Proteomic survey of metabolic pathways in rice

Abstract

A systematic proteomic analysis of rice (Oryza sativa) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissue-specific and subcellular compartment-specific isoforms of ADP-glucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.

Figure 1

Functional class analysis of the identified proteins. Each of the combined total number of unique proteins (see Table 1) was functionally classified according to the categories described by Schoof et al. (31). After functional class assessment of each protein, the total number and percentage of proteins in each class were determined. (A) The percentage of proteins from each functional class from the total combined number of proteins from all tissues is shown. These values correspond to the Leaf Root Seed (LRS) column in B. (B) The table of numbers and percentages of proteins from each functional class from each grouping of tissue datasets is shown. LRS stands for the complete pool of proteins from Leaf, Root, and Seed. LRSINT are the shared proteins identified in Leaf, Root, and Seed. OL are the proteins found Only in Leaf. OR are the proteins found Only in Root. OS are the proteins found Only in Seed.

Figure 2

Tissue localization of proteins identified from the starch biosynthesis and degradation pathways. On functional classification of each uniquely identified protein, the tissue-specific protein expression was analyzed from a metabolic pathway perspective (Fig. 5). A focused portion of Fig. 5 is shown where the tissue-specific expression of the starch biosynthesis and degradation pathways is detailed. Enzymes involved in each pathway are in blue. The boxes show in which tissue the enzymes have been found, with green representing detection in leaf, red representing detection in root, and yellow representing detection in seed.

Figure 3

Tissue-specific expression of AGPase isozymes. (A) clustalw alignment (52) of the small subunits (ss under subunit) and large subunits (ls under subunit) of AGPase isozymes detected and identified in the current study. The tissues in which each enzyme were detected and identified are specified under the localization column. The yellow boxes designate the peptides that were detected and identified from each isozyme that passed the sequest (21) Xcorr and ΔCn criteria described in Materials and Methods. The proteinid listed for each AGPase isozyme corresponds to the proteinid listed in Table 2. A subset of the tandem mass spectra (MS/MS) that uniquely identified each of the AGPase isozymes is shown in B–D. (B) The sequest-interpreted MS/MS of the +2 peptide YAELHDFGSEILPR (m/z of 824.4; Xcorr = 2.97; ΔCn = 0.30) from the AGPase isozyme with the proteinid 09904. (C) The sequest-interpreted MS/MS of the +2 peptide YAHLQDFGSEILPR (m/z of 823.9; Xcorr = 3.28; ΔCn = 0.25) from the AGPase isozyme with the proteinid 34550. (D) The sequest-interpreted MS/MS of the +2 peptide WRFPTANDFGSEIIPASAK (m/z of 1,054.8; Xcorr = 3.95; ΔCn = 0.36) from the AGPase isozyme with the proteinid 50182.

Figure 4

Proteomic identification of allergens in rice seed. (A) clustalw alignment (52) of the four allergens detected and identified in rice seed via proteomic methods in the current study. The proteinid listed for each allergen corresponds to the proteinid listed in Table 2. The yellow boxes designate the peptides that were detected and identified from each protein that passed the sequest (21) Xcorr and ΔCn criteria described in Materials and Methods. A tandem mass spectra (MS/MS) that uniquely identifies each allergenic protein is shown in B. (B) The sequest-interpreted MS/MS of the +2 peptide GTAAAAEQVR (m/z of 487.3; Xcorr = 3.17; ΔCn = 0.11) from the allergen with the proteinid 18831. The sequest-interpreted MS/MS of the +2 peptide GAPALSHMLVGMYK (m/z of 738.0; Xcorr = 3.47; ΔCn = 0.31) from the allergen with the proteinid 33143. The sequest-interpreted MS/MS of the +2 peptide DHHQEQVVYTPGQL (m/z of 826.2; Xcorr = 2.84; ΔCn = 0.23) from the allergen with the proteinid 33575. The sequest-interpreted MS/MS of the +2 peptide SALNHMVGGIYR (m/z of 616.3; Xcorr = 3.44; ΔCn = 0.34) from the allergen with the proteinid 55653.