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The article presents a study on breeding a disease-resistant plant using genomics and proteomics. Technological advances in genomics and proteomics has provided insights into disease-resistance pathways. A combination of structural genomics and functional genomics help in identifying regions that carry genes controlling resistance pathways and provide a means in cloning the genes involved, while phosphoproteomics help in discovering proteins involved in the signaling pathways.

The effect of genotype and environment on oat protein composition was analyzed through size exclusion-high-performance liquid chromatography (SE-HPLC) and liquid chromatography-mass spectrometry (LC-MS) to characterize oat protein isolate (OPI) extracted from three genotypes grown at three locations in the Canadian Prairies. SE-HPLC identified four fractions in OPI, including polymeric globulins, avenins, glutelins, and albumins, and smaller proteins. The protein composition was dependent on the environment, rather than the genotype. The proteins identified through LC-MS were grouped into eight categories, including globulins, prolamins/avenins, glutelins, enzymes/albumins, enzyme inhibitors, heat shock proteins, grain softness proteins, and allergenic proteins. Three main globulin protein types were also identified, including the P14812|SSG2-12S seed storage globulin, the Q6UJY8_TRITU-globulin, and the M7ZQM3_TRIUA-Globulin-1 S. Principal component analysis indicated that samples from Manitoba showed a positive association with the M7ZQM3_TRIUA-Globulin-1 S allele and Q6UJY8_TRITU-globulin, while samples from Alberta and Saskatchewan had a negative association with them. The results show that the influence of G × E on oat protein fractions and their relative composition is crucial to understanding genotypes' behavior in response to different environments.

Background: Screening and developing novel antifungal agents with minimal environmental impact are needed to maintain and increase crop production, which is constantly threatened by various pathogens. Small peptides with antimicrobial and antifungal activities have been known to play an important role in plant defense both at the pathogen level by suppressing its growth and proliferation as well as at the host level through activation or priming of the plant's immune system for a faster, more robust response against fungi. Rust fungi (Pucciniales) are plant pathogens that can infect key crops and overcome resistance genes introduced in elite wheat cultivars., Results: We performed an in vitro screening of 18 peptides predominantly of plant origin with antifungal or antimicrobial activity for their ability to inhibit leaf rust (Puccinia triticina, CCDS-96-14-1 isolate) urediniospore germination. Nine peptides demonstrated significant fungicidal properties compared to the control. Foliar application of the top three candidates, β-purothionin, Purothionin-α2 and Defensin-2, decreased the severity of leaf rust infection in wheat (Triticum aestivum L.) seedlings. Additionally, increased pathogen resistance was paralleled by elevated expression of defense-related genes., Conclusions: Identified antifungal peptides could potentially be engineered in the wheat genome to provide an alternative source of genetic resistance to leaf rust., (Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. Published by Elsevier Inc. All rights reserved.)

Background: Pseudochlorella pringsheimii (Ppr) is a green unicellular alga rich with chlorophyll, carotenoids, and antioxidants. As a widespread organism, Ppr must face, and adapt to, many environmental stresses and these are becoming more frequent and more extreme under the conditions of climate change. We therefore focused on salinity induced by NaCl and iron (Fe) variation stresses, which are commonly encountered by algae in their natural environment., Results: The relatively low stress levels improved the biomass, growth rate, and biochemical components of Ppr. In addition, the radical-scavenging activity, reducing power, and chelating activity were stimulated by lower iron concentrations and all NaCl concentrations. We believe that the alga has adapted to the stressors by increasing certain biomolecules such as carotenoids, phenolics, proteins, and carbohydrates. These act as antioxidants and osmoregulators to protect cell membranes and other cellular components from the harmful effects of ions. We have used SDS-PAGE and 2D-PAGE in combination with tandem mass spectrometry to identify responsive proteins in the proteomes of stressed vs. non-stressed Ppr. The results of 2D-PAGE analysis showed a total of 67 differentially expressed proteins, and SDS-PAGE identified 559 peptides corresponding to 77 proteins. Of these, 15, 8, and 17 peptides were uniquely identified only under the control, iron, and salinity treatments, respectively. The peptides were classified into 12 functional categories: energy metabolism (the most notable proteins), carbohydrate metabolism, regulation, photosynthesis, protein synthesis, stress proteins, oxido-reductase proteins, transfer proteins, ribonucleic-associated proteins, hypothetical proteins, and unknown proteins. The number of identified peptides was higher under salinity stress compared to iron stress., Conclusions: A proposed mechanism for the adaptation of Ppr to stress is discussed based on the collected data. This data could serve as reference material for algal proteomics and the mechanisms involved in mediating stress tolerance., (© 2024. The Author(s).)

Pyrenophora teres f. maculata (Ptm) is a fungal pathogen that causes the spot form of net blotch on barley and leads to economic losses in many of the world's barley-growing regions. Isolates of Ptm exhibit varying levels of aggressiveness that result in quantifiable changes in the severity of the disease. Previous research on plant-pathogen interactions has shown that such divergence is reflected in the proteome and secretome of the pathogen, with certain classes of proteins more prominent in aggressive isolates. Here we have made a detailed comparative analysis of the secretomes of two Ptm isolates, GPS79 and E35 (highly and mildly aggressive, respectively) using a proteomics-based approach. The secretomes were obtained in vitro using media amended with barley leaf sections. Secreted proteins therein were harvested, digested with trypsin, and fractionated offline by HPLC prior to LC-MS in a high-resolution instrument to obtain deep coverage of the proteome. The subsequent analysis used a label-free quantitative proteomics approach with relative quantification of proteins based on precursor ion intensities. A total of 1175 proteins were identified, 931 from Ptm and 244 from barley. Further analysis revealed 160 differentially abundant proteins with at least a two-fold abundance difference between the isolates, with the most enriched in the aggressive GPS79 secretome. These proteins were mainly cell-wall (carbohydrate) degrading enzymes and peptidases, with some oxidoreductases and other pathogenesis-related proteins also identified, suggesting that aggressiveness is associated with an improved ability of GPS79 to overcome cell wall barriers and neutralize host defense responses., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Aslihan Gunel reports financial support was provided by The Scientific and Technological Research Council of Turkey., (Copyright © 2022. Published by Elsevier GmbH.)

Fusarium graminearum is a fungal pathogen that causes Fusarium head blight in cereal crops. The identification of proteins secreted from pathogens to overcome plant defenses and cause disease, collectively known as effectors, can reveal the etiology of a disease process. Proximity-dependent biotin identification (BioID) was used to identify potential effector proteins secreted in planta by F. graminearum during the infection of Arabidopsis. Mass spectrometry analysis of streptavidin affinity-purified proteins revealed over 300 proteins from F. graminearum, of which 62 were candidate effector proteins (CEPs). An independent analysis of secreted proteins from axenic cultures of F. graminearum showed a 42% overlap with CEPs, thereby assuring confidence in the BioID methodology. The analysis also revealed that 19 out of 62 CEPs (approx. 30%) had been previously characterized with virulence function in fungi. The functional characterization of additional CEPs was undertaken through deletion analysis by the CRISPR/Cas9 method, and by overexpression into Triticum aestivum (wheat) leaves by the Ustilago hordei delivery system. Deletion studies of 12 CEPs confirmed the effector function of three previously characterized CEPs and validated the function of another four CEPs on wheat inflorescence or vegetative tissues. Lastly, overexpression in wheat showed that all seven CEPs enhanced resistance against the bacterial pathogen Pseudomonas syringae DC3000., (© 2022 Her Majesty the Queen in Right of Canada. The Plant Journal © 2022 Society for Experimental Biology and John Wiley & Sons Ltd. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.)

Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analog misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury., (© 2021. The Author(s).)

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance. Abbreviations: BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3β: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta.

The Gram-positive bacterium Clavibacter nebraskensis (Cn) causes Goss's wilt and leaf blight on corn in the North American Central Plains with yield losses as high as 30%. Cn strains vary in aggressiveness on corn, with highly aggressive strains causing much more serious symptoms and damage to crops. Since Cn inhabits the host xylem, we investigated differences in the secreted proteomes of Cn strains to determine whether these could account for phenotypic differences in aggressiveness. Highly and a weakly aggressive Cn strains (Cn14-15-1 and DOAB232, respectively) were cultured, in vitro, in the xylem sap of corn (CXS; host) and tomato (TXS; non-host). The secretome of the Cn strains were extracted and processed, and a comparative bottom-up proteomics approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine their identities and concentration. Relative quantitation of peptides was based on precursor ion intensities to measure protein abundances. In total, 745 proteins were identified in xylem sap media. In CXS, a total of 658 and 396 proteins were identified in strains Cn14-5-1 and DOAB232, respectively. The unique and the differentially abundant proteins in the secretome of strain Cn14-5-1 were higher in either sap medium compared to DOAB232. These proteins were sorted using BLAST2GO and assigned to 12 cellular functional processes. Virulence factors, e.g., cellulase, β-glucosidase, β-galactosidase, chitinase, β-1,4-xylanase, and proteases were generally higher in abundance in the aggressive Cn isolate. This was corroborated by enzymatic activity assays of cellulase and protease in CXS. These proteins were either not detected or detected at significantly lower abundance levels in Cn strains grown in non-host xylem sap (tomato), suggesting potential factors involved in Cn-host (corn) interactions.

Reactive oxygen species (ROS) play an important role during host-pathogen interactions and are often an indication of induced host defence responses. In this study, we demonstrate for the first time that Puccinia triticina (Pt) generates ROS, including superoxide, H 2 O 2 and hydroxyl radicals, during wheat infection. Through pharmacological inhibition, we found that ROS are critical for both Pt urediniospore germination and pathogenic development on wheat. A comparative RNA-Seq analysis of different stages of Pt infection process revealed 291 putative Pt genes associated with the oxidation-reduction process. Thirty-seven of these genes encode known proteins. The expressions of five Pt genes, including PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod, were subsequently verified using RT-qPCR analysis. The results show that the expressions of PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod are up-regulated during urediniospore germination. In comparison, the expressions of PtNoxA, PtNoxB, PtNoxR and PtCat are down-regulated during wheat infection from 12 to 120 h after inoculation (HAI), whereas the expression of PtSod is up-regulated with a peak of expression at 120 HAI. We conclude that ROS are critical for the full virulence of Pt and a coordinate down-regulation of PtNox genes may be important for successful infection in wheat., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Wheat leaf rust caused by the pathogenic fungus, Puccinia triticina , is a serious threat to bread wheat and durum production in many areas of the world. This plant-pathogen interaction has been studied extensively at the molecular genetics level however, proteomics data are still relatively scarce. The present study investigated temporal changes in the abundance of the apoplastic fluid proteome of resistant and susceptible wheat leaves infected with P. triticina race-1, using a label-free LC-MS-based approach. In general, there was very little difference between inoculated and control apoplastic proteomes in either host, until haustoria had become well established in the susceptible host, although the resistant host responds to pathogen challenge sooner. In the earlier samplings (up to 72 h after inoculation) there were just 46 host proteins with significantly changing abundance, and pathogen proteins were detected only rarely and not reproducibly. This is consistent with the biotrophic lifestyle of P. triticina , where the invading pathogen initially causes little tissue damage or host cell death, which occur only later during the infection cycle. The majority of the host proteins with altered abundance up to 72 h post-inoculation were pathogen-response-related, including peroxidases, chitinases, β-1-3-endo-glucanases, and other PR proteins. Five days after inoculation with the susceptible apoplasm it was possible to detect 150 P. triticina proteins and 117 host proteins which had significantly increased in abundance as well as 33 host proteins which had significantly decreased in abundance. The latter represents potential targets of pathogen effectors and included enzymes which could damage the invader. The pathogen-expressed proteins-seen most abundantly in the incompatible interaction-were mostly uncharacterized proteins however, many of their functions could be inferred through homology-matching with pBLAST. Pathogen proteins also included several candidate effector proteins, some novel, and some which have been reported previously. All MS data have been deposited in the PRIDE archive (www.ebi.ac.uk/pride/archive/) under Project PXD012586., (Copyright © 2019 Rampitsch, Huang, Djuric-Cignaovic, Wang and Fernando.)

NADPH oxidase (NOX) is one of the sources of reactive oxygen species (ROS) that modulates the activity of proteins through modifications of their cysteine residues. In a previous study, we demonstrated the importance of NOX in both the development and pathogenicity of the phytopathogen Fusarium graminearum. In this article, comparative proteomics between the wild-type and a Nox mutant of F. graminearum was used to identify active cysteine residues on candidate redox-sensing proteins. A two-dimensional gel approach based on labelling with monobromobimane (mBBR) identified 19 candidate proteins, and was complemented with a gel-free shotgun approach based on a biotin switch method, which yielded 99 candidates. The results indicated that, in addition to temporal regulation, a large number of primary metabolic enzymes are potentially targeted by NoxAB-generated ROS. Targeted disruption of these metabolic genes showed that, although some are dispensable, others are essential. In addition to metabolic enzymes, developmental proteins, such as the Woronin body major protein (FGSG_08737) and a glycosylphosphatidylinositol (GPI)-anchored protein (FGSG_10089), were also identified. Deletion of either of these genes reduced the virulence of F. graminearum. Furthermore, changing the redox-modified cysteine (Cys 325 ) residue in FGSG_10089 to either serine or phenylalanine resulted in a similar phenotype to the FGSG_10089 knockout strain, which displayed reduced virulence and altered cell wall morphology; this underscores the importance of Cys 325 to the function of the protein. Our results indicate that NOX-generated ROS act as intracellular signals in F. graminearum and modulate the activity of proteins affecting development and virulence in planta., (© 2018 BSPP and John Wiley & Sons Ltd.)

Background: Members of the Alternaria genus produce various toxins whose occurrence in agricultural commodities is a major concern for humans and the environment. The present study developed a simple and efficient matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the rapid detection of Alternaria toxins., Results: A new method for the detection of alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) by MALDI-TOF MS was developed. Different solid phase extraction (SPE) clean-up methods were tried to optimize the purification of wheat matrix, and an optimal extraction method was designed to recover the three Alternaria toxins. In addition, various MALDI matrices were examined and α-cyano-4-hydroxycinnamic acid (CHCA) matrix gave good repeatability for all three Alternaria toxins., Conclusion: This is the first study to report the detection of three important Alternaria toxins concurrently using MALDI-TOF MS and opens up the possibility of rapid screening of Alternaria toxins in several other cereals and food products. © 2016 Her Majesty the Queen in Right of Canada Journal of the Science of Food and Agriculture © 2016 Society of Chemical Industry., (© 2016 Her Majesty the Queen in Right of Canada Journal of the Science of Food and Agriculture © 2016 Society of Chemical Industry.)

Protein phosphorylation is a key signaling mechanism during the plant biotic and abiotic stress response. Signaling cascades communicate between the cell surface, where the stress is perceived, and the nucleus, where a response can be enacted. Many of these signals involve the specific, transient phosphorylation of proteins by kinases, a signal which is usually amplified through cascades. The advent of high-throughput phosphoproteomics, pioneered mainly in yeast and mammalian cells, has made it possible to discover novel phosphorylation events rapidly and efficiently in a data-dependent manner and this has greatly enlarged our understanding of the plant's response to stress. This chapter describes a simple gel-free protocol for high-throughput phosphoproteomics, which is amenable to most labs engaged in plant stress research.

Ergot is a common disease of wheat and other cereal grains that is predominantly caused by Claviceps purpurea in the field, often affecting crop yield in addition to the environment. Infected grain can be contaminated with dark sclerotia, which contain fungal metabolites such as ergot alkaloids. The occurrence of ergot alkaloids in cereal grain is a major health concern for humans and livestock. Effective and rapid screening of these mycotoxins is crucial for producers, processors, and consumers of cereal-based food and feed grain. Established methods of ergot alkaloid screening based on LC-MS or GC-MS require laborious processes. A novel method using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS was developed to identify four ergot alkaloids. Using dihydroxybenzoic acid as the matrix, ergosine, ergocornine, ergocryptine, and ergocristine were readily detected in individual sclerotia of C. purpurea. The accuracy of the identified ergot alkaloids was further confirmed by tandem MS analysis. MALDI-TOF MS is suitable for high-throughput screening of ergot alkaloids because it permits rapid and accurate identification, simple sample preparation, and no derivatization or chromatographic separation.

Abiotic and biotic stresses constrain plant growth and development negatively impacting crop production. Plants have developed stress-specific adaptations as well as simultaneous responses to a combination of various abiotic stresses with pathogen infection. The efficiency of stress-induced adaptive responses is dependent on activation of molecular signaling pathways and intracellular networks by modulating expression, or abundance, and/or post-translational modification (PTM) of proteins primarily associated with defense mechanisms. In this review, we summarize and evaluate the contribution of proteomic studies to our understanding of stress response mechanisms in different plant organs and tissues. Advanced quantitative proteomic techniques have improved the coverage of total proteomes and sub-proteomes from small amounts of starting material, and characterized PTMs as well as protein-protein interactions at the cellular level, providing detailed information on organ- and tissue-specific regulatory mechanisms responding to a variety of individual stresses or stress combinations during plant life cycle. In particular, we address the tissue-specific signaling networks localized to various organelles that participate in stress-related physiological plasticity and adaptive mechanisms, such as photosynthetic efficiency, symbiotic nitrogen fixation, plant growth, tolerance and common responses to environmental stresses. We also provide an update on the progress of proteomics with major crop species and discuss the current challenges and limitations inherent to proteomics techniques and data interpretation for non-model organisms. Future directions in proteomics research toward crop improvement are further discussed.

Puccinia triticina causes leaf rust, a disease that causes annual yield losses in wheat. It is an obligate parasite that invades the host leaf and forms intracellular structures called haustoria, which obtain nutrients and suppress host immunity using secreted proteins called effectors. Since effector proteins act at the frontier between plant and pathogen and help determine the outcome of the interaction, it is critical to understand their functions. Here, we used a direct proteomics approach to identify effector candidates from P. triticina Race 1 haustoria isolated with a specific monoclonal antibody. Haustoria were >95% pure and free of host contaminants. Using high resolution MS we have identified 1192 haustoria proteins. These were quantified using normalized spectral counts and spanned a dynamic range of three orders of magnitude, with unknown proteins and metabolic enzymes as the most highly represented. The dataset contained 140 candidate effector proteins, based on the presence of a signal peptide and the absence of a known function for the protein. Some of these candidates were significantly enriched with cysteine, with up to 13 residues per protein and up to 6.8% cysteine in composition., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Pyrenophora tritici-repentis is a phytopathogenic fungus which causes tan spot on wheat. Some races of P. tritici-repentis produce host-specific toxins which present symptoms of chlorosis or necrosis on susceptible wheat cultivars. One such toxin is Ptr ToxA, which enters mesophyll cells through a putative toxin-receptor and localizes with chloroplasts, ultimately causing damage and necrosis on leaves. These symptoms can occur even in the absence of the pathogen. Insensitive cultivars lack the receptor and Ptr ToxA cannot enter cells. The molecular mechanisms surrounding this plant-pathogen interaction are still largely unknown, although some details have begun to emerge., Results: Using 2-D electrophoresis, fifteen protein changes were identified reproducibly in the leaf proteomes of a sensitive and an insensitive cultivar over three days after inoculation of purified Ptr ToxA. Functional analysis of the proteins indicated that senescence signals may be induced in the sensitive cultivar. In the insensitive cultivar proteins involved in some features of senescence inhibition were seen. Complementary responses at the biochemical level may be actively promoting a localized senescence-like response in sensitive wheat cultivars whilst actively inhibiting this response in insensitive cultivars., Conclusion: This is the first report of a biochemical response in an insensitive cultivar in this plant-pathogen interaction. Findings support the involvement of ethylene, and the activation of complementary pathways in sensitive versus insensitive wheat cultivars responding to Ptr ToxA. The nature of the system permits using purified toxin to mimic disease, which eliminates the pathogen proteome and ensures a synchronous response in inoculated leaves.

To our knowledge, this study represents the first high-throughput characterization of a stigma proteome in the Triticeae. A total of 2184 triticale mature stigma proteins were identified using three different gel-based approaches combined with mass spectrometry. The great majority of these proteins are described in a Triticeae stigma for the first time. These results revealed many proteins likely to play important roles in stigma development and pollen-stigma interactions, as well as protection against biotic and abiotic stresses. Quantitative comparison of the triticale stigma transcriptome and proteome showed poor correlation, highlighting the importance of having both types of analysis. This work makes a significant contribution towards the elucidation of the Triticeae stigma proteome and provides novel insights into its role in stigma development and function., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Wheat seeds can be released from a dormant state by after-ripening; however, the underlying molecular mechanisms are still mostly unknown. We previously identified transcriptional programmes involved in the regulation of after-ripening-mediated seed dormancy decay in wheat (Triticum aestivum L.). Here, we show that seed dormancy maintenance and its release by dry after-ripening in wheat is associated with oxidative modification of distinct seed-stored mRNAs that mainly correspond to oxidative phosphorylation, ribosome biogenesis, nutrient reservoir and α-amylase inhibitor activities, suggesting the significance of post-transcriptional repression of these biological processes in regulating seed dormancy. We further show that after-ripening induced seed dormancy release in wheat is mediated by differential expression of specific proteins in both dry and hydrated states, including those involved in proteolysis, cellular signalling, translation and energy metabolism. Among the genes corresponding to these proteins, the expression of those encoding α-amylase/trypsin inhibitor and starch synthase appears to be regulated by mRNA oxidation. Co-expression analysis of the probesets differentially expressed and oxidized during dry after-ripening along with those corresponding to proteins differentially regulated between dormant and after-ripened seeds produced three co-expressed gene clusters containing more candidate genes potentially involved in the regulation of seed dormancy in wheat. Two of the three clusters are enriched with elements that are either abscisic acid (ABA) responsive or recognized by ABA-regulated transcription factors, indicating the association between wheat seed dormancy and ABA sensitivity., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Tremendous progress in plant proteomics driven by mass spectrometry (MS) techniques has been made since 2000 when few proteomics reports were published and plant proteomics was in its infancy. These achievements include the refinement of existing techniques and the search for new techniques to address food security, safety, and health issues. It is projected that in 2050, the world's population will reach 9-12 billion people demanding a food production increase of 34-70% (FAO, 2009) from today's food production. Provision of food in a sustainable and environmentally committed manner for such a demand without threatening natural resources, requires that agricultural production increases significantly and that postharvest handling and food manufacturing systems become more efficient requiring lower energy expenditure, a decrease in postharvest losses, less waste generation and food with longer shelf life. There is also a need to look for alternative protein sources to animal based (i.e., plant based) to be able to fulfill the increase in protein demands by 2050. Thus, plant biology has a critical role to play as a science capable of addressing such challenges. In this review, we discuss proteomics especially MS, as a platform, being utilized in plant biology research for the past 10 years having the potential to expedite the process of understanding plant biology for human benefits. The increasing application of proteomics technologies in food security, analysis, and safety is emphasized in this review. But, we are aware that no unique approach/technology is capable to address the global food issues. Proteomics-generated information/resources must be integrated and correlated with other omics-based approaches, information, and conventional programs to ensure sufficient food and resources for human development now and in the future., (© 2013 Wiley Periodicals, Inc.)

To understand early events in plant-pathogen interactions, it is necessary to explore the pathogen secretome to identify secreted proteins that help orchestrate pathology. The secretome can be obtained from pathogens grown in vitro, and then characterized using standard proteomic approaches based on protein extraction and subsequent identification of tryptic peptides by LC-MS. A subset of the secretome is composed of proteins whose presence is required to initiate infection and their removal from the secretome would result in pathogens with reduced or no virulence. We present here comparative secretome from Fusarium graminearum. This filamentous fungus causes Fusarium head blight on wheat, a serious cereal disease found in many cereal-growing regions. Affected grain is contaminated with mycotoxins and cannot be used for food or feed. We used label-free quantitative MS to compare the secretomes of wild-type with two nonpathogenic deletion mutants of F. graminearum, Δtri6, and Δtri10. These mutations in mycotoxin-regulating transcription factors revealed a subset of 29 proteins whose relative abundance was affected in their secretomes, as measured by spectral counting. Proteins that decreased in abundance are potential candidate virulence factors and these included cell wall-degrading enzymes, metabolic enzymes, pathogenesis-related proteins, and proteins of unknown function., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hormone signaling crosstalk plays a major role in plant defense against a wide range of both biotic and abiotic stresses. While many reviews on plant-microbe interactions have well described the general trends of signaling pathways in shaping host responses to pathogens, few discussions have considered a synthesis of positive versus negative interactions among such pathways, or variations in the signaling molecules themselves. This review deals with the interaction trends between salicylic, jasmonic, and abscisic acids in the signaling pathways, as well as exceptions to such trends. Here we focused on antagonistic versus cooperative interactions between salicylic and jasmonic acids, two major disease resistance signaling molecules, and some interactions with abscisic acid, a known abiotic stress hormone, and another player in plant defense mechanisms. We provide a set of examples materializing either antagonism or cooperation for each interaction between two pathways, thereby showing the trends and pinpointing the exceptions. Such analyses are practical for researchers working on the subject and essential for a better exploitation of the data already available in plant disease resistance signaling, both in Arabidopsis and crop species, toward the development of better disease management strategies for economically important crops., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Mitogen-activated protein kinase (MAPK) pathways represent a crucial regulatory mechanism in plant development. The ability to activate and inactivate MAPK pathways rapidly in response to changing conditions helps plants to adapt to a changing environment. AtMKK1 is a stress response kinase that is capable of activating the MAPK proteins AtMPK3, AtMPK4 and AtMPK6. To elucidate its mode of action further, several tests were undertaken to examine the response of AtMKK1 to salt stress using a knockout (KO) mutant of AtMKK1. We found that AtMKK1 mutant plants tolerated elevated levels of salt during both germination and adulthood. Proteomic analysis indicated that the level of the α subunit of mitochrondrial H(+)-ATPase, mitochrondial NADH dehydrogenase and mitochrondrial formate dehydrogenase was enhanced in AtMKK1 knockout mutants upon high salinity stress. The level of formate dehydrogenase was further confirmed by immunoblotting and enzyme assay. The possible involvement of these enzymes in salt tolerance is discussed.

Systems biology is a scientific approach that integrates many scientific disciplines to develop a comprehensive understanding of biological phenomena, thus allowing the prediction and accurate simulation of complex biological behaviors. It may be presumptuous to write about toxin regulation at the level of systems biology, but the last decade of research is leading us closer than ever to this approach. Past research has delineated multiple levels of regulation in the pathways leading to the biosynthesis of secondary metabolites, including mycotoxins. At the top of this hierarchy, the global or master transcriptional regulators perceive various environmental cues such as climatic conditions, the availability of nutrients, and the developmental stages of the organism. Information accumulated from various inputs is integrated through a complex web of signalling networks to generate the eventual outcome. This review will focus on adapting techniques such as chemical and other genetic tools available in the model system Saccharomyces cerevisiae, to disentangle the various biological networks involved in the biosynthesis of mycotoxins in the Fusarium spp.

It has been clearly demonstrated that plants redox control can be exerted over virtually every cellular metabolic pathway affecting metabolic homeostasis and energy balance. Therefore, a tight link exists between cellular/compartmental steady-state redox level and cellular metabolism. Proteomics offers a powerful new way to characterize the response and regulation of protein oxidation in different cell types and in relation to cellular metabolism. Compelling evidence revealed in proteomics studies suggests the integration of the redox network with other cellular signaling pathways such as Ca(2+) and/or protein phosphorylation, jasmonic, salicylic, abscisic acids, ethylene, and other phytohormones. Here we review progress in using the various proteomics techniques and approaches to answer biological questions arising from redox signaling and from changes in redox status of the cell. The focus is on reversible redox protein modifications and on three main processes, namely oxidative and nitrosative stress, defense against pathogens, cellular redox response and regulation, drawing on examples from plant redox proteomics studies., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Seed dormancy is an important trait in wheat (Trticum aestivum L.) and it can be released by germination-stimulating treatments such as after-ripening. Previously, we identified proteins specifically associated with after-ripening mediated developmental switches of wheat seeds from the state of dormancy to germination. Here, we report seed proteins that exhibited imbibition induced co-regulation in both dormant and after-ripened seeds of wheat, suggesting that the expression of these specific proteins/protein isoforms is not associated with the maintenance or release of seed dormancy in wheat.

Enzymatic antioxidant system plays an important role in maintaining seed vigor and regulating plant growth and development. It involves a number of enzymes that scavenge excessive reactive oxygen species (ROS) produced during seed aging and also modulate the level of these compounds during plant developmental processes. This study investigated the transcriptional regulation of enzymatic antioxidative capacity in pea during the pre- and post-germinative phases and in response to seed aging by analyzing the spatio-temporal expression of five antioxidative genes: PsAPX, PsSOD, PsGRcyt, PsGRcm and PsCAT. Transcripts of all these genes were found in mature dry seeds, embryo axes and cotyledons of germinating seeds, and cotyledons, roots and shoots of young seedlings. However, PsAPX and PsSOD were predominant and exhibited developmental regulation, suggesting that these genes play important roles in controlling the intracellular homeostasis of ROS for promoting cell elongation, and thereby embryo axis expansion and early seedling growth in pea. Accelerated aging of pea seeds led to reduction in seed viability and seedling growth, and this effect was correlated with substantial decrease in the transcriptional activation of the prominent antioxidative genes. Furthermore, our proteomic analysis indicated the association of seed aging with changes in the abundance of specific proteins, revealing additional mechanisms underlying seed aging in pea., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

This review examines why a knowledge of plant protein phosphorylation events is important in devising strategies to protect crops from both biotic and abiotic stresses, and why proteomics should be included when studying stress pathways. Most of the achievements in elucidating phospho-signaling pathways in biotic and abiotic stress are reported from model systems: while these are discussed, this review attempts mainly to focus on work done with crops, with examples of achievements reported from rice, maize, wheat, grape, Brassica, tomato, and soy bean after cold acclimation, hormonal and oxidative hydrogen peroxide treatment, salt stress, mechanical wounding, or pathogen challenge. The challenges that remain to transfer this information into a format that can be used to protect crops against biotic and abiotic stresses are enormous. The tremendous increase in the speed and ease of DNA sequencing is poised to reveal the whole genomes of many crop species in the near future, which will facilitate phosphoproteomics and phosphogenomics research.

This study presents a high-throughput proteomic analysis of phosphopeptides from Fusarium graminearum strain DAOM 233423 grown in vitro without nutritional limitation. Using a combination of strong cation exchange (SCX) and immobilized metal affinity chromatography (IMAC) followed by LC-MS, we identified 2902 putative phosphopeptides with homologous matches to 1496 different proteins. Functional classification of the annotated protein set revealed that phosphopeptides from nuclear proteins with ATP-binding function were the most abundant. There are indications that phosphorylation sites from well-characterized phosphoproteins representing diverse biological processes are conserved in F. graminearum: sequences of three phosphopeptides from known phosphoproteins (transcription elongation factor 1β, acidic ribosomal proteins, and glycogen synthase) revealed phosphorylation site conservation., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The study of plant disease and immunity is benefiting tremendously from proteomics. Parallel streams of research from model systems, from pathogens in vitro and from the relevant pathogen-crop interactions themselves have begun to reveal a model of how plants succumb to invading pathogens and how they defend themselves without the benefit of a circulating immune system. In this review, we discuss the contribution of proteomics to these advances, drawing mainly on examples from crop-fungus interactions, from Arabidopsis-bacteria interactions, from elicitor-based model systems and from pathogen studies, to highlight also the important contribution of non-crop systems to advancing crop protection., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The thiol redox-sensitive and the total proteome in harvest-ripe grains of closely related genotypes of wheat (Triticum aestivum L.), with either a dormant or a non-dormant phenotype, were investigated using hybrid lines of spring wheat double haploid population segregating transgressively, to gain further insight into seed dormancy controlling events. Redox signalling by reactive oxygen species has been shown to play a role in seed dormancy alleviation. Thiol-disulfide proteins are of particular importance in the context of redox-dependent regulation as a central and flexible mechanism to control metabolic and developmental activities of the cells. Here we describe functional proteomic profiling of reversible oxidoreductive changes and characterize in vivo intrinsic reactivity of cysteine residues using thiol-specific fluorescent labelling, solubility-based protein fractionation, two-dimensional electrophoresis, and mass spectrometry analysis in conjunction with wheat EST sequence libraries. Quantitative differences between genotypes were found for 106 spots containing 64 unique proteins. Forty seven unique proteins displayed distinctive abundance pattern, and among them 31 proteins contained 78 unique redox active cysteines. Seventeen unique proteins with 19 reactive modified cysteines were found to have differential post-translational thiol redox modification. The results provide an insight into the alteration of thiol-redox profiles in proteins that function in major processes in seeds and include groups of redox- and stress-responsive, genetic information processing and cell cycle control, transport and storage proteins, enzymes of carbohydrate metabolism, proteases and their inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Puccinia triticina (Pt) is a representative of several cereal-infecting rust fungal pathogens of major economic importance world wide. Upon entry through leaf stomata, these fungi establish intracellular haustoria, crucial feeding structures. We report the first proteome of infection structures from parasitized wheat leaves, enriched for haustoria through filtration and sucrose density centrifugation. 2-D PAGE MS/MS and gel-based LC-MS (GeLC-MS) were used to separate proteins. Generated spectra were compared with a partial proteome predicted from a preliminary Pt genome and generated ESTs, to a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) and to various plant resources. We identified over 260 fungal proteins, 16 of which matched peptides from Pgt. Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six have effector protein signatures, some are related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. Measuring gene expression over several life cycle stages of ten representative candidates using quantitative RT-PCR, all were shown to be strongly upregulated and four expressed solely upon infection., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oxidative signalling by ROS has been demonstrated to play a role in seed dormancy alleviation, but the detailed molecular mechanisms underlying this process remain largely unknown. Here, we show dynamic differences in redox-sensitive proteome upon wheat seed dormancy release. Using thiol-specific fluorescent labelling, solubility-based protein fractionation, 2-D IEF PAGE, and MS analysis in conjunction with wheat EST sequence libraries, proteins with reversible oxidoreductive changes were characterized. Altogether, 193 reactive Cys were found in 79 unique proteins responding differentially in dormant, non-dormant, abscisic, or gibberellic acid-treated seed protein extracts from RL4137, a wheat cultivar with extreme dormancy. The identified proteins included groups that are redox-, stress-, and pathogen-responsive, involved in protein synthesis and storage, are enzymes of carbohydrate metabolism, proteases, and those involved in transport and signal transduction. Two types of redox response could be detected: (i) a dramatic increase in protein thiol redox state in seeds during imbibition and hormonal treatment; (ii) higher antioxidant capacity related to sensing of a threshold redox potential and balancing the existing redox pools, in dry dormant versus non-dormant seeds. These results highlight occurrence of the antioxidant defence mechanisms required for the protection of seed during a dormancy stage., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To gain more insights into the translational and PTM that occur in rat offspring exposed to alcohol in utero, 2-D PAGE with total, phospho- and glycoprotein staining and MALDI-MS/MS and database searching were conducted. The results, based on fold-change expression, revealed a down-regulation of total protein expression by prenatal alcohol exposure in 7-day-old and 3-month-old rats. There was an up-regulation of protein phosphorylation but a down-regulation of glycosylation by prenatal alcohol exposure in both age groups. Of 31 protein spots examined per group, differentially expressed proteins were identified as ferritin light chain, aldo-keto reductase, tumor rejection antigen gp96, fructose-1,6-bisphosphatase, glycerol-3-phosphate dehydrogenase, malate dehydrogenase, and gamma-actin. Increased phosphorylation was observed in proteins such as calmodulin, gluthatione S-transferase, glucose regulated protein 58, alpha-enolase, eukaryotic translation elongation factor 1 beta-2, riboprotein large P2, agmatinase, ornithine carbamoyltransferase, quinolinate phosphoribosyltransferase, formimidoyltransferase cyclodeaminase, and actin. In addition, glycosylation of adenosine kinase, adenosylhomocysteine hydrolase, and 3-hydroxyanthranilate dioxygenase was reduced. Pathways affected by these protein alterations include cell signaling, cellular stress, protein synthesis, cytoskeleton, as well as glucose, aminoacid, adenosine and energy metabolism. The activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase was elevated by prenatal alcohol. The observations may have important physiological implications.

Fusarium graminearum grown under stress, such as nutrient deprivation, activates, among others, the trichothecene pathway that produces the mycotoxin deoxynivalenol and its derivatives. The kinase inhibitor staurosporine reduced the production of trichothecenes by 39% compared with control in vitro. On the other hand, phosphatase inhibitor okadaic acid increased the amount by 72% compared with the control in vitro. This suggests that phosphorylation events are involved in the signalling pathway, leading to the activation of the trichothecene pathway. Three approaches were used to study the phosphoproteome of F. graminearum under nitrogen-limiting conditions: 2-DE (2-DE: IEFxSDS-PAGE) in combination with MS protein identification; SDS-PAGE in combination with off-line IMAC and TiO(2) enrichment and gel electrophoresis LC-MS analysis; and a gel-free approach using strong anion exchange chromatography, IMAC and LC-MS. A total of 348 phosphorylation sites localized in 301 peptides from 241 proteins were identified. By 2-DE, 20 phosphoproteins were identified, nine of which underwent changes during the time course examined. Using gel electrophoresis LC-MS 231 phosphopeptides were identified from three samples (ten gel slices each) at time points of nitrogen starvation t=0, 6, and 12 h. The gel-free analysis added 70 peptides from 65 proteins to the total. Proteins of unknown function and enzymes of known function comprised the largest groups overall. Ten protein kinases and seven transcription factors were identified. This is the first reported phosphoproteome of F. graminearum.

Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396-9) and weakly (Vs06-14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty-five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC-ESI-MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant-defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics-based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.

The term 'Proteomics' was introduced in 1997 to describe a growing interest in the study of the proteome - the expressed protein set of an organism. As this new discipline evolved, it quickly became obvious that proteomics would be a very complex and ambitious undertaking, perhaps even more so than genomics, which had engendered it. New techniques for both the separation and analysis/identification of proteins were emerging or being refined, and these facilitated the development of this new field. Many proteomics experiments are now routine in some laboratories. In this chapter we describe a typical proteomics experiment, using examples from our laboratory: the separation of complex mixtures of proteins by 2-dimensional electrophoresis and subsequent identification of a protein spot by mass spectrometry with two commonly used instruments: MALDI-QqTOF and ESI-ion trap.

Callus is an important intermediate tissue for the propagation of plants through tissue culture, as it can form new shoots, and hence plants, through somatic embryogenesis, a process whose underlying biochemical mechanisms are poorly understood. The involvement of kinases suggests that signalling through phosphorylation cascades may play an important role. In this report methods for labelling wheat (Triticum aestivum L.) callus phosphoproteins in vivo through the uptake of [(32)P]orthophosphate and subsequently revealing the phosphoproteome of this tissue by two-dimensional electrophoresis and autoradiography are described. Labelled proteins from 2D gels were identified by tandem mass spectrometry analysis using the 'Mascot' search engine, and by de novo sequencing in combination with BLAST and MS-BLAST, using a cross-species protein identification approach. Eight putative phosphoproteins from 10 spots were identified, confirming the potential utility of this method; three of these have not previously shown to be phosphorylated. Furthermore, the phosphorylation site for one of the proteins was identified. Protein synthesis functions were tentatively assigned to five of the phosphoproteins, one reactive oxygen species (ROS)-detoxifying enzyme ascorbate peroxidase, and two could not be identified reliably from current database matches., (Crown Copyright © 2006. Published by Elsevier Ireland Ltd. All rights reserved.)

Flax's recent popularity in human and animal foods is mostly due to its desirable FA composition. Flax is an excellent source of omega-3 FA, which have been shown to have many health benefits. To date, little is known about the genetic and environmental factors that control the FA composition of flax seeds. To elucidate some of the important genetic components, reverse transcriptase (RT)-PCR and real-time PCR were used to determine the expression profiles of two key FA biosynthetic genes during seed development. Plants of flax cultivar AC McDuff were grown under field conditions, and RNA was extracted from ovaries and developing bolls collected from 2 d after anthesis (DAA) to maturity. Desaturation enzymes stearoyl-ACP desaturase (SAD) and delta12 FA desaturase 2 (FAD2) were both expressed in ovaries, and their expression was differentially modulated throughout seed development. SAD was most highly expressed in ovaries. Its expression quickly decreased until 4 DAA; this was followed by a slight peak at 8 DAA, only to return to relatively low levels of expression in maturing bolls, ranging from 2.1% to 4.5% relative to the level observed in ovaries. FAD2 expression displayed a different temporal pattern. While expression of FAD2 did decrease in the early stages of seed development, expression increased starting at 8 DAA, peaking at 16 DAA, when it was 158% relative to the level observed in ovaries. FAD2, which desaturates oleic acid (18:1cisdelta9) into linoleic acid (18:2cisdelta9,12), is therefore controlled at the transcription level. To relate enzyme expression with FA profile, GC was performed on the same subsamples used for RT-PCR and real-time PCR, and proportions of palmitic, stearic, oleic, linoleic, and linolenic acids were determined for the same developmental stages. Although FAD2 expression increased from 8 to 16 DAA, relative changes in linoleic acid (18:2cis delta9,12) were not observed. However, linolenic acid (ALA; alpha-18:3; 18:3cisdelta9,12,15) levels increased steadily, meaning that linoleic acid (18:2cisdelta9,12) is a transient substrate converted by FAD3 as quickly as it is produced by FAD2. Phenotypes are the result of genotypes, environment, and the interaction of the two. To evaluate the environmental impact on the production of FA in flax, FA profiles were assessed in a total of four environments (two locations, two years). Warm and dry environmental conditions resulted in lower levels of PUFA 18:2cisdelta9,12 and 18:3cisdelta9,12,15, and higher levels of 18:1 cisdelta9. FAD2 expression and/or activity may therefore be affected by the environment.

Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing two chaotropes (urea and thiourea), two detergents (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate and N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate), two reducing agents (dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride) and two types of carrier ampholytes (BioLyte pH 4-6 and pH 3-10) solubilized the most acidic proteins in the pH range between 4 and 7. The buffer made up of urea, thiourea, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, DeStreak reagent (Amersham Biosciences, Uppsala, Sweden) and immobilized pH gradient buffer, pH 6-11 (Amersham Biosciences) solubilized the most basic proteins in the pH range between 6 and 11. These two buffers produced two-dimensional gels with high resolution, superior quality and maximum number of detectable protein (1425 acidic protein and 897 basic protein) spots.

Wheat leaf rust is caused by the fungus Puccinia triticina. The genetics of resistance follows the gene-for-gene hypothesis, and thus the presence or absence of a single host resistance gene renders a plant resistant or susceptible to a leaf rust race bearing the corresponding avirulence gene. To investigate some of the changes in the proteomes of both host and pathogen during disease development, a susceptible line of wheat infected with a virulent race of leaf rust were compared to mock-inoculated wheat using 2-DE (with IEF pH 4-8) and MS. Up-regulated protein spots were excised and analyzed by MALDI-QqTOF MS/MS, followed by cross-species protein identification. Where possible MS/MS spectra were matched to homologous proteins in the NCBI database or to fungal ESTs encoding putative proteins. Searching was done using the MASCOT search engine. Remaining unmatched spectra were then sequenced de novo and queried against the NCBInr database using the BLAST and MS BLAST tools. A total of 32 consistently up-regulated proteins were examined from the gels representing the 9-day post-infection proteome in susceptible plants. Of these 7 are host proteins, 22 are fungal proteins of known or hypothetical function and 3 are unknown proteins of putative fungal origin.

MALDI tandem mass spectrometry analysis on a hybrid quadrupole-quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a novel glycoprotein, pathogenesis-related subtilisin-like proteinase P69B from tomato. Glycopeptide fractions from microcolumn reversed-phase HPLC deposited on MALDI targets were identified from MS by their specific m/z spacing patterns (203, 162, 146 u) between glycoforms. In most cases, MS/MS spectra of [M + H]+ ions of glycopeptides featured peaks useful for determining sugar compositions, peptide sequences, and thus probable glycosylation sites. Furthermore, peptide-related product ions could readily be used in database search procedures to identify the glycoprotein. Four out of five predicted glycosylation sites were biologically relevant and occupied by five N-linked glycan side chains each. In addition, the fragmentation efficiency allowed detection of further modification of methionine-containing glycoforms with either oxidized or iodoacetamide alkylated methionine. The high resolution furnished by MALDI-Qq-TOF allowed rapid and sensitive structural characterization of site-specific N-glycosylation from a limited quantity of material and revealed heterogeneity at different levels, including different glycan side-chain modifications, and heterogeneity of oligosaccharide structures on the same glycosylation site.

A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.