This Article Abstract Full Text (PDF) All Versions of this Article: 140/1/311    most recent pp.105.070219v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (39) Citing Articles via Google Scholar Google Scholar Articles by Zhu, J. Articles by Sharp, R. E. Search for Related Content PubMed PubMed Citation Articles by Zhu, J. Articles by Sharp, R. E. Agricola Articles by Zhu, J. Articles by Sharp, R. E.

DEVELOPMENT AND HORMONE ACTION

Cell Wall Proteome in the Maize Primary Root Elongation Zone. I. Extraction and Identification of Water-Soluble and Lightly Ionically Bound Proteins¹

Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211 (J.Z., R.E.S.); Donald Danforth Plant Science Center, St. Louis, Missouri 63132 (S.C., S.A., V.S.A., D.P.S.); and Department of Plants, Soils and Biometeorology, Utah State University, Logan, Utah 84322 (Y.W.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Cell wall proteins (CWPs) play important roles in various processes, including cell elongation. However, relatively little is known about the composition of CWPs in growing regions. We are using a proteomics approach to gain a comprehensive understanding of the identity of CWPs in the maize (Zea mays) primary root elongation zone. As the first step, we examined the effectiveness of a vacuum infiltration-centrifugation technique for extracting water-soluble and loosely ionically bound (fraction 1) CWPs from the root elongation zone. The purity of the CWP extract was evaluated by comparing with total soluble proteins extracted from homogenized tissue. Several lines of evidence indicated that the vacuum infiltration-centrifugation technique effectively enriched for CWPs. Protein identification revealed that 84% of the CWPs were different from the total soluble proteins. About 40% of the fraction 1 CWPs had traditional signal peptides and 33% were predicted to be nonclassical secretory proteins, whereas only 3% and 11%, respectively, of the total soluble proteins were in these categories. Many of the CWPs have previously been shown to be involved in cell wall metabolism and cell elongation. In addition, maize has type II cell walls, and several of the CWPs identified in this study have not been identified in previous cell wall proteomics studies that have focused only on type I walls. These proteins include endo-1,3;1,4--D-glucanase and -L-arabinofuranosidase, which act on the major polysaccharides only or mainly present in type II cell walls.

Previous studies with maize (Zea mays) primary roots have indicated that CWPs play an important role in controlling cell wall extensibility and thus cell expansion rate. Spatial analysis of the profile of elongation rate within the root elongation zone under well-watered conditions revealed that variation in cell wall extensibility is a key factor in determining the rate of elongation because turgor pressure is relatively constant along the root (Spollen and Sharp, 1991). Under water stress, elongation rates are maintained in the apical region of the elongation zone despite reduced turgor pressure (Sharp et al., 1988; Spollen and Sharp, 1991). These results suggested that water stress results in an increase in cell wall extensibility in the apical region, which was confirmed by direct measurement of acid-induced extension properties (Wu et al., 1996). Further studies demonstrated that activities of two CWPs, expansins and xyloglucan endotransglycosylase, were increased specifically in the apical few millimeters of water-stressed compared to well-watered roots (Wu et al., 1994, 1996), providing a biochemical basis for the increase in cell wall extensibility.

To gain a more comprehensive understanding of the involvement of CWPs in controlling cell elongation in the maize primary root, we have initiated a proteomics project to identify CWPs from the root elongation zone. CWPs can be divided into three major categories based on their chemical and physical association with cell walls and available extraction methods: water-soluble and loosely ionically bound (fraction 1), tightly ionically bound (fraction 2), and covalently bound (fraction 3) CWPs. As the first step, we studied fraction 1 CWPs from the elongation zone of well-watered roots.

Fraction 1 CWP-related proteomics analysis has been performed on cultured cells of dicot species (Robertson et al., 1997; Borderies et al., 2003). The proteins were extracted by washing cultured cells with buffer or low concentrations of salt solutions. The use of cultured cells is advantageous for the convenience of protein extraction and the simplicity of protein composition from undifferentiated cells. However, root elongation depends on the coordinated regulation of growth processes in differentiated cell types within a complex of tissues. The method used for extracting fraction 1 CWPs from cultured cells is not suitable for complex tissues. A method involving vacuum infiltration with buffer containing a low concentration of salts followed by low-speed centrifugation was developed to collect CWPs from living tissues (Terry and Bonner, 1980). The vacuum infiltration-centrifugation technique has been used to isolate apoplastic fluid with little detectable cytosolic contamination (assessed from the activity of cytosolic marker enzymes) from tissues of leaves, stems, and roots (Morrow and Jones, 1986; MacAdam et al., 1992a, 1992b; Cordoba-Pedregosa et al., 1996; de Souza and MacAdam, 2001). However, previous studies of fraction 1 CWPs from tissues have mostly focused on a single protein or protein class, such as peroxidases. Two recent reports used the technique to examine the cell wall proteome of leaf tissues (Boudart et al., 2005; Dani et al., 2005), but to our knowledge no proteomics studies have focused on roots or specifically on growing regions.

In this study, we examined the effectiveness of the vacuum infiltration-centrifugation technique for extracting fraction 1 CWPs from the maize primary root elongation zone. CWPs were analyzed using a proteomics approach to identify the protein composition and also to use this information to evaluate the purity of the fraction 1 CWP population. Maize plants have type II cell walls due to a different polysaccharide composition from the walls of dicot and most monocot plants, which have type I cell walls (Fry, 1988; Carpita and Gibeaut, 1993). To our knowledge, no proteomics work has been reported to address the protein composition in type II cell walls. Thus, this study also provides valuable information on the differences in CWP composition between cell wall types.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Optimization of the Vacuum Infiltration-Centrifugation Method for Extracting Fraction 1 CWPs from the Root Elongation Zone

To effectively extract fraction 1 CWPs from the root elongation zone, we first tested the effect of different concentrations of KCl, as an extractant, on CWP yield and cytosolic protein contamination. Because the yield of CWPs proved to be very low, the feasibility and efficiency of sequential extractions from the same tissue were examined. In these initial tests, cytosolic protein contamination in the extracts was assessed by assay of Glc-6-P dehydrogenase (G6PDH) activity. Infiltration with 0.2 M KCl yielded a similar amount of protein in each of three sequential extractions, without detectable cytosolic contamination (Table I). Decreasing the KCl concentration below 0.2 M greatly reduced the extraction efficiency (Fig. 1 ). Infiltration with 0.4 M KCl yielded a greater amount of protein, but cytosolic contamination was detected after the first infiltration (Table I). Further increase in KCl concentration to 1.0 or 1.5 M decreased protein yield. This may have resulted from shrinkage (due to dehydration) of the cell walls and thus a reduction in wall pore sizes that hindered protein release.

View larger version (11K): [in this window] [in a new window]   Figure 1. Effect of KCl concentration on the yield of fraction 1 CWPs from the root elongation zone. Fraction 1 CWPs were extracted from elongation zone segments (approximately 100 segments/replicate) by vacuum infiltration with different concentrations of KCl solution followed by low-speed centrifugation. Three sequential extractions of the same samples were combined. Data are means ± SE of four replicates. No G6PDH activity, a marker of cytosolic contamination, was detected in any of the samples.

Two-Dimensional Gel Analysis and Protein Identification of Fraction 1 CWPs and Total Soluble Proteins

To analyze and identify fraction 1 CWPs, proteins were extracted from the root elongation zone using the infiltration-centrifugation method. Three replicate samples of 50-µg CWPs (extracted from approximately 100 12-mm segments per sample) were collected in independent experiments and separated by two-dimensional electrophoresis (2-DE). One gel was stained with Coomassie Blue (Fig. 2A ) and the others with SyproRuby (for increased staining sensitivity; data not shown). The total number of spots on the gels varied depending on the staining method, from 149 with Coomassie staining to 225 with SyproRuby staining. Of the 149 spots on the Coomassie-stained gel, 144 were present on both of the SyproRuby-stained gels. For comparison, three replicate samples of 50 µg of total soluble proteins extracted from the elongation zone after tissue homogenization were separated by 2-DE and stained with Coomassie Blue. One hundred thirty-six spots were common to the three replicates; a representative 2-DE gel image is shown in Figure 2B.

View larger version (30K): [in this window] [in a new window]   Figure 2. Images of 2-DE gels for fraction 1 CWP (A) and total soluble proteins (B). Fifty micrograms of fraction 1 CWPs or total soluble proteins from the root elongation zone were separated on each gel. Gels were stained with Coomassie Blue and processed by PDQuest 7.2 (Bio-Rad). The 53 most abundant spots in A and 42 of the most abundant spots (randomly selected) in B were labeled and picked for MS analysis.

View larger version (47K): [in this window] [in a new window]   Figure 3. Mass spectra of two selected protein samples. Top, Nanoelectrospray quadrupole TOF MS of tryptic peptides from spot 11 in Table II. TOF MS precursor ion spectrum shows all the peptide ions at different charge states present in the tryptic digest. Two representative product ion spectra of m/z 492.7999 and m/z 563.8418 and their derived sequences are shown as insets. Database searching using the MS/MS data unambiguously identified the spot as chain B of -glucosidase with a score of 434 and a sequence coverage of 16%. Bottom, MALDI-TOF MS of tryptic peptides eluted from spot t5 in Figure 2B. After baseline correction, noise removal, and peak deisotoping, 42 ions were submitted to Protein-Prospector. Twenty-four of the submitted ions were matched to theoretical tryptic peptides from aconitate hydratase (inset).

The difference between the two profiles was also obvious when the number of proteins carrying predicted signal peptides that lead to protein targeting into the secretory pathway was analyzed (Nielsen et al., 1997). Seventeen of 43 protein identifications from fraction 1 CWPs had signal peptides (Table II), whereas only one protein from the total soluble protein gel had a signal peptide (Table III). Increasing evidence indicates that many proteins lacking signal peptides can be secreted into the extracellular matrix through an unknown mechanism. A program was recently developed to predict these so-called nonclassical secretory proteins for the mammalian system (Bendtsen et al., 2004). We explored the use of this program to analyze plant proteins. Fourteen of 26 proteins from the CWP gel that did not contain signal peptides were predicted to be nonclassical secretory proteins (Table II), whereas only four of 35 proteins from the total soluble protein gel were identified as nonclassical secretory proteins (Table III), further illustrating that these two protein populations were different.

To identify CWP of lower abundance, 150 µg of fraction 1 CWPs (combined from three independent experiments) were extracted from the root elongation zone and analyzed by 2-DE (stained with Coomassie Blue; Fig. 4 ). Image comparison between the 50- and the 150-µg CWP gels showed that all 53 of the spots on the 50-µg gel that were selected for protein identification matched the spots on the 150-µg gel. Because of the high reproducibility of the CWP gel pattern and the large amount of work involved in collecting CWPs, the 150-µg CWP gel was only performed once. Thirty-five protein spots with reasonable staining intensity that were not identified from the 50-µg CWP gel were excised for MS analysis, resulting in 31 protein identifications with high confidence (Table IV). All of these spots were also present on both of the 50-µg SyproRuby-stained CWP gels (note that because of the lower sensitivity of protein staining with Coomassie Blue, not all of these spots are present on the 50-µg CWP gel in Fig. 2A). Among the 31 additional proteins identified, five had signal peptides and 12 were predicted to be nonclassical secretory proteins (Table IV). However, some cytosolic proteins were found on the 150-µg gel, including nuclear transport factor 2, mitochondrial chaperonin-60, actin depolymerization factor, and cytosolic glyceraldehyde-3-P dehydrogenase (GAPC3).

View larger version (19K): [in this window] [in a new window]   Figure 4. Image of 2-DE gel loaded with 150 µg of fraction 1 CWPs from the root elongation zone. The gel was stained with Coomassie Blue and processed by PDQuest 7.2 (Bio-Rad). Thirty-five spots with reasonable intensity that were not identified on the 50-µg CWP gel (Fig. 2A) were labeled and picked for MS analysis.

To better understand the biological processes encompassed by the proteins identified using the 2-DE proteomics approach, fraction 1 CWPs and total soluble proteins were classified in functional categories (Fig. 5 ). It should be noted that these classifications are provisional because the biological role of many of the proteins identified has not been established experimentally (Tatusov et al., 1997). The largest proportion of the CWPs, 38%, was categorized in carbohydrate metabolism. In comparison, 24% of the total soluble proteins were in this category. For the CWPs, the second largest group (21%) was related to defense mechanisms, whereas only 2.8% of the total soluble proteins were in this category. A small number of the CWPs (7.0%) were classified as functionally unknown. The categories of total soluble proteins included translation, ribosomal structure and biogenesis, cytoskeleton, coenzyme transport and metabolism, lipid transport and metabolism, and RNA processing and modification. Proteins in these categories were not present in fraction 1 CWPs.

View larger version (55K): [in this window] [in a new window]   Figure 5. Comparison of the functional classifications of the 43 identified proteins from the 50-µg fraction 1 CWP gel (A; Table II) and 36 of the most abundant total soluble proteins (B; Table III) as determined using the KOG classification.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

One of the great challenges when working with the subproteome in cell walls is to isolate proteins free of contamination from membrane and cytosolic proteins. In this study, the vacuum infiltration-centrifugation technique was optimized to isolate the fraction 1 CWPs from the maize primary root elongation zone. Several lines of evidence indicate that this method can effectively enrich for CWPs. First, the infiltrate showed no detectable enzymatic activity of G6PDH, a cytosolic protein marker. Second, analyses of the gel images (Fig. 2) and protein identities (Tables II and III) showed that the protein profile of the CWP extract was clearly different from that of the total soluble protein extract. Only about 16% of the proteins identified from the 50-µg fraction 1 CWP gel overlapped with those from the total soluble protein gel. In addition, the differences in protein functional categories (Fig. 5), such as lacking translation and cytoskeleton groups in fraction 1 CWPs, further indicated the effectiveness of the extraction method. Third, about 40% of the proteins identified from fraction 1 CWPs have an N-terminal signal peptide, whereas only 3% of the proteins identified from the total soluble protein extract have signal peptides. Fourth, the majority of signal peptide-carrying proteins from the 50-µg fraction 1 CWP gel are related to cell wall structure, metabolism, and modification, including -xylosidase, -D-glucan exohydrolase, -L-arabinofuranosidase, -galactosidase, -galactosidase, endoxyloglucan transferase, chitinase, endo-1,3;1,4--D-glucanase, and peroxidases (see Fry, 1988). Many of these proteins have also been isolated from cell walls using other approaches (Huber and Nevins, 1981; MacAdam et al., 1992a, 1992b; Nishitani and Tominaga, 1992; Kim et al., 2000), supporting their identification as true CWPs. None of these proteins appeared in the total soluble protein list.

In contrast to the major differences between the CWP and total soluble protein gels, highly similar profiles were observed on replicate CWP gels. Since the replicate samples were extracted from independent experiments, the results indicate a high reproducibility of the seedling culture system, the extraction protocol, and 2-DE. The reproducibility of CWP identification was also rigorously examined in this study. Seventeen protein spots were identified individually from three independent samples using MS analysis, and 94% of the proteins were reproducibly identified.

Cytosolic protein contamination was not obvious from the identities of the proteins from the 50-µg CWP gel (Table II). However, some cytosolic contamination was evident from the additional 31 spots identified from the 150-µg CWP gel (Table IV). Several proteins were annotated as cytosolic proteins, such as cytoplasmic malate dehydrogenase, nuclear transport factor 2, and mitochondrial chaperonin. It is possible that cytosolic protein contamination occurred on the 50-µg gel also, but in very low quantities that were not detected. However, when the gel was loaded with a larger amount of protein, the contamination became sufficient to be detected. It is also possible that the apparent cytosolic proteins could in fact be true CWPs for reasons discussed below.

Fraction 1 CWPs Include Proteins without a Traditional Signal Peptide Sequence

A high percentage of the identified CWPs from the 50-µg gel (26/43; 60%), and an even higher percentage of the additional proteins identified from the 150-µg gel (26/31; 84%), did not appear to contain an N-terminal signal peptide. This seems to be common based on other cell wall proteomics studies (Chivasa et al., 2002; Borderies et al., 2003; Canovas et al., 2004; Slabas et al., 2004; Watson et al., 2004). Increasing evidence suggests that proteins can be secreted into cell walls without a classic N-terminal signal peptide. For example, malate dehydrogenase, which is proposed to be involved in H₂O₂ production in cell walls (Gross, 1977; Fry, 1988), and -glucosidase, both of which were identified in this study, were found in apoplastic fluid in barley (Hordeum vulgare) and oat (Avena sativa) primary leaves (Li et al., 1989). Enolase, which was also identified in this study, was detected in the cell walls of Candida albicans, Arabidopsis, and alfalfa (Medicago sativa) (Chivasa et al., 2002; Pitarch et al., 2002; Watson et al., 2004). Using immunolocalization, enolase was shown to be secreted to the cell wall or extracellular space even though it lacked a signal peptide (Edwards et al., 1999). Glyoxalase 1 was identified in our study and was also present in a cell wall proteomics study of mature stems of alfalfa (Watson et al., 2004).

In mammalian systems, it has also been observed that many proteins that do not contain signal peptides can be secreted into the extracellular matrix. Bendtsen et al. (2004) have classified this group of proteins as nonclassical or leadless secretory proteins and have developed a program (SecretomeP 1.0b; http://www.cbs.dtu.dk/services/SecretomeP-1.0) based on a group of known extracellular localized proteins to predict this type of protein. Using this software with the proteins identified from the CWP gels that did not contain signal peptides revealed that 54% (14/26) from the 50-µg gel and 46% (12/26) from the 150-µg gel were predicted to be nonclassical secretory proteins. Interestingly, malate dehydrogenase, enolase, and -glucosidase were included in this group of proteins, suggesting that this software could potentially be used for predicting nonclassical secretory proteins in plants. In contrast, only four of 35 proteins identified from the total soluble protein gel were predicted to be nonclassical secretory proteins.

Twelve proteins identified from the 50-µg CWP gel did not have a signal peptide sequence nor were they predicted to be nonclassical secretory proteins. However, they could still be true CWPs because the protein identification process currently available has limitations for distinguishing family members. Most CWPs belong to multiprotein families, and proteins in the same family can have different cellular localizations. The 14-3-3 proteins were thought to be cytosolic or organelle-localized proteins involved in transcriptional regulation of ATP synthesis in plants (Voigt and Frank, 2003). Recently, it was found that some 14-3-3 proteins are constituents of the insoluble glycoprotein framework of Chlamydomonas cell walls (Voigt and Frank, 2003). In another example, a citrate synthase (At2g44350) was identified in a cell wall proteomics study, but was annotated as a mitochondria or peroxisome or glyoxysome protein (Slabas et al., 2004). By analyzing all the members in the citrate synthase family in Arabidopsis, Slabas et al. (2004) found one of the members (At3g58750) not only contained an N-terminal signal peptide but also contained peptide sequence information for peroxisome targeting, indicating the proteins can be targeted to two different locations. Due to the limitation of current protein identification technology in distinguishing slight differences among members in the same protein family, it is possible that At2g44350 identified in the proteomics work was actually At3g58570 (Slabas et al., 2004). It is even more challenging to work with maize proteins because the maize genome information is incomplete. Accordingly, the CWP identifications in this study, based on limited peptide sequences, may have matched proteins in the database that represent other members in the protein families.

Fraction 1 CWPs from Maize Roots Include Proteins Associated with Type II Cell Walls

Maize plants have type II cell walls (Fry, 1988; Carpita and Gibeaut, 1993) and are expected to have differences in composition and abundance of CWPs from type I cell walls. This study confirmed this notion by identifying nine proteins (indicated by asterisks in Table II) that were not reported in previous cell wall proteomics studies, which have focused only on type I walls. (Additional potentially novel CWPs have not been indicated in Table IV because of the indication of greater cytosolic contamination on the 150-µg CWP gel.) These proteins include endo-1,3;1,4--D-glucanase and -L-arabinofuranosidase, both of which were reproducibly identified (Table V). -1,3;1,4-Mixed-linkage glucan is a unique polysaccharide for type II cell walls of grass species and is considered to be of major importance in cell wall metabolism or modification (Buckeridge et al., 2004). In maize coleoptiles or whole seedlings, it was found that mixed-linkage glucan was developmentally regulated and was associated with the cell elongation process (Carpita, 1996; Kim et al., 2000). Another major component in type II primary cell walls, but a minor fraction in type I cell walls, is glucuronoarabinoxylan (GAX), which can make up 30% of the wall mass (Fry, 1988). GAXs are considered as the counterpart of xyloglucan molecules in type I cell walls and the primary microfibril-tethering molecules in type II cell walls (Carpita, 1996; Buckeridge et al., 2004). -L-Arabinofuranosidase is probably one of the major enzymes responsible for cleavage and modification of GAX (Fry, 1988). In contrast, no major pectin-related CWPs were identified in this study, while the pectin-related enzymes were always identified as one of the major constituents in cell wall proteomics work with type I cell walls. This finding is in line with the fact that pectin is a minor component in type II cell walls, whereas pectin can make up >30% of cell wall mass in type I cell walls (Fry, 1988; Carpita et al., 2001).

Involvement of Fraction 1 CWPs in Cell Elongation

Many of the CWPs identified in this study have been shown to be involved in the cell elongation process in maize or rice (Oryza sativa) coleoptiles. Both exo- and endoglucanase activities were found to be associated with cell walls of maize seedlings (Huber and Nevins, 1981; Kim et al., 2000). Exogenous application of an exo--D-glucanase (probably the -D-glucan exohydrolase in this study) purified from cell walls of maize coleoptiles was capable of inducing cell elongation in the same tissue (Labrador and Nevins, 1989). Antibodies raised against exo- and endoglucanases (with high specificity to -1,3;1,4-mixed-linkage glucan) from cell walls of maize seedlings inhibited auxin-induced cell elongation of maize and rice coleoptiles (Inouhe and Nevins, 1991; Thomas et al., 2000). An obvious question is whether similar proteins identified in this study, as well as the putative CWPs that have not been identified in previous studies, are associated with cell elongation in maize roots. We are currently utilizing the methods developed here to study the changes in fraction 1 CWP composition in regions of the root elongation zone that exhibit maintenance or inhibition of cell elongation in response to water deficits (Sharp et al., 1988, 2004).

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials

Maize (Zea mays L. cv FR697) seeds were surface sterilized in 0.3% NaOCl solution for 15 min, rinsed with distilled water, and imbibed for 24 h in aerated 1 mM CaSO₄. The seeds were germinated for 28 h in vermiculite (grade 3; Strong-Lite), well moistened with 1 mM CaSO₄ at 29°C and near-saturation humidity in the dark (Spollen et al., 2000). Seedlings with primary roots approximately 10 mm in length were then transplanted to a plastic container containing vermiculite well moistened with 1 mM CaSO₄ and grown under the same conditions. At 48 h after transplanting, the apical 12 mm were harvested (using a green safelight; Saab et al., 1990) for either CWP or total soluble protein extraction. The elongation zone constitutes the apical 12 mm in the primary root of well-watered seedlings of this cultivar (Sharp et al., 2004). In additional experiments, specific regions within the elongation zone (3–7 and 7–12 mm from the root apex) were harvested for extraction of CWPs. These samples were used to create region-specific gels that were utilized to test for reproducibility of CWP identification.

Extraction of Fraction 1 CWPs

Fraction 1 CWPs were extracted from the root elongation zone segments according to the methods described by Fry (1988) and MacAdam et al. (1992a, 1992b). Immediately after harvest, the segments were transferred into 20 mM ice-cold K₂HPO₄ solution (pH 6.0). The segments were then rinsed twice with 0.01 M MES buffer and oriented vertically with the root apex at the top in filter-free baskets of tared microfilterfuge tubes (approximately 33 segments per tube; Rainin Instrument Co.). The baskets with root segments were weighed and then placed in a scintillation vial where they were held in place by a stainless steel wire screen molded to fit inside the vial above the basket. Five milliliters of ice-cold, degassed 0.01 M MES buffer (pH 5.5) containing various concentrations of KCl plus protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 5 µL of protease inhibitor cocktail; Sigma) were added to the vial, submerging the tissue. The whole assembly containing the root segments was vacuum infiltrated at –50 kPa for 15 min and for another 5 min without vacuum. Baskets were removed, drained, and excess buffer was blotted away from the segments through the perforated base of the baskets. Baskets with segments were then transferred to microfuge tubes and centrifuged for 15 min at 1,000g. All steps were conducted on ice or in a cold room at 4°C. Infiltration and centrifugation were then repeated twice. Apoplastic fluid from the three successive extractions was pooled (except in the initial optimization studies) in Centricon-10 Microconcentrators (Millipore), and the extract was desalted to less than 0.005 M KCl by adding 0.005 M MES buffer (pH 5.5, containing 1 µL mL^–1 of protease inhibitor cocktail) and centrifuging for 80 min at 12,000g. The sample reservoirs were inverted inside new microfuge tubes and centrifuged at 12,000g for 20 min to collect the desalted apoplastic solution.

Extraction of Total Soluble Proteins

Total soluble proteins were extracted according to the method described by Tsugita et al. (1994). Immediately after harvest, the root elongation zone segments (approximately 50 segments/sample) were ground into fine powder in liquid N₂. Proteins were precipitated overnight at –70°C with 10% (w/v) TCA in acetone containing 0.07% 2-mercaptoethanol. The mixture was centrifuged at 35,000g at 4°C for 15 min, and the precipitate was washed three times with ice-cold acetone with 0.07% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA. Pellets were dried by vacuum centrifugation.

Protein Quantification

The desalted CWPs and total soluble proteins were quantified using the Bradford (1976) method with IgG (Bio-Rad Laboratories) as a standard.

G6PDH Assay

The activity of G6PDH (EC 1.1.1.49), a cytosolic protein marker (MacAdam et al., 1992b), was assayed in the fraction 1 CWP extracts as previously described (Li et al., 1989). Ten micrograms of protein were used for the G6PDH assay. The reaction mixture contained 0.1 M Tricine buffer (pH 8.0), 0.1 M MgCl₂, 10 mg mL^–1 Glc-6-P, and 10 mg mL^–1 NADP (Sigma).

Protein Separation by 2-DE

Prior to 2-DE, the CWP samples were precipitated overnight at –70°C with 10% (w/v) TCA, and the pellets were washed three times with ice-cold methanol and dried briefly by vacuum centrifugation. Total soluble protein (50 µg) or fraction 1 CWP (50 and 150 µg) samples were solubilized in an isoelectric focusing (IEF) buffer containing 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB3-10, 20 mM dithiothreitol, 40 mM Tris, and 0.2% Bio-Lyte 3/10 ampholytes (Bio-Rad Laboratories), and were loaded onto Bio-Rad ReadStrip 11-cm immobilized pH gradient gel strips (pH range 3–10 NL). After the strips were rehydrated overnight, IEF was performed for approximately 10 h, reaching a total of 40 kVh at 20°C on a flat-bed electrophoresis unit (Protean IEF cell; Bio-Rad). After electrophoresis in the first dimension, the immobilized pH gradient strips were equilibrated for 10 min in reducing buffer containing 6 M urea, 2% SDS, 0.375 M Tris-Cl (pH 8.8), 20% glycerol, and 2% dithiothreitol, followed by equilibration for 10 min in alkylation buffer containing 6 M urea, 2% SDS, 0.375 M Tris-Cl (pH 8.8), 20% glycerol, and 2.5% iodoacetamide. Second-dimension SDS-PAGE gels (8%–16% linear gradient, 8.7 x 13.3 cm) were run on a Bio-Rad criterion cell at 60 V for 15 min and then at 180 V for 1 h. Gels were stained with either Bio-Rad BioSafe Coomassie Blue or SyproRuby (Molecular Probes) according to the manufacturer's recommendations. Additional gels were loaded with approximately 50 µg of CWP extracted from the 3- to 7- and 7- to 12-mm regions of the elongation zone, and stained with SyproRuby.

Image Analysis of 2-DE Gels

Coomassie Blue-stained gels were scanned with an Epson flat-bed scanner equipped with Adobe Photoshop 7.0. SyproRuby-stained gels were analyzed using a Typhoon laser scanner 9410 (GE Healthcare). Image analysis was carried out with Bio-Rad PDQuest version 7.2 software. After background subtraction and spot detection, spots were matched and normalized using the method of total density in gel image. Spots of interest were identified and excised using a Genetix GelPix protein spot excision system (Genetix USA).

Protein In-Gel Digestion and Peptide Clean-Up

Gel plugs were destained extensively with 50% (v/v) acetonitrile and 50 mM NH₄HCO₃, followed by dehydration in acetonitrile for 5 min. Proteins were then digested at 37°C in 25 µL of 50 mM NH₄HCO₃ containing 6 µg mL^–1 trypsin (sequencing grade, modified; Promega) for 10 h. To extract the digested peptides from the gel matrix, 30 µL 1% formic acid/2% acetonitrile was added to the digest and incubated at 30°C for 30 min on a shaking platform. The digested supernatant was transferred to a clean tube. The gel pieces were re-extracted with 25 µL of 60% acetonitrile for 15 min and the supernatant was combined with the first extraction. The pooled digest was lyophilized dry in a Speedvac. For whole elongation zone samples, the peptides were resuspended into 10 µL of 1% formic acid/2% acetonitrile, followed by solid-phase extraction using ZipTip microC18 (Millipore) columns according to the manufacturer's instructions, except the peptides were eluted into 4 µL of 60% acetonitrile in water with 0.1% formic acid. For the region-specific samples, peptides were resuspended in 8 µL of 0.1% formic acid/5% acetonitrile for nanoflow HPLC-tandem mass spectrometry (MS/MS).

MALDI-TOF and Electrospray Quadrupole TOF MS

Total soluble protein samples were analyzed using MALDI-TOF MS. Aliquots of protein digest (0.5 µL) were mixed with 0.5 µL of 5 mg mL^–1 (w/v) matrix-cyano-4-hydroxycinnamic acid (purified by recrystallization from ethanol) in 50% (v/v) acetonitrile and 0.1% (v/v) TCA for spotting onto a 96 x 2 stainless steel target for MALDI-TOF MS (Voyager DE STR biochemistry workstation; Applied Biosystems). Standard peptide calibration mixtures (Applied Biosystems) were used as a close external lockmass for instrument and spectrum calibration. MALDI-TOF MS was run in reflector-positive mode for peptide mass fingerprinting.

An ABI QSTAR XL (Applied Biosystems) hybrid quadrupole TOF MS/MS system equipped with a nanoelectrospray source (Protana XYZ manipulator) was used for protein identification analysis. For whole elongation zone samples, the peptides were directly infused into the mass spectrometer and the nanoelectrospray was generated from a borosilicate nanoelectrospray needle with an Au/Pd coating (2-µm tip; Proxeon Biosystems) usually at a voltage of 1,500 V. The instrument m/z response was calibrated daily with standards from the manufacturer. This calibration procedure provides a molecular mass measurement accuracy of <5 ppm for peptides. For nanoflow HPLC (Ultimate) analysis of the region-specific samples, 5 µL of protein digests were loaded onto a C18 precolumn (LC Packings) for desalting and concentrating. Peptides were then eluted from the precolumn and separated on a nanoflow analytical C18 column (PepMap 75-µm i.d., 3 µm, 100 A; LC Packings). The nanoelectrospray was generated from a PicoTip needle (10-µm i.d.; New Objectives) at a voltage of 2,400 V. TOF MS spectra and product ion spectra were acquired using the information-dependent data acquisition feature in the Analyst QS software; mass ranges for TOF MS and MS/MS were 300 to 1,500 and 65 to 1,500, respectively. Every 1 s a TOF MS spectrum was accumulated followed by three product ion spectra, each for 3 s. The DP, DP2, and FP settings were 50, 10, and 200, respectively, and the collision energy was dependent on the m/z values of the ions.

Data Analysis and Database Searching

MALDI-TOF peptide mass fingerprints were used to gain an initial indication of the identity and homogeneity of the proteins. MALDI-TOF MS analyses provide a set of 15 to 50 abundant peptide masses from trypsin-digested protein samples. These masses were used via Protein-Prospector (prospector.ucsf.edu) to identify hits in several different maize-related protein and nucleic acid databases, including NCBInr, Swiss-Pro, dbESTothers, and Unigenes of maize, rice (Oryza sativa), wheat (Triticum aestivum), sorghum (Sorghum bicolor), and potato (Solanum tuberosum), with the maximum miscleavage of 1 and mass tolerance of 50 ppm. Cys carbamidomethylation was chosen as fixed modification, and acetylation of the N terminus, oxidation of Met, and pyroGlu formation of N-terminal Gln were considered as possible modifications. Matching was performed by the number of peptide matches and the coverage (typically greater than 20%), with consideration of Protein-Prospector scores, PMFQ (Watson et al., 2003), and the predicted MW and pI, which were compared with observed MW and pI values on the 2-DE gels. The peptide electrospray tandem mass spectra were searched against the above databases using the Mascot search engine (http://www.matrixscience.com) with a mass tolerance of 100 ppm and one allowed trypsin miscleavage. Search parameters allowed for the fixed Cys carbamidomethylation and the variable Met oxidation and charge state from 2 to 3. Unambiguous identification was judged by the number of peptide sequence tags, sequence coverage, Mowse score, and the quality of MS/MS spectra.

	ACKNOWLEDGMENTS

 
We thank Dr. Jennifer MacAdam for technical advice and training on the vacuum infiltration-centrifugation technique, Ellen Marsh for excellent 2-DE work, Dr. William Spollen for help with protein functional categorization, Dr. Georgia Davis for use of PDQuest software, and Dr. Mary LeNoble for useful discussions.

Received August 19, 2005; returned for revision October 4, 2005; accepted November 7, 2005.

	FOOTNOTES

 
¹ This work was supported by the National Science Foundation, Plant Genome Program (grant no. DBI–0211842); the Missouri Agricultural Experiment Station (project no. MO–PSFCO355); and the Utah Agricultural Experimental Station (project no. UTA00366).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yajun Wu (yajun.wu{at}usu.edu).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.070219.

^* Corresponding author; e-mail yajun.wu{at}usu.edu; fax 435–797–3376.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815[CrossRef][Medline]

Bendtsen JD, Jensen LJ, Blom N, Von Heijne G, Brunak S (2004) Feature-based prediction of non-classical and leaderless protein secretion. Protein Eng Des Sel 17: 349–356[Abstract/Free Full Text]

Blee KA, Wheatley ER, Bonham VA, Mitchell GP, Robertson D, Slabas AR, Burrell MM, Wojtaszek P, Bolwell GP (2001) Proteomic analysis reveals a novel set of cell wall proteins in a transformed tobacco cell culture that synthesises secondary walls as determined by biochemical and morphological parameters. Planta 212: 404–415[CrossRef][Web of Science][Medline]

Borderies G, Jamet E, Lafitte C, Rossignol M, Jauneau A, Boudart G, Monsarrat B, Esquerre-Tugaye MT, Boudet A, Pont-Lezica R (2003) Proteomics of loosely bound cell wall proteins of Arabidopsis thaliana cell suspension cultures: a critical analysis. Electrophoresis 24: 3421–3432[CrossRef][Web of Science][Medline]

Boudart G, Jamet E, Rossignol M, Lafitte C, Borderies G, Jauneau A, Esquerre-Tugaye MT, Pont-Lezica R (2005) Cell wall proteins in apoplastic fluids of Arabidopsis thaliana rosettes: identification by mass spectrometry and bioinformatics. Proteomics 5: 212–221[CrossRef][Web of Science][Medline]

Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254[CrossRef][Web of Science][Medline]

Buckeridge MS, Rayon C, Urbanowicz B, Tine MAS, Carpita NC (2004) Mixed linkage (1-3),(1-4)--D-glucans of grasses. Cereal Chem 81: 115–127

Canovas FM, Dumas-Gaudot E, Recorbet G, Jorrin J, Mock HP, Rossignol M (2004) Plant proteome analysis. Proteomics 4: 285–298[CrossRef][Web of Science][Medline]

Carpita NC (1996) Structure and biogenesis of the cell walls of grasses. Annu Rev Plant Physiol Plant Mol Biol 47: 445–476[CrossRef][Web of Science]

Carpita NC, Defernez M, Findlay K, Wells B, Shoue DA, Catchpole G, Wilson RH, McCann MC (2001) Cell wall architecture of the elongating maize coleoptile. Plant Physiol 127: 551–565[Abstract/Free Full Text]

Carpita NC, Gibeaut DM (1993) Structural models of primary-cell walls in flowering plants: consistency of molecular-structure with the physical-properties of the walls during growth. Plant J 3: 1–30[CrossRef][Web of Science][Medline]

Cassab GI, Varner JE (1988) Cell wall proteins. Annu Rev Plant Physiol Plant Mol Biol 39: 321–353[CrossRef][Web of Science]

Chen S (2005) Rapid protein identification using direct infusion nanoelectrospray ionization mass spectrometry. Proteomics (in press)

Chivasa S, Ndimba BK, Simon WJ, Robertson D, Yu XL, Knox JP, Bolwell P, Slabas AR (2002) Proteomic analysis of the Arabidopsis thaliana cell wall. Electrophoresis 23: 1754–1765[CrossRef][Web of Science][Medline]

Cordoba-Pedregosa M, Gonzalez-Reyes JA, Canadillas M, Navas P, Cordoba F (1996) Role of apoplastic and cell-wall peroxidases on the stimulation of root elongation by ascorbate. Plant Physiol 112: 1119–1125[Abstract]

Cosgrove DJ (1999) Enzymes and other agents that enhance cell wall extensibility. Annu Rev Plant Physiol Plant Mol Biol 50: 391–417[CrossRef][Web of Science][Medline]

Dani V, Simon WJ, Duranti M, Croy RRD (2005) Changes in the tobacco leaf apoplast proteome in response to salt stress. Proteomics 5: 737–745[CrossRef][Web of Science][Medline]

de Souza IRP, MacAdam JW (2001) Gibberellic acid and dwarfism effects on the growth dynamics of B73 maize (Zea mays L.) leaf blades: a transient increase in apoplastic peroxidase activity precedes cessation of cell elongation. J Exp Bot 52: 1673–1682[Abstract/Free Full Text]

Edwards SR, Braley R, Chaffin WL (1999) Enolase is present in the cell wall of Saccharomyces cerevisiae. FEMS Microbiol Lett 177: 211–216[CrossRef][Web of Science][Medline]

Fry SC (1988) The Growing Plant Cell Wall: Chemical and Metabolic Analysis. John Wiley & Sons, New York

Gross GG (1977) Cell wall-bound malate dehydrogenase from horseradish. Phytochemistry 16: 319–321[CrossRef]

Huber DJ, Nevins DJ (1981) Partial purification of endo- and exo--D-glucanase enzymes from Zea mays L. seedlings and their involvement in cell-wall autohydrolysis. Planta 151: 206–214[CrossRef]

Inouhe M, Nevins DJ (1991) Inhibition of auxin-induced cell elongation of maize coleoptiles by antibodies specific for cell wall glucanases. Plant Physiol 96: 426–431[Abstract/Free Full Text]

Keller B (1993) Structural cell wall proteins. Plant Physiol 101: 1127–1130[Medline]

Kim JB, Olek AT, Carpita NC (2000) Cell wall and membrane-associated exo--D-glucanases from developing maize seedlings. Plant Physiol 123: 471–486[Abstract/Free Full Text]

Labrador E, Nevins DJ (1989) An exo--D-glucanase derived from Zea coleoptile walls with a capacity to elicit cell elongation. Physiol Plant 77: 479–486[CrossRef]

Li ZC, McClure JW, Hagerman AE (1989) Soluble and bound apoplastic activity for peroxidase, -D-glucosidase, malate dehydrogenase, and nonspecific arylesterase, in barley (Hordeum vulgare L.) and oat (Avena sativa L.) primary leaves. Plant Physiol 90: 185–190[Abstract/Free Full Text]

MacAdam JW, Nelson CJ, Sharp RE (1992a) Peroxidase activity in the leaf elongation zone of tall fescue. I. Spatial distribution of ionically bound peroxidase activity in genotypes differing in length of the elongation zone. Plant Physiol 99: 872–878[Abstract/Free Full Text]

MacAdam JW, Sharp RE, Nelson CJ (1992b) Peroxidase activity in the leaf elongation zone of tall fescue. II. Spatial distribution of apoplastic peroxidase activity in genotypes differing in length of the elongation zone. Plant Physiol 99: 879–885[Abstract/Free Full Text]

McCann MC, Roberts K (1994) Changes in cell wall architecture during cell elongation. J Exp Bot 45: 1683–1691[Web of Science]

Morrow DL, Jones RL (1986) Localization and partial characterization of the extracellular proteins centrifuged from pea internodes. Physiol Plant 67: 397–407

Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10: 1–6[Abstract/Free Full Text]

Nishitani K, Tominaga R (1992) Endoxyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule. J Biol Chem 267: 21058–21064[Abstract/Free Full Text]

Pappin DJC, Rahman D, Hansen HF, Bartlet-Jones M, Jeffery W, Bleasby AJ (1996) Chemistry, mass spectrometry and peptide-mass databases: evolution of methods for the rapid identification and mapping of cellular proteins. In AL Burlingame, SA Carr, eds, Mass Spectrometry in the Biological Sciences. Humana Press, Totowa, NJ, pp 135–150

Pitarch A, Sanchez M, Nombela C, Gil C (2002) Sequential fractionation and two-dimensional analysis unravels the complexity of the dimorphic fungi Candida albicans cell wall proteome. Mol Cell Proteomics 1: 967–982[Abstract/Free Full Text]

Robertson D, Mitchell GP, Gilroy JS, Gerrish C, Bolwell GP, Slabas AR (1997) Differential extraction and protein sequencing reveals major differences in patterns of primary cell wall proteins from plants. J Biol Chem 272: 15841–15848[Abstract/Free Full Text]

Saab IN, Sharp RE, Pritchard J, Voetberg GS (1990) Increased endogenous abscisic acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials. Plant Physiol 93: 1329–1336[Abstract/Free Full Text]

Sharp RE, Poroyko V, Hejlek LG, Spollen WG, Springer GK, Bohnert HJ, Nguyen HT (2004) Root growth maintenance during water deficits: physiology to functional genomics. J Exp Bot 55: 2343–2351[Abstract/Free Full Text]

Sharp RE, Silk WK, Hsiao TC (1988) Growth of the maize primary root at low water potentials. I. Spatial distribution of expansive growth. Plant Physiol 87: 50–57[Abstract/Free Full Text]

Showalter AM (1993) Structure and function of plant-cell wall proteins. Plant Cell 5: 9–23[Free Full Text]

Slabas AR, Ndimba B, Simon WJ, Chivasa S (2004) Proteomic analysis of the Arabidopsis cell wall reveals unexpected proteins with new cellular locations. Biochem Soc Trans 32: 524–528[Medline]

Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J, Osborne E, Paredez A, Persson S, Raab T, et al (2004) Toward a systems approach to understanding plant cell walls. Science 306: 2206–2211[Abstract/Free Full Text]

Spollen WG, LeNoble ME, Samuels TD, Bernstein N, Sharp RE (2000) ABA accumulation maintains primary root elongation at low water potentials by restricting ethylene production. Plant Physiol 122: 967–976[Abstract/Free Full Text]

Spollen WG, Sharp RE (1991) Spatial distribution of turgor and root growth at low water potentials. Plant Physiol 96: 438–443[Abstract/Free Full Text]

Sun W, Xu J, Yang J, Kieliszewski MJ, Showalter AM (2005) The lysine-rich arabinogalactan-protein subfamily in Arabidopsis: gene expression, glycoprotein purification and biochemical characterization. Plant Cell Physiol 46: 975–984[Abstract/Free Full Text]

Tatusov RL, Koonin EV, Lipman DJ (1997) A genomic perspective on protein families. Science 278: 631–637[Abstract/Free Full Text]

Terry ME, Bonner BA (1980) An examination of centrifugation as a method of extracting an extracellular solution from peas, and its use for the study of indoleacetic acid-induced growth. Plant Physiol 66: 321–325[Abstract/Free Full Text]

Thomas BR, Inouhe M, Simmons CR, Nevins DJ (2000) Endo-1,3;1,4--glucanase from coleoptiles of rice and maize: role in the regulation of plant growth. Int J Biol Macromol 27: 145–149[Medline]

Tsugita A, Kawakami T, Uchiyama Y, Kamo M, Miyatake N, Nozu Y (1994) Separation and characterization of rice proteins. Electrophoresis 15: 708–720[CrossRef][Medline]

Voigt J, Frank R (2003) 14-3-3 proteins are constituents of the insoluble glycoprotein framework of the Chlamydomonas cell wall. Plant Cell 15: 1399–1413[Abstract/Free Full Text]

Watson BS, Asirvatham VS, Wang L, Sumner LW (2003) Mapping the proteome of barrel medic (Medicago truncatula). Plant Physiol 131: 1104–1123[Abstract/Free Full Text]

Watson BS, Lei Z, Dixon RA, Sumner LW (2004) Proteomics of Medicago sativa cell walls. Phytochemistry 65: 1709–1720[CrossRef][Web of Science][Medline]

Wu Y, Cosgrove DJ (2000) Adaptation of roots to low water potentials by changes in cell wall extensibility and cell wall proteins. J Exp Bot 51: 1543–1553[Abstract/Free Full Text]

Wu Y, Sharp RE, Durachko DM, Cosgrove DJ (1996) Growth maintenance of the maize primary root at low water potentials involves increases in cell-wall extension properties, expansin activity, and wall susceptibility to expansins. Plant Physiol 111: 765–772[Abstract]

Wu Y, Spollen WG, Sharp RE, Hetherington PR, Fry SC (1994) Root growth maintenance at low water potentials: increased activity of xyloglucan endotransglycosylase and its possible regulation by abscisic acid. Plant Physiol 106: 607–615[Abstract]

This article has been cited by other articles:

				C. Albenne, H. Canut, G. Boudart, Y. Zhang, H. San Clemente, R. Pont-Lezica, and E. Jamet Plant Cell Wall Proteomics: Mass Spectrometry Data, a Trove for Research on Protein Structure/Function Relationships Mol Plant, September 1, 2009; 2(5): 977 - 989. [Abstract] [Full Text] [PDF]

				A. Laohavisit, J. C. Mortimer, V. Demidchik, K. M. Coxon, M. A. Stancombe, N. Macpherson, C. Brownlee, A. Hofmann, A. A.R. Webb, H. Miedema, et al. Zea mays Annexins Modulate Cytosolic Free Ca2+ and Generate a Ca2+-Permeable Conductance PLANT CELL, February 1, 2009; 21(2): 479 - 493. [Abstract] [Full Text] [PDF]

				G. Lycett The role of Rab GTPases in cell wall metabolism J. Exp. Bot., November 1, 2008; 59(15): 4061 - 4074. [Abstract] [Full Text] [PDF]

				T. Tokunaga, Y. Miyata, Y. Fujikawa, and M. Esaka RNAi-Mediated Knockdown of the XIP-Type Endoxylanase Inhibitor Gene, OsXIP, Has No Effect on Grain Development and Germination in Rice Plant Cell Physiol., July 1, 2008; 49(7): 1122 - 1127. [Abstract] [Full Text] [PDF]

				R. Tuberosa, S. Salvi, S. Giuliani, M. C. Sanguineti, M. Bellotti, S. Conti, and P. Landi Genome-wide Approaches to Investigate and Improve Maize Response to Drought Crop Sci., December 18, 2007; 47(Supplement_3): S-120 - S-141. [Abstract] [Full Text] [PDF]

				J. Zhu, S. Alvarez, E. L. Marsh, M. E. LeNoble, I.-J. Cho, M. Sivaguru, S. Chen, H. T. Nguyen, Y. Wu, D. P. Schachtman, et al. Cell Wall Proteome in the Maize Primary Root Elongation Zone. II. Region-Specific Changes in Water Soluble and Lightly Ionically Bound Proteins under Water Deficit Plant Physiology, December 1, 2007; 145(4): 1533 - 1548. [Abstract] [Full Text] [PDF]

				D. Bhushan, A. Pandey, M. K. Choudhary, A. Datta, S. Chakraborty, and N. Chakraborty Comparative Proteomics Analysis of Differentially Expressed Proteins in Chickpea Extracellular Matrix during Dehydration Stress Mol. Cell. Proteomics, November 1, 2007; 6(11): 1868 - 1884. [Abstract] [Full Text] [PDF]

				Z. Minic, E. Jamet, L. Negroni, P Arsene der Garabedian, M. Zivy, and L. Jouanin A sub-proteome of Arabidopsis thaliana mature stems trapped on Concanavalin A is enriched in cell wall glycoside hydrolases J. Exp. Bot., July 1, 2007; 58(10): 2503 - 2512. [Abstract] [Full Text] [PDF]

				R. Shin, S. Alvarez, A. Y. Burch, J. M. Jez, and D. P. Schachtman Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes PNAS, April 10, 2007; 104(15): 6460 - 6465. [Abstract] [Full Text] [PDF]

				F. Wen, H. D. VanEtten, G. Tsaprailis, and M. C. Hawes Extracellular Proteins in Pea Root Tip and Border Cell Exudates Plant Physiology, February 1, 2007; 143(2): 773 - 783. [Abstract] [Full Text] [PDF]

				V Poroyko, W. Spollen, L. Hejlek, A. Hernandez, M. LeNoble, G Davis, H. Nguyen, G. Springer, R. Sharp, and H. Bohnert Comparing regional transcript profiles from maize primary roots under well-watered and low water potential conditions J. Exp. Bot., January 1, 2007; 58(2): 279 - 289. [Abstract] [Full Text] [PDF]

				S. Guillaumie, H. San-Clemente, C. Deswarte, Y. Martinez, C. Lapierre, A. Murigneux, Y. Barriere, M. Pichon, and D. Goffner MAIZEWALL. Database and Developmental Gene Expression Profiling of Cell Wall Biosynthesis and Assembly in Maize Plant Physiology, January 1, 2007; 143(1): 339 - 363. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 140/1/311    most recent pp.105.070219v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (39) Citing Articles via Google Scholar Google Scholar Articles by Zhu, J. Articles by Sharp, R. E. Search for Related Content PubMed PubMed Citation Articles by Zhu, J. Articles by Sharp, R. E. Agricola Articles by Zhu, J. Articles by Sharp, R. E.