Determining the Impact of Genotype × Environment on Oat Protein Isolate Composition Using HPLC and LC-MS Techniques.

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.

Foliar application of plant-derived peptides decreases the severity of leaf rust (Puccinia triticina) infection in bread wheat (Triticum aestivum L.).

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, ß-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.

Biochemical and proteomic response of the freshwater green alga Pseudochlorella pringsheimii to iron and salinity stressors.

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.

Increased levels of cell wall degrading enzymes and peptidases are associated with aggressiveness in a virulent isolate of Pyrenophora teres f. maculata.

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.

Proximity-dependent biotinylation identifies a suite of candidate effector proteins from Fusarium graminearum.

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.

Misoprostol treatment prevents hypoxia-induced cardiac dysfunction through a 14-3-3 and PKA regulatory motif on Bnip3.

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.

Secretome Analysis of Clavibacter nebraskensis Strains Treated with Natural Xylem Sap In Vitro Predicts Involvement of Glycosyl Hydrolases and Proteases in Bacterial Aggressiveness.

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, ß-glucosidase, ß-galactosidase, chitinase, ß-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.

BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation.

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/14-3-3ß: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta.

The role of reactive oxygen species in the virulence of wheat leaf rust fungus Puccinia triticina.

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, H2 O2 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.

Temporal Quantitative Changes in the Resistant and Susceptible Wheat Leaf Apoplastic Proteome During Infection by Wheat Leaf Rust (Puccinia triticina).

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, ß-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.

Redox signalling from NADPH oxidase targets metabolic enzymes and developmental proteins in Fusarium graminearum.

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 (Cys325 ) 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 Cys325 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.

Phosphoproteomics Analysis for Probing Plant Stress Tolerance.

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.

Advances in plant proteomics toward improvement of crop productivity and stress resistancex.

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.

Proteome changes induced by Pyrenophora tritici-repentis ToxA in both insensitive and sensitive wheat indicate senescence-like signaling.

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.

Proteome of monoclonal antibody-purified haustoria from Puccinia triticina Race-1.

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.

Proteomic profiling reveals insights into Triticeae stigma development and function.

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.

Wheat seed proteins regulated by imbibition independent of dormancy status.

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.

Integrated analysis of seed proteome and mRNA oxidation reveals distinct post-transcriptional features regulating dormancy in wheat (Triticum aestivum L.).

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.

Towards systems biology of mycotoxin regulation.

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.