Digging deeper into the plant cell wall proteome

Plant Physiology and Biochemistry 42 (2004) 979–988 www.elsevier.com/locate/plaphy

Review article

Digging deeper into the plant cell wall proteome Sang-Jik Lee a,b, Ramu S. Saravanan a, Cynthia M.B. Damasceno a, Hisayo Yamane a, Byung-Dong Kim b, Jocelyn K.C. Rose a,* b

a Department of Plant Biology, 228 Plant Science Building, Cornell University, Ithaca, NY 14853, USA Center for Plant Molecular Genetics and Breeding Research, Seoul National University, Seoul 151-742, South Korea

Received 30 August 2004; accepted 18 October 2004 Available online 19 December 2004

Abstract The proteome of the plant cell wall/apoplast is less well characterized than those of other subcellular compartments. This largely reflects the many technical challenges involved in extracting and identifying extracellular proteins, many of which resist isolation and identification, and in capturing a population that is both comprehensive and relatively uncontaminated with intracellular proteins. However, a range of disruptive techniques, involving tissue homogenization and subsequent sequential extraction and non-disruptive approaches has been developed. These approaches have been complemented more recently by other genome-scale screens, such as secretion traps that reveal the genes encoding proteins with N-terminal signal peptides that are targeted to the secretory pathway, many of which are subsequently localized in the wall. While the size and complexity of the wall proteome is still unresolved, the combination of experimental tools and computational prediction is rapidly expanding the catalog of known wall-localized proteins, suggesting the unexpected extracellular localization of other polypeptides and providing the basis for further exploration of plant wall structure and function. © 2005 Elsevier SAS. All rights reserved. Keywords: Plant cell wall; Secreted; Proteome; Signal sequence; Apoplast; Extracellular

1. Introduction The plant cell wall can be viewed as the structural matrix that encapsulates all plant cells, forming the mechanical framework that dictates plant architecture. In this context, plant walls are frequently categorized as either primary or secondary; the former being associated with young, growing and differentiating tissues and the latter with a terminal developmental phase in which cell size and shape are permanently established. However, a broader perspective of ‘the wall’, and one that is assumed in this review, is of a highly complex compartment that also comprises the middle lamella and apo-

Abbreviations: AGPs, arabinogalactan proteins; CDTA, 1,2-cyclohexanediaminetetraacetic acid; cTP, chloroplast transit peptide; 2DE, 2-dimensional gel electrophoresis; DTT, dithiothreitol; ER, endoplasmic reticulum; GFP, green fluorescent protein; GUS, b-glucuronidase; mTP, mitochondrial targeting peptide; PR, pathogenesis-related; Rubisco, ribulose bisphosphate decarboxylase/oxygenase; SP, secretory pathway signal peptide; YSST, yeast signal sequence trap. * Corresponding author. Tel.: +1-607-225-4781; fax: +1-607-255-5407. E-mail address: [email protected] (J.K.C. Rose). 0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.10.014

plastic continuum. The last decade or so has seen remarkable progress in not only elucidating the structure and organization of the extracellular matrix, but also in revealing the importance of the wall as the interface with the biotic and abiotic environment [33,74]. Moreover, research across the spectrum of plant biology is resulting in an ever-growing list of developmental processes that are directly or indirectly influenced by wall-localized molecular interactions and signaling pathways. Several cell wall-related websites are listed in Table 1 that provide examples of recent progress in elucidating plant wall structure and wall-related phenomena. It is therefore not surprising that a substantial portion of the plant proteome is localized in the cell wall/apoplast, although in comparison with the protein complements of other subcellular compartments, such as chloroplasts or mitochondria, the wall proteome has received far less attention and is less well defined. This review outlines some of the unique challenges that are encountered when studying the plant extracellular proteome, or ‘secretome’, and summarizes a range of experimental approaches that have been developed to address those chal-

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Table 1 Signal peptide prediction programs and plant cell wall-related web sites Software for predicting N-terminal signal peptides Programs Prediction TargetP 1.01 cTP, mTP or SP iPSORT cTP, mTP or SP SignalP 3.0 SP

URLs http://www.cbs.dtu.dk/services/TargetP http://www.hypothesiscreator.net/iPSORT/ http://www.cbs.dtu.dk/services/SignalP

Plant cell wall-related web sites Web site http://www.xyloglucan.prl.msu.edu/ http://www.cellwall.stanford.edu http://www.bio.psu.edu/expansins http://www.afmb.cnrs-mrs.fr/CAZY/ http://www.labs.plantbio.cornell.edu/XTH/ http://www.ccrc.uga.edu/~mao/cellwall/main.htm http://www.cellwall.genomics.purdue.edu/

Research target Plant cell wall biosynthesis research network Cellulose synthases and callose synthases Plant cell wall loosening proteins known as expansins Carbohydrate-active enzymes that degrade, modify, or create glycosidic bonds Xyloglucan transglucosylase/hydrolases The role of the cell wall in plant growth and development Cell wall biogenesis-related genes

References [21,54] [2] [5,54]

cTP, chloroplast transit peptide; mTP, mitochondrial targeting peptide; SP, secretory pathway signal peptide.

lenges. The many classes of cell wall-localized proteins are often described as belonging to one of two basic categories: so-called structural proteins, that are typically immobilized within the wall [76] and which comprise approximately 5–10% of the wall dry weight [12], or soluble apoplastic proteins, including many enzymes, that are usually more readily extracted from plant tissues. However, it is an oversimplification to use extractability to define the two categories of ‘wall proteins’ and ‘apoplastic proteins’. For example, arabinogalactan proteins (AGPs) are often described as structural proteins [76] but they are typically highly soluble and appear to associate only loosely with the wall [55]. In contrast, a-expansins are proteins with no proposed structural role that have several characteristics of enzymes and yet are relatively insoluble and resistant to extraction [15]. For the purpose of this review, ‘extracellular’, ‘apoplastic’ and ‘wallassociated’ are used as synonymous terms when describing proteins that are secreted from the protoplast, implying localization outside the plasma membrane and therefore a physical proximity to the wall, rather than only those with a direct or biologically significant interaction with the extracellular matrix itself.

2. The plant cell wall: a multifunctional subcellular compartment The perception of plant cell walls has evolved rapidly from early descriptions of inert, somewhat featureless boxes, to the currently accepted view of intricate highly dynamic structures with substantial spatial and temporal variation in architecture and composition. Similarly, the concept of a cell wall ‘proteome’ has matured from a time when it was sincerely doubted that plant cell walls might contain any protein, as elegantly reviewed in Lamport [42], to recent genome-scale assessments that predict the existence of many hundreds of extracellular proteins. The size and complexity of the wall proteome underlines its multifunctional nature and indeed, the tightly regulated expression of cell wall proteins has been

observed in association with a broad range of developmental events, metabolic processes and in response to many external stimuli. These include cell expansion and differentiation, where wall assembly, deposition, reorganization and selective disassembly require the activities of a complex battery of cell wall-localized enzymes including polysaccharide hydrolases, transglycosylases, lyases and other proteins that influence wall structure [16,25,70,71]. Additional enzymes, such as peroxidases, are believed to play a role in cross-linking polysaccharides at the cessation of expansion to rigidify the wall [1,63,66] Cell expansion is regulated by various environmental stimuli, such as light, gravity, anoxia, water stress, and hormones [33] and numerous reports describe correlations between these regulatory factors and changes in the structure of primary wall polysaccharides and the expression of wall-localized proteins, enzymatic activities or corresponding genes. As would be expected, a growing number of extracellular proteins are believed to play a crucial role in plant defense against microbes [19,34,74] and several studies have described changes in the population of secreted proteins in response to wounding [45], insect infestation [85], fungal or oomycete infection [31,34,52,58,67,92], fungal elicitors [53] and the defense-related hormone jasmonic acid [28]. These proteins include several of the well known pathogenesis-related (PR) proteins [79] that directly interact with pathogens, such as chitinases and endo-b-1,3-glucanases. However, plants also deploy a repertoire of proteins in the wall that act as a surveillance system to allow the early detection of an impending pathogen assault. An increasing number of extracellular proteins and peptides are being identified that contribute to signaling and recognition of not only pathogens [18,36,37,78,84], but also other cell types, such as proteins associated with pollen–pistil interactions [24,38,75,82,90]. Yet other apoplastic proteins mediate intercellular communication as part of developmental programs [11,44,48,49,62,69,86]. In addition to its important role as a protective barrier against microbial pathogens, the cell wall is also the site of many responses to environmental stresses. Quantitative or

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qualitative changes in cell wall-associated protein populations have been detected following exposure to ozone or sulfur dioxide [64], heavy metals [7,23,40], osmotic stress and water deficit [47] and cold stress or acclimation [46,80]. These examples represent only a subset of the developmental and environmental factors that are known to influence the protein composition of the apoplast and any perturbation of plant stasis appears likely to result in secretion or turnover of cell wall/extracellular proteins [7,33]. The apoplast is also an important compartment for the transport and delivery of ions, assimilates and other metabolites [13,43] and a range of wall-localized enzymes involved in the generation and translocation of assimilates have been described [10,56]. Fig. 1 summarizes some of the processes and physiological events that are associated with the plant cell wall. This list is not designed to be comprehensive, but rather to illustrate the diversity of the biology that underlies the wall/apoplast proteome, together with some specific examples of affiliated classes of wall-localized proteins.

3. Extraction and identification of cell wall proteins Substantial progress has been made over the last few years in characterizing a range of plant subcellular proteomes, such as those of chloroplasts and mitochondria, as detailed in two other reviews in this issue. In comparison, far less is known about the cell wall proteome, which likely reflects some major technical challenges that have to be surmounted. Firstly, and perhaps most importantly, unlike many intracellular or-

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ganelles, the cell wall is not conveniently bounded by a single discrete membrane, but is instead a continuum that extends throughout the plant corpus. An essential goal of subcellular fractionation prior to proteomic analysis is to capture the most comprehensive representation of the protein complement, whilst minimizing contamination with proteins from other subcellular locations. For this purpose, highly effective protocols have been developed to isolate intracellular organelles that typically involve controlled tissue disruption, followed by density gradient centrifugation to separate the spectrum of organelles into highly enriched fractions. In these cases the membrane acts both to contain the organellar proteins and to exclude extraneous protein contaminants. In contrast, any tissue disruption that compromises the integrity of the plasma membrane instantly leads to contamination of the cell wall fraction with intracellular proteins, many of which bind to the wall matrix with high affinity (see below). The second related technical hurdle is that proteins in the wall/apoplast exhibit a remarkably wide range of biochemical characteristics and affinities with the polysaccharide matrix. Many are highly soluble with no apparent interaction with the wall polysaccharides and are therefore combined with the cytosolic protein fraction during tissue homogenization and are effectively ‘lost’ from the wall extract. However, other walllocalized proteins, such as extensins [59,89], can be covalently linked into the cell wall architecture and are consequently highly resistant to extraction, even with harsh solvents. In addition, many wall proteins are glycosylated, which can complicate extraction, isolation and identification, while yet other extracellular proteins have domains that anchor them into the plasma membrane [9,20,60,81], or that span the membrane.

Fig. 1. Schematic representation of four adjacent plant cells, depicting the cell wall, middle lamella, apoplastic continuum and wall-localized proteins; some of which are freely soluble while others are covalently bound to the extracellular matrix or membrane-associated. Various cell wall functions and related processes are listed together with the names of some associated classes of proteins. The bottom right hand cell wall is shown as fragmented, representing degradation by pathogens: a process associated with numerous defense-related apoplastic proteins and a variety of signaling pathways.

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In conclusion, there is no one single protocol that can be used to extract all cell wall proteins and so techniques must be used that are tailored to a specific subset of proteins of interest. These can broadly be divided into two categories, disruptive and non-disruptive.

4. Non-disruptive approaches to isolate wall-localized proteins One strategy to obtain proteins that are localized in the apoplast/cell wall that minimizes contamination with proteins from within the protoplast involves the use of suspension cultured plant cells. Robertson et al. [68] used this system in a pioneering study of secreted proteins from five plant species that represented the first attempt to systematically separate and identify large numbers of cell wall-associated proteins. Protein populations were isolated from the suspension cell culture medium and from intact cells that were sequentially washed with a series of solvents that were designed to leave the plasma membrane intact. Specifically, the first solution was 200 mM calcium chloride, which has been shown to effectively liberate many wall-bound proteins, followed by 50 mM 1,2-cyclohexanediaminetetraacetic acid (CDTA) that might be expected to extract proteins that associate with pectins. The third buffer contained 2 mM dithiothreitol (DTT) to disrupt intermolecular disulfide bonds, followed by 1 M sodium chloride, to extract proteins that were strongly ionically associated with the wall, and finally a 200 mM borate buffer, which was included to perturb interactions between the wall and glycoproteins. The proteins were separated by 1-dimensional gel electrophoresis and more than 200 protein bands subjected to N-terminal amino acid sequencing, which yielded sequence information for approximately two thirds. Numerous families of known cell walllocalized proteins were identified, although a large proportion could not be assigned to a protein functional class based on sequence homology. In many cases, this probably reflected the scarcity of DNA sequence information for some of the species that were studied (e.g. carrot). Approximately 30% of the proteins from Arabidopsis could not be classified but since publication of this paper, the Arabidopsis genome sequence has become available. A ‘post-genome’ reevaluation of the published N-terminal sequences now allows a considerably greater proportion, but not all, of the proteins to be assigned a putative biochemical function (S.-J. Lee, J.K.C. Rose, unpublished data). The sequential washing approach was recently re-evaluated, using Arabidopsis suspension cells, in order to assess the likely degree of contamination with intracellular proteins and to identify additional loosely bound wall proteins [8]. The authors used two basic protocols to extract the proteins: one being essentially the same extractant series as that described in Robertson et al. [68] and the other a variant that included 2 M lithium chloride but omitted the calcium chloride wash. This was coupled with microscopic analysis to evaluate the integrity of the intra-

cellular compartments. It was concluded that the calcium chloride and chelator extractions were particularly likely to induce contamination of the wall-bound protein population with intracellular proteins. Furthermore, it was concluded that the cellular integrity deceased substantially after more than two sequential extractions. Since the plasma membrane is easily ruptured, great care must be taken, even when using this supposedly non-destructive approach. The authors also noted that individual protein isoforms were not exclusively eluted in one solvent, which further complicates analysis. Contamination with intracellular proteins was also observed in a study of cell wall-associated proteins in the green algae Haematococcus pluvialis that also used sequential washing of the cell walls [87], again using the same reagents as described in Robertson et al. [68]. A similar strategy has been used to study secondary cell wall synthesis using tobacco cells expressing high cytokinin levels [6], which consequently have highly thickened cell walls and exhibit many of the expected characteristics of cells that are actively synthesizing secondary walls. The spectrum of proteins that were extracted from the walls of this tobacco line, with either 200 mM calcium chloride or 40 mM CDTA, appeared substantially different from that seen in the equivalent study of tobacco primary wall proteins reported by Robertson et al. [68]. While many new proteins were identified, other sequences indicated the presence of proteins that are related to secondary wall formation, such as peroxidase and polyphenol oxidase/laccase, a lysine-rich protein and extensin. A related study of proteins in the culture medium and cell wall fractions of Norway spruce suspension cells that were actively synthesizing lignin, also identified proteins associated with secondary wall formation [39]. Suspension cultured cells also represent a convenient experimental system in which to study processes such as plant defense, since stimuli may be readily applied and studies of secreted protein populations have been described following the application of fungal elicitors [53] and to identify new secreted PR proteins [57] from Arabidopsis and tobacco, respectively. The use of cell cultures has also been used to study cell wall protein populations in the fungus Candida albicans [65] and cell wall construction and reorganization in yeast by identifying the proteins that are secreted into the culture medium of Saccharomyces cerevisiae protoplasts that are actively regenerating a cell wall [61]. The authors reported the identification of several proteins that are known to participate in wall construction. Similar proteomic analyses of wall assembly during cell wall regeneration in plants have yet to be reported. While suspension cells provide a useful source of homogenous plant material that can be rapidly regenerated and from which cell walls and wall proteins may easily be obtained, they represent an artificial biological system. The complement of wall proteins in intact complex plant tissues is likely to be significantly different, given the associated cellular heterogeneity, and to exhibit substantial spatial and temporal vari-

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ability. Different tissues and cell types will have distinct subsets of wall proteins with diverse functions and this variability will emerge through careful proteomic analysis of subtypes of plant material. Similarly, little is currently known about the dynamic aspects of wall protein populations during cell growth and differentiation, although some preliminary results have recently started to emerge. For example, an examination of cell wall-associated proteins from the developing xylem of compression and non-compression wood of Sitka spruce [50] resulted in the identification of several differentially expressed proteins, including oxidases that may contribute to secondary wall formation. An alternative experimental approach to isolate cell wall/apoplastic proteins from complex tissues, and one that can be adapted to minimize contamination with cytosolic proteins, is to use pressure-rehydration and vacuum infiltration protocols to extract the apoplastic fluid from the target sample. This approach has been used successfully to extract extracellular proteins from several tissue or organ types, including roots, leaves, stems, fruit, tubers, xylem and phloem [7,29,31,32,40,58,67,72,73,91]. In addition, a range of solutions can be infiltrated into the tissues to release different subsets of proteins, such as buffers containing relatively highsalt concentrations that liberate proteins that are ionically bound to the wall. The disadvantages of this technique are that the protein yield is typically low and great care has to be taken to avoid cell lysis. Consequently, throughput is generally slow and many wall-localized proteins cannot be recovered without rupturing the plasma membrane. Assessment of contamination is an important factor that should ideally be assessed to obtain an estimation of purity or enrichment. Two common ways to do this are to assay the enzyme activity of a cytosolic protein in the apoplastic protein extracts, or to perform a Western analysis with an antiserum to an intracellular protein. In some plant tissues, such as leaves, the plastidlocalized protein ribulose bisphosphate decarboxylase/ oxygenase (Rubisco) is highly abundant and readily identifi-

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able when protein extracts are separated on an acrylamide gel if substantial contamination has occurred. Fig. 2 shows a 2-dimensional gel electrophoresis (2DE) analysis of total protein extracts (Fig. 2A) and apoplast protein extracts (Fig. 2B) from tomato leaves. The large subunit of Rubisco is shown boxed in panel A and while it represents the most abundant protein in the total protein sample, it is barely detectable in the apoplastic protein extract. This was also verified by Western analysis using an anti-Rubisco antibody. Since the patterns of protein spots in the two gels are very different, with few spots in common, this further indicates a successful enrichment with minimal contamination. 5. Disruptive approaches to isolate wall-bound proteins Many reports describe the isolation of a “cell wall protein fraction” by tissue homogenization in a buffer containing a low-salt concentration, followed by sequential washing of the cell wall pellet with a low-salt buffer to remove cytosolic protein contaminants and then with solutions containing high concentrations of salt (e.g. 1.5 M NaCl) to release proteins that are ionically bound to the wall. However, since the polygalacturonate component of cell wall pectin can essentially act as a polyanionic matrix, positively charged proteins from within the protoplast have the potential to bind to the wall once the plasma membrane has been ruptured, contaminating the sample. In some cases a cytosolic protein can associate so strongly with the wall that a high-salt buffer does not disrupt the interaction and a detergent such as SDS is subsequently required to re-solubilize the protein from the wallenriched pellet (R.S. Saravanan, J. Rose, unpublished data). Therefore, considerable caution should be used when classifying proteins that are isolated using this disruptive technique as cell wall proteins and ideally other approaches should be used to verify their subcellular localization. This type of strategy was used in an analysis of wallassociated proteins extracted from Arabidopsis thaliana sus-

Fig. 2. Two-dimensional gel electrophoretic analysis of apoplastic fluid. The two gels show a total protein extract of homogenized tomato leaves (A) or proteins in the apoplastic fluid (B). The boxed abundant protein spots in (A) correspond to the large subunit of Rubisco. The proteins were focused in the first dimension using a pI range of 4–7 and the second dimension gels were stained with colloidal Coomassie blue.

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pension cells [14], where a cell wall fraction was removed from disrupted cells and sequentially extracted with calcium chloride and urea. The authors used 2DE followed by mass spectrometry analysis to identify 69 different proteins, which included numerous known wall proteins with well-established biochemical functions, a number of unclassified proteins and several polypeptides whose location in the wall was unexpected. These latter two classes of proteins demonstrate the potential value of this approach for identifying new cell wall/apoplastic proteins and wall-localized biochemical pathways. Such experiments will provide a platform for subsequent functional studies. The authors also acknowledged the possibility of contamination of the cell wall protein fraction and the need for additional confirmatory analyses using techniques such as immunolocalization [77]. A similar analysis was recently reported of cell wallassociated proteins from alfalfa stems [88], where about 100 proteins were identified from LiCl and CaCl2 extracts. Many corresponded to known extracellular proteins with well defined biochemical functions, such as wall modification or defense; however, others were termed non-classical wall proteins, in that they are not typically thought to be secreted. Such identification of supposedly intracellular proteins in wall extracts is not unique [14,39] and the authors presented a thoughtful discussion of the potential for contamination, which was suggested by the detection of Rubisco polypeptides in the gels, versus the possibility of proteins with multiple functions localized in different subcellular compartments. This disruptive “grind and find” approach is clearly effective for many classes of wall proteins, particularly those that are more tightly associated with the extracellular matrix. However, ideally, contamination with non-wall localized proteins should be rigorously assessed, as has been described in analyses of Arabidopsis suspension cells [3,14]. The in vivo localization should subsequently be confirmed using other techniques, such as immunolocalization or green fluorescent protein (GFP)-fusion protein analysis.

6. Additional approaches to dissect the cell wall proteome In addition to the more conventional strategies to characterize the cell wall proteome, involving protein extraction, gel-based separation and identification by mass spectrometry, a number of genome-scale screens have been developed in the last few years that either directly or indirectly reveal populations of secreted proteins. For example, highthroughput screening of plant protein subcellular localization has been described following the fusion of plant cDNA libraries with the DNA sequence encoding GFP and screening transformed plants by fluorescence microscopy. The first such large-scale study in plants was performed with cDNAs from Arabidopsis seedlings and callus and was designed such that GFP was expressed at the N-termini of the fusion pro-

teins [17]. Consequently, the native N-terminal targeting sequences that are required for entry into the secretory pathway were masked and cell wall-localized proteins were not identified. A more recent report used both 5′- and 3′-GFP fusions with a cDNA library from Nicotiana benthamiana roots in a screen that revealed a number of new extracellular proteins, as well as details of localization of certain proteins in specific microdomains within the wall matrix [22]. A more focused approach to identify extracellular proteins involves “secretion traps” that specifically target secreted and plasma membrane-spanning proteins. Groover et al. [27] described an ingenious strategy that involved the analysis of Arabidopsis transposon insertion lines expressing b-glucuronidase (GUS):protein fusions. If the fusion proteins are routed through the secretory pathway, they can be identified by growing the plants in the presence or absence of a glycosylation inhibitor, since glycosylation of GUS in the endoplasmic reticulum (ER) alters GUS enzyme activity. This analysis allowed the identification of a range of both known and new wall-localized proteins and membrane-bound proteins, in addition to revealing their expression patterns in specific tissues. Another high-throughput screen that targets secreted proteins is termed the yeast signal sequence trap (YSST) [35,41,83]. Fig. 3 summarizes this approach, which involves ligating cDNAs in frame at the 5′ end of the DNA sequence encoding a truncated yeast invertase gene without the initiator methionine and signal peptide. This library is then transformed into an invertase-deficient yeast mutant, which is grown on a medium with sucrose as the sole carbon source. Any yeast transformant containing a plant-derived cDNA encoding a N-terminal secretory pathway signal peptide (SP) sequence for targeting to the ER and the secretory pathway has the potential to secrete the polypeptide as an invertase fusion protein, resulting in reconstitution of extracellular invertase activity and the rescue of the mutant. Plates of yeast transformants containing constructs with or without an N-terminal SP are shown in Fig. 3A. This secretion trap has now been used to identify substantial populations of genes encoding secreted plant proteins [4,26,34] and while it has some limitations, such as redundancy and a low level of false positives, it represents an effective and rapid screen that importantly allows the identification of some wall-localized proteins that are not found by traditional protein extraction techniques.

7. Concluding remarks A range of experimental approaches and tools has now been developed that is incrementally revealing the complexity and dynamics of the cell wall proteome. It is worth noting that most studies of the wall protein populations, whether isolating and sequencing native proteins from plant tissues, or performing secretion trap screens, result in the identification of proteins that are not generally thought to be secreted. In

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Fig. 3. Summary of the YSST screen for secreted proteins. A, invertase-deficient mutant yeast strains were transformed with a plasmid containing a gene encoding a protein without (left plate) or with (right plate) a secretion SP, fused to invertase, as shown in the cartoon above the photographs. Many distinct colonies are visible on the right plate but no colonies are present on the left. B, schematic diagram of the steps involved in the YSST screen. When used in combination with a computational screen, this approach can be a highly effective means to survey a population of secreted proteins. In some cases, additional verification may be desirable, either by retransformation into yeast or by using other approaches such as immunolocalization or GFP analysis.

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some cases, as described above, this may be attributed to contamination, or an artifact of the screen; however, in other cases these may represent legitimate localization. Indeed, rapidly increasing numbers of proteins and members of protein families are being identified that show targeting to multiple subcellular compartments [30,51]. Another common observation from the wall protein isolation studies and secretion trap screens is that in a significant proportion of cases, proteins are identified that are not predicted to be secreted by the webbased programs that are used to determine the presence of an N-terminal signal sequence or to suggest subcellular localization (listed in Table 1). These results may be interpreted to reflect the contamination issue, but another explanations is that there exist currently unknown non-classical secretory signal sequences and pathways, as has been shown in yeast and bacteria (described in [14]), but that has yet to be demonstrated in plants. However, another factor that most certainly explains the paradoxical localization in many cases is simply that prediction software occasionally incorrectly assigns location. For example, we have identified a number of cases in which existing software incorrectly distinguishes between chloroplast transit peptides (cTP) and N-terminal signal sequences (S.-J. Lee, J.K.C. Rose, unpublished data). While prediction programs are a valuable tool to help suggest the subcellular destination of a protein, they are no more than this and should be used judiciously and the predictions should not taken as incontrovertible evidence. Improvements to the prediction algorithms are likely to result from larger training data sets, such as can be derived from the secretion trap screens (as indicated in Fig. 3). Unambiguous demonstration of protein localization is a challenging goal as each technique has specific limitations, whether immunolocalization or GFP-fusion studies. While progress is incremental, it appears that the combination of complementary screens, protein isolation and in silico analysis is generating a more comprehensive catalog of the wall proteome, although it difficult to estimate how many wall proteins remain to be identified. Future milestones will include developing a picture of protein dynamics and localization within individual walls and wall micro-domains, the identification of protein complexes and interaction networks in the wall/apoplast and an assessment of post-translational modification. Doubtless new technologies and techniques will continue to emerge that will propel the field towards achieving these goals and will continue to provide remarkable insights into cell wall structure and function.

Acknowledgements

United States Department of Agriculture (2001-521001137). Dr. Carmen Catalá and Dr. Montserrat Saladié are thanked for their helpful suggestions and criticism.

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Support in this research area was provided by a JSPS Postdoctoral Fellowship for Research Abroad to H.Y. from the Japan Society for the Promotion of Science, by a grant from KOSEF to S.J.L. and B.D.K. of the Center for Plant Molecular Genetics and Breeding Research and by grants to J.K.C.R. from the National Science Foundation (IBN-009109) and the

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