compartment such as the cell wall, plasma membrane, vacuole membrane, Golgi membrane,mitochondrion, chloroplast, nucleus, and cyto-sol. Some of these results are described below. The plasma,

vacuolar and Golgi membranes, isolated from rice seed- lings and suspension-cultured cells, were solubilized in lysis

 buffer (O’Farrell, 1975), and the proteins were separated by 2D-PAGE and analysed with Image-Master 2D Elite software. The 2D maps of the various subcellular compartments resolved 464 proteins in the plasma mem-

 brane, 141 in the vacuolar membrane, and 361 in the Golgimembrane (Tanaka et al., 2004). The most abundant

 proteins on 2D-PAGE were analysed  by Edman sequencing and mass spectrometry. Edman sequencing showed that thenumber of N-terminally blocked proteins varied widely among the subcellular compartments. In mitochondria and

chloroplasts, respectively, 73% and 60% of the proteins were N-terminally blocked (Tanaka et al., 2004). A much larger 

 proportion of the proteins were N-terminally blockedin the plasma membrane (96%), vacuolar membrane (89%),

and Golgi membrane (98%). The SOSUI system software, developed for the classification and secondary structure

 prediction of membrane proteins (http://sosui.proteome. bio.tuat.ca.jp/), was used to analyse the proteins identified inthe three membrane samples. This software predicted transmembrane helices for 14 of the 58 plasma mem- brane

 proteins, 6 of the 43 vacuolar membrane proteins,and 7 of the 46 Golgi membrane proteins sequenced inthis study.

To narrow down the possible role of the more abundant proteins in subcellular membrane fractions of rice, the

identified proteins were categorized by criteria used by Bevan et al. (1998). In the plasma membrane of rice, pro- teins

with functions associated with metabolism, energy, signal transduction, and defence categories were abundant.By contrast, no proteins in the signal transduction and

defence categories were identified in the plasma membrane proteome of A. thaliana, but proteins involved in metab-

olism and energy were present (Santoni et al., 1998).

The vacuolar membrane proteome is still poorly under- stood, but the vacuolar membrane of plant cells is known to

contain two electrogenic proton pumps, H+-ATPase

Proteomics of plant cell membranes 107

and H+-translocating inorganic pyrophosphatase (PPase) (Hedrich and Schroeder, 1989). In the Rice Proteomics Project,

the b subunit of ATPase was identified in the vacuolar membrane proteome, but not the PPase. The a2 subunit of the20S proteasome was detected in the vacuolar membrane fraction, providing evidence that vacuoles might participate in thedegradation of denatured proteins. The vacuolar membrane proteome also included a water channel protein, a member of afamily of vacuolar and plasma membrane proteins that transport water molecules with high efficiency and selectivity

(Maurel, 1997). Signal transduction proteins were abundant in the vacuolar membrane and included a calmodulin-likeCa

2+-binding protein. Ca

2+ pumps are widely distributed across plant membranes, including the vacuolar membrane

(Sze et al.,2000).

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The Golgi complex is a multifunctional organelle re- sponsible for the biosynthesis of complex cell-surface

 polysaccharides, the processing and modification of glyco- proteins, and the sorting of polysaccharides and proteins

destined for various locations (Staehelin and Moore, 1995). To understand better how these functions are carried out, itwill be necessary to survey the proteome of Golgi. For proteins from the Golgi membrane fraction of rice, thefunctional categories of metabolism, energy, and defence

were represented in abundance (Tanaka et al., 2004). A reversibly glycosylated polypeptide, previously identified as a

 protein localized to the Golgi membrane and involved in the synthesis of xyloglucan and possibly other hemi- celluloses(Dhugga et al., 1997), was detected in the Golgi membrane proteome.

Data on the proteomics of rice membranes will be valuable for resolving questions in functional genomicsas well as for genome-wide exploration of plant cell function (Fig. 1).

Vacuolar  membrane proteomics changes in rice treated with gibberellin

Plant cells with defects in vacuole expansion cannot expand (Schumacher et al., 1999). In these defective cells, thevacuolar membrane cannot regulate the rapid uptake of water by expanding vacuoles for a rapid osmoregulation betweenthe cytosol and the vacuole (Chaumont et al.,1998). To analyse proteins that affect vacuolar functions, the vacuolar membrane fraction was isolated using a dis-

continuous sucrose/sorbitol system. The purity of the fraction was examined by assaying for H+-ATPase activity. The

sensitivity of H+-ATPase activity to nitrate, vanadate, and azide was used to distinguish between vacuolar  membrane,

 plasma membrane, and mitochondrial enzymes, respectively (Sze, 1985). The proportions of the total activity that weresensitive to nitrate, vanadate, and azide amounted to 60%, 10%, and 2%, respectively. The nitrate- sensitive fractionwas thus enriched in the vacuolar 

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Fig. 1. Numbers and percentages of proteins from each functional category found in the plasma membrane, vacuolar, and Golgi mem- branes of rice. Proteins were categorized using the criteria of Bevan et al. (1998).

membrane fraction, but it is clear that the preparation also contained traces of plasma membrane and mitochondrialcontaminants.

Proteins were extracted from vacuolar membrane frac- tions of rice leaf sheath from plants treated with 5 lM GA3 for 48 h and from untreated control plants. Proteins were separated in the first dimension by IEF or IPG and in the

second dimension by SDS-PAGE (Fig. 2; S Komatsu, unpublished data). The abundance of 20 proteins increased withGA3 treatment. These proteins were identified by MALDI-TOF MS analysis. A number of them could be identified by

their sequence similarity with known plant proteins such as ribosomal protein L (AF526214), gluta- thione S-transferase(AF402795), hypothetical protein T20K18.50 (T06628), glyceraldehyde-3-phosphate dehy- drogenase (AB106691 andX78307), Arabidopsis thaliana36716 (AY087569), inorganic  pyrophosphatase (AY153213), RuBisCO SSU (X07515), 3-methylcrotonyl CoA carbox-

ylase (AF251074), connectin/titin N2A-PEVK (AB100271), clathrin assembly protein AP17-like protein (AF443601), anddihydrolipoamide acetyltransferase (D21086). These proteins contribute to vacuolar function in rice leaf sheaths.

To examine changes in vacuolar membrane proteins in

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response to GA3 in the root, proteins were extracted from vacuolar membrane fractions of rice roots treated or not with

0.1 lM GA3 for 48 h, and separated  by 2D-PAGE. The abundance of 10 proteins increased in the root vacuolar 

membranes  by GA3 treatment (Konishi et al., 2005). These proteins were identified by MALDI-TOF MS analysis.

Some of them were similar  to known plant  proteins, such as V-ATPase subunit B (AF375052), ubiquitin RiP-20(AF216530), V-ATPase subunit A (P31450), ferredoxin (AF010320), proteinase 2 precursor (S53952), and fruc- tose-1,6-bisphosphate aldolase C-1 (D50301). Aldolase C-1 increased in GA3-treated roots at low levels in roots of Tan-

ginbozu, a mutant of GA biosynthesis, and this increase is reduced in roots treated with uniconazole-P or ABA ascompared with the control (Konishi et al., 2005). These results suggest that aldolase C-1 may act as a mediator  between GA signalling and root growth. In rice, three cytoplasmic aldolases (aldolase C-1, C-2, and C- a) and onechloroplastic aldolase (aldolase P) have been reported (Hidaka et al., 1990; Tsutsumi et al., 1994; Nakamura et al.,

1996). Internal amino acid sequences of protein 09 (Fig. 2) of the root vacuolar membrane (aldolase C-1) weredetermined by sequence analysis of peptides obtained using the Cleveland peptide mapping method (Cleveland et al.,1977). Aldolase C-1 specific amino acid residues N (115), S (191), E (195), S (222), E (234), S (235), Q (241), andL (242) were detected, clearly indicating that protein 09 identified was neither aldolase C-2, aldolase C-a, nor aldolase P. Thus, aldolase C-1 is likely to influence specifically vacuolar function in rice roots.

Since Aldolase C-1 is involved in glycolysis, these results suggest that GA3 enhances the metabolic rate of glycolysis in rice roots. Based on immunoprecipitation experiments (Konishi et al., 2005), aldolase C-1 activates V-

ATPase through physiological interactions. As a result, the rate of cell growth of seedling roots may be efficientlyenhanced.

Proteomics of plant cell membranes 109

Fig. 2. Changes in the protein patterns of vacuolar membranes of rice leaf sheaths or roots treated with GA3. Proteins were extracted from thevacuolar membranes of leaf sheaths treated or not with 5 lM GA3 and roots treated or not with 0.1 lM GA3 for 48 h, separated by 2D-PAGE withIEF and IPG in the first dimension and SDS-PAGE in the second dimension, and detected by silver staining. The isoelectric point and relativemolecular mass of each protein were determined using 2-D markers (Bio-Rad). Circles show the positions of 20 and 10 proteins whose abundanceincreased in the vacuolar membrane of leaf sheaths and roots, respectively, treated with GA3 as compared with control.

Plasma membrane proteomics changes in cold-treated rice  plants

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Cold stress is one of the most severe environmental stresses affecting plant growth and development. Plants that are

subjected to low temperature induce specific physiological responses. Low-temperature-responsive  proteins have been

identified using proteomic approaches. Such cold-response proteins were participants in various metabolic pathways such

as protein biosynthesis, folding, and degradation, bio- synthesis of cell wall components, and the energy pathway in rice

leaf tissue (Cui et al., 2005). Freezing-stress provokes injury to the plasma membrane and induces the expression of 

stress-related proteins. Kawamura and Uemura et al. (2003) reported that the acidic protein dehyrin, which increased

during cold-acclimation, associ- ates with the plasma membrane resulting in increased freezing-tolerance.

Lipoprotein-like proteins are also asso- ciated with acquired resistance to osmotic stress caused by freezing in

Arabidopsis leaves.Plasma membrane fractions were prepared by an

aqueous-polymer two-phase partitioning method. The

 purity of these fractions were determined by assaying for H+-ATPase activity. The sensitivity of H

+-ATPase activity to

vanadate and nitrate was used to distinguish between plasma membrane and vacuolar enzymes, respectively (Sze, 1985).

The proportion of the total activity that was

sensitive to vanadate and nitrate amounted to 93% and

44%, respectively. The vanadate-sensitive fraction was thus enriched in the plasma membrane fraction; however, the preparation contained traces of vacuolar membrane con- taminants. A number of plasma membrane proteomicsstudies have been carried out in plants, but there are few plasma membrane proteomic analyses in rice, except for leaf blade plasma membranes, and only a very few of them are

related to cold stress. In this study, plasma membranes, not only of leaf blades but also of leaf sheaths and roots, were purified from cold-stressed rice seedlings (Fig. 3).

Plasma membrane proteins from rice roots were sepa- rated and many different proteins were identified. However,

many of these proteins were aquaporins. Proteins that changed in abundance after cold stress were also detected. Inleaf blades of rice seedlings, more than 100 proteins were determined as plasma membrane proteins by MALDI-TOF MS

and ESI-MS/MS. Chlorophyll a/b binding protein, the large and small subunits of ribulose-bisphosphatecarboxylase/oxygenase (RuBisCO), putative photosystem Ireaction centre subunit II, glyceraldehyde-3-phosphate

dehydrogenase, and several unknown proteins were de- termined in the same fractions. Whether these are contam- inants

of the plasma membrane preparation or not cannot be definitely determined before tagged fusion proteins have beenmade or immunogold localizations have been done. Only a few obvious contaminants, such as highly

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Fig. 3. Changes in the protein patterns of the plasma membrane from rice leaf blades, leaf sheaths or roots upon exposure to cold. After coldtreatment at 5 8C, leaf blades, leaf sheaths and roots were collected, and plasma membranes were purified. Plasma membrane proteins were separated

 by SDS-PAGE, and stained with CBB. The protein bands that change with cold treatment are marked on the figure with closed circles.

abundant soluble proteins like the RuBisCO large subunit and chlorophyll a/b binding protein which are chloroplast proteins, were found. It might be difficult to detect leaf blade plasma membrane proteins in the absence of chloroplastmembrane contamination. On the other hand, glyceraldehyde-3-phosphate dehydrogenase has been im- munolocalizedoutside fungal cells and may be similarly localized in plants (Gozalbo et al., 1998).

Among the proteins found differentially regulated in rice leaf blades subjected to cold stress, several functionallyunknown proteins and the RuBisCO small subunit were detected. The RuBisCO small subunit is generally trans- lated asa precursor from a nuclear gene including a signal peptide for chloroplast targeting (Dean et al., 1985); however, theTargetP program (http://www.cbs.dtu.dk/ services/TargetP) predicted that the product of this cDNA, for example,the mature RuBisCO small subunit identified in this study, clearly has no signal peptide (Kawamura and Uemura,2003), suggesting that this pro- tein may remain in the cytoplasm and become associated with the plasma membraneduring cold stress.

Conclusions

Due to the wide variety of physiological and biochemical reactions carried out in different membrane systems, the

 proteomes of plant subcellular compartments should be fully described. This requires the ability to separate and purify

membranes successfully. However, no methods are currently available to purify particular membranes from plant cells to

homogeneity, and cross-contamination with other membranes and/or cytosolic constituents must be expected. One wayto circumvent this problem is to cor- relate the  presence of  particular proteins with the abundance of different cell

membranes and/or to confirm the location of  particular  proteins using complementary techniques such as fluorescence-tagging or immunochemical approaches.

 Numerous membrane-associated proteins in plants are still to be discovered and characterized (Ephritikhine et al.,2004). To facilitate proteomic analyses, more powerful

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 bioinformatics tools capable of analysing large sets of proteins have been developed, including tools devoted to the

systematic analysis of membrane protein structures (Schwacke et al., 2004). In other respects, the functional

characterization of proteomes still lacks completely accu- rate and reliable programmes for functional annotation,

especially in the case of rice membrane proteins. Progress should come from a combination of proteomic strategies

applied to membrane sub-proteomes. Taking into account the dynamic nature of membrane proteomes arising from the

diversity between organs and organelles, and the functional specificity of membrane systems, it is predicted that post-

translational modifications in response to stresses should bring the first clues about protein functions.

Analysis of membrane proteins remains a major chal- lenge for proteomics techniques based on 2D-PAGE. For 

this reason, alternative methods based either on the use of SDS–PAGE (Bell et al., 2001), or on the separation of 

digested peptides have been described (von Haller et al.,

2001; Wolters et al., 2001). Although these methods have been successful for the identification of membrane proteins, they do not achieve the combination of quantitative analysis

and degree of separation of protein variants available through the use of 2D-PAGE. The solubilizing power of various

nonionic and zwitterionic detergents as membrane protein solubilizers for  2D-PAGE has been reported (Luche et al.,

2003). These results suggest that additional strategies must be used in order to gain insight into the characteriza- tion of 

membrane proteins. Furthermore, many plasma membrane proteins are probably only expressed in certain cell types, at

discrete development stages, or in response to a particular stress. This dynamic nature means that the large majority of 

 plasma membrane proteins remain to be identified. In addition, the functions of many of these pro- teins are  presentlyunknown, many  proteins identified in the present study are functionally unclassified, and more than half of these are

 predicted to be integral membrane proteins.

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