Cell wall proteome in the maize primary root elongation zone. I. Extraction and identification of water-soluble and lightly ionically bound proteins

Abstract

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-beta-D-glucanase and alpha-L-arabinofuranosidase, which act on the major polysaccharides only or mainly present in type II cell walls.

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.

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.

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 β-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).

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.

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.