The absence of heat shock protein HSP101 affects the proteome of mature and germinating maize embryos.

Maize heat shock protein HSP101 accumulates during embryo maturation and desiccation and persists at high levels during the first 24 h following kernel imbibition in the absence of heat stress. This protein has a known function in disaggregation of high molecular weight complexes and has been proposed to be a translational regulator of specific mRNAs. Here, a global proteomic approach was used to identify changes in the maize proteome due to the absence of HSP101 in embryos from mature-dry or 24 h-imbibed kernels. A total of 26 protein spots from the mature dry embryo exhibited statistically significant expression changes in the L10 inbred hsp101 mutant (hsp101-m5::Mu1/hsp101-m5::Mu1) line as compared to the corresponding wild type (Hsp101/Hsp101). Additional six spots reproducibly showed qualitative changes between the mutant and wild-type mature and germinating embryos. Several chaperones, translation-related proteins, actin, and enzymes participating in cytokinin metabolism were identified in these spots by tandem mass-spectrometry (MS). The proteomic changes partially explain the altered root growth and architecture observed in young hsp101 mutant seedlings. In addition, specific protein de novo synthesis was altered in the 24 h-imbibed mutant embryos indicating that maize HSP101 functions as both chaperone and translational regulator during germination. Supporting this, HSP101 was found as part of Cap-binding and translation initiation complexes during early kernel imbibition. Overall, these findings expose the relevance of maize HSP101 for protein synthesis and balance mechanisms during germination.

Genomically biased accumulation of seed storage proteins in allopolyploid cotton.

Allopolyploidy is an important process during plant evolution that results in the reunion of two divergent genomes into a common nucleus. Many of the immediate as well as longer-term genomic and epigenetic responses to polyploidy have become appreciated. To investigate the modifications of gene expression at the proteome level caused by allopolyploid formation, we conducted a comparative analysis of cotton seed proteomes from the allopolyploid Gossypium hirsutum (AD genome) and its model A-genome and D-genome diploid progenitors. An unexpectedly high level of divergence among the three proteomes was found, with about one-third of all protein forms being genome specific. Comparative analysis showed that there is a higher degree of proteomic similarity between the allopolyploid and its D-genome donor than its A-genome donor, reflecting a biased accumulation of seed proteins in the allopolyploid. Protein identification and genetic characterization of high-abundance proteins revealed that two classes of seed storage proteins, vicilins and legumins, compose the major component of cotton seed proteomes. Analyses further indicate differential regulation or modification of homoeologous gene products, as well as novel patterns in the polyploid proteome that may result from the interaction between homoeologous gene products. Our findings demonstrate that genomic merger and doubling have consequences that extend beyond the transcriptome into the realm of the proteome and that unequal expression of proteins from diploid parental genomes may occur in allopolyploids.

Comparative proteomics of seed maturation in oilseeds reveals differences in intermediary metabolism.

Proteomics is increasingly being used to understand enzyme expression and regulatory mechanisms involved in the accumulation of storage reserves in crops with sequenced genomes. During the past six years, considerable progress has been made to characterize proteomes of both mature and developing seeds, particularly oilseeds - plants which accumulate principally oil and protein as storage reserves. This review summarizes the emerging proteomics data, with emphasis on seed filling in soy, rapeseed, castor and Arabidopsis as each of these oilseeds were analyzed using very similar proteomic strategies. These parallel studies provide a comprehensive view of source-sink relationships, specifically sucrose assimilation into organic acid intermediates for de novo amino acid and fatty acid synthesis. We map these biochemical processes for seed maturation and illustrate the differences and similarities among the four oilseeds. For example, while the four oilseeds appear capable of producing cytosolic phosphoenolpyruvate as the principal carbon intermediate, soybean and castor also express malic enzymes and malate dehydrogenase, together capable of producing malate that has been previously proposed to be the major intermediate for fatty acid synthesis in castor. We discuss these and other differences in the context of intermediary metabolism for developing oilseeds.

Quantitation of soybean allergens using tandem mass spectrometry.

Soybean (Glycine max) seed contain some proteins that are allergenic to humans and animals. However, the concentration of these allergens and their expression variability among germplasms is presently unknown. To address this problem, 10 allergens were quantified from 20 nongenetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches. Relative quantitation was performed by spectral counting and absolute quantitation was performed using multiple reaction monitoring (MRM) with synthetic, isotope-labeled peptides as internal standards. During relative quantitation analysis, 10 target allergens were identified, and five of these allergens showed expression levels higher than technical variation observed for bovine serum albumin (BSA) internal standard (∼11%), suggesting expression differences among the varieties. To confirm this observation, absolute quantitation of these allergens from each variety was performed using MRM. Eight of the 10 allergens were quantified for their concentration in seed and ranged from approximately 0.5 to 5.7 µg/mg of soy protein. MRM analysis reduced technical variance of BSA internal standards to approximately 7%, and confirmed differential expression for four allergens across the 20 varieties. This is the first quantitative assessment of all major soybean allergens. The results show the total quantity of allergens measured among the 20 soy varieties was mostly similar.

Evolution of seed allergen quantification--from antibodies to mass spectrometry.

Development of accurate, high-throughput approaches for protein allergen quantification is important for the seed industry as a means to monitor natural variability in expression and ensure introduced transgenes do not collaterally alter the expression of any known allergen. Analytical approaches for protein quantification have undergone a renaissance in recent years with the emergence of soft-ionization approaches and advanced mass spectrometers capable of achieving low attomolar sensitivity. These advances coupled with bioinformatic tools to mine mass spectral data are collectively referred to as proteomics, and allow for the large-scale study of proteins with high precision and quantitative accuracy. In this review, we discuss differential and quantitative proteomics workflows that proceed from discovery profiling to targeted, quantitative analysis of specific proteins using stable isotopically-labeled, synthetic peptide doping standards. These synthetic peptide standards, also referred to as AQUA peptides, are synthetic mimics to proteotypic peptides and allow for absolute quantification of proteins in complex biological mixtures. The approaches discussed herein are ideal for the analysis of prominently expressed proteins such as protein allergens from plant seed, as no gels or sample pre-fractionation is required. We discuss these new techniques in the context of traditional, antibody-based technologies for allergen detection and quantification.

A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity.

The Homo sapiens and Arabidopsis thaliana genomes are believed to encode more than 500 and 1000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Here we describe a quantitative mass spectrometry (MS)-based approach for identifying kinase-client proteins. During method development, we used the dedicated kinase pyruvate dehydrogenase kinase (PDK) for the in vitro assays. As kinase substrate, we used synthetic peptide cocktails and, in the process, demonstrated that the assay is both sensitive and specific. The method is also useful for characterizing protein kinase-substrate kinetics once the peptide substrate is detected. Applying a label-free spectral counting method, the activity of PDK was determined using the peptide substrate YHGH(292)SMSDPGSTYR derived from the pyruvate dehydrogenase E1alpha subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We also measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications.

Quantitative proteomics of seed filling in castor: comparison with soybean and rapeseed reveals differences between photosynthetic and nonphotosynthetic seed metabolism.

Seed maturation or seed filling is a phase of development that plays a major role in the storage reserve composition of a seed. In many plant seeds photosynthesis plays a major role in this process, although oilseeds, such as castor (Ricinus communis), are capable of accumulating oil without the benefit of photophosphorylation to augment energy demands. To characterize seed filling in castor, a systematic quantitative proteomics study was performed. Two-dimensional gel electrophoresis was used to resolve and quantify Cy-dye-labeled proteins expressed at 2, 3, 4, 5, and 6 weeks after flowering in biological triplicate. Expression profiles for 660 protein spot groups were established, and of these, 522 proteins were confidently identified by liquid chromatography-tandem mass spectrometry by mining against the castor genome. Identified proteins were classified according to function, and the most abundant groups of proteins were involved in protein destination and storage (34%), energy (19%), and metabolism (15%). Carbon assimilatory pathways in castor were compared with previous studies of photosynthetic oilseeds, soybean (Glycine max) and rapeseed (Brassica napus). These comparisons revealed differences in abundance and number of protein isoforms at numerous steps in glycolysis. One such difference was the number of enolase isoforms and their sum abundance; castor had approximately six times as many isoforms as soy and rapeseed. Furthermore, Rubisco was 11-fold less prominent in castor compared to rapeseed. These and other differences suggest some aspects of carbon flow, carbon recapture, as well as ATP and NADPH production in castor differs from photosynthetic oilseeds.

X-ray structure of ILL2, an auxin-conjugate amidohydrolase from Arabidopsis thaliana.

The plant hormone indole-3-acetic acid (IAA) is the most abundant natural auxin involved in many aspects of plant development and growth. The IAA levels in plants are modulated by a specific group of amidohydrolases from the peptidase M20D family that release the active hormone from its conjugated storage forms. Here, we describe the X-ray crystal structure of IAA-amino acid hydrolase IAA-leucine resistantlike gene 2 (ILL2) from Arabidopsis thaliana at 2.0 A resolution. ILL2 preferentially hydrolyses the auxin-amino acid conjugate N-(indol-3-acetyl)-alanine. The overall structure of ILL2 is reminiscent of dinuclear metallopeptidases from the M20 peptidase family. The structure consists of two domains, a larger catalytic domain with three-layer alpha beta alpha sandwich architecture and aminopeptidase topology and a smaller satellite domain with two-layer alphabeta-sandwich architecture and alpha-beta-plaits topology. The metal-coordinating residues in the active site of ILL2 include a conserved cysteine that clearly distinguishes this protein from previously structurally characterized members of the M20 peptidase family. Modeling of N-(indol-3-acetyl)-alanine into the active site of ILL2 suggests that Leu175 serves as a key determinant for the amino acid side-chain specificity of this enzyme. Furthermore, a hydrophobic pocket nearby the catalytic dimetal center likely recognizes the indolyl moiety of the substrate. Finally, the active site of ILL2 harbors an absolutely conserved glutamate (Glu172), which is well positioned to act as a general acid-base residue. Overall, the structure of ILL2 suggests that this enzyme likely uses a catalytic mechanism that follows the paradigm established for the other enzymes of the M20 peptidase family.

Phylogenetic analyses identify 10 classes of the protein disulfide isomerase family in plants, including single-domain protein disulfide isomerase-related proteins.

Protein disulfide isomerases (PDIs) are molecular chaperones that contain thioredoxin (TRX) domains and aid in the formation of proper disulfide bonds during protein folding. To identify plant PDI-like (PDIL) proteins, a genome-wide search of Arabidopsis (Arabidopsis thaliana) was carried out to produce a comprehensive list of 104 genes encoding proteins with TRX domains. Phylogenetic analysis was conducted for these sequences using Bayesian and maximum-likelihood methods. The resulting phylogenetic tree showed that evolutionary relationships of TRX domains alone were correlated with conserved enzymatic activities. From this tree, we identified a set of 22 PDIL proteins that constitute a well-supported clade containing orthologs of known PDIs. Using the Arabidopsis PDIL sequences in iterative BLAST searches of public and proprietary sequence databases, we further identified orthologous sets of 19 PDIL sequences in rice (Oryza sativa) and 22 PDIL sequences in maize (Zea mays), and resolved the PDIL phylogeny into 10 groups. Five groups (I-V) had two TRX domains and showed structural similarities to the PDIL proteins in other higher eukaryotes. The remaining five groups had a single TRX domain. Two of these (quiescin-sulfhydryl oxidase-like and adenosine 5'-phosphosulfate reductase-like) had putative nonisomerase enzymatic activities encoded by an additional domain. Two others (VI and VIII) resembled small single-domain PDIs from Giardia lamblia, a basal eukaryote, and from yeast. Mining of maize expressed sequence tag and RNA-profiling databases indicated that members of all of the single-domain PDIL groups were expressed throughout the plant. The group VI maize PDIL ZmPDIL5-1 accumulated during endoplasmic reticulum stress but was not found within the intracellular membrane fractions and may represent a new member of the molecular chaperone complement in the cell.