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1
Noreen, Sadaf. "Plant-microbe interactions : metabolite analysis and proteomics". Thesis, University of Manchester, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488230.
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Wang, Shengbing y 王聖兵. "A proteomic study of the green microalga haematococcus pluvialis". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31246047.
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Abeysinghe, Arachchige Jayami Kaushalya Abeysinghe. "Mechanism of WRKY transcription factors-mediated defense and heterosis in Arabidopsis polyploids". HKBU Institutional Repository, 2018. https://repository.hkbu.edu.hk/etd_oa/596.
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WRKY transcription factors (TFs) belong to a large family of regulatory proteins in plants that modulate many plant processes. Extensive studies have been conducted on WRKY-mediated defense response in Arabidopsis thaliana and many crop species. This study aims to investigate the potential roles and contributions of WRKY TFs regulation in improving defense response in the resynthesized Arabidopsis allotetraploids (Arabidopsis suecica) from two related autotetraploid progenitors, Arabidopsis thaliana (At4) and Arabidopsis arenosa (Aa). Upon infection by Pseudomonas syringae (Pst), the allotetraploids has showed enhanced resistance against the pathogen when compared to the parents. Rapid induction of WRKY18, WRKY40, WRKY38, WRKY53, WRKY6; MAP kinase pathway related genes, WRKY33, PAD3; SA-pathway related genes, ICS1, EDS1, PBS3, MYB31; was evident in response to Pst and salicylic acid treatment in the allotetraploids. Cleaved amplified polymorphic sequences analysis further revealed that the AtWRKY18, AaWRKY40, AtWRKY33, and AtWRKY60 alleles expressed at higher levels when compared to their respective homoeologs in the allotetraploids, suggesting potential altered protein-protein interaction networks in the hybrids. Therefore, a split-luciferase complementation assay was used to characterize and quantify protein-protein interaction among these homoeologous WRKYs in the allotetraploids. Results showed that preferential protein-protein interactions exist for the cis-interacting AtWRKY18/AtWRKY18 homodimer or trans-interacting AtWRKY18/AaWRKY40 heterodimer when compared to the respective interacting complexes. In addition, differential affinities of WRKY18 and WRKY40 homo- and hetero- dimers toward the W-boxes at the WRKY60 promoter were observed. In the allotetraploids, PR1 expression was repressed under basal state when compared to the progenitors. Although PR1 is expressed at a higher level in A. thaliana, its expression fold change was higher and faster in the all otetraploids upon salicylic acid treatment. Transient expression of WRKY18 or WRKY40 homodimer in various combinations induced differential expression of PR1 gene in their respective wrky18 and wrky40 Arabidopsis thaliana mutants. In contrast, similar PR1 induction by homodimer in various combinations was observed when they were transiently expressed in the allotetraploids. In addition, transgenic AtWRKY18 overexpression plant displayed enhanced disease resistance against Pst when compared to AaWRKY18 overexpression lines. Such enhanced disease resistance was found to associate with the higher expression of PR1 and PR2 in AtWRKY18 transgenic lines. Moreover, differential Pst-induced expression of the direct targets (ICS1, EDS1 and PBS3) of WRKY18 in the Arabidopsis AtWRKY18 and AaWRKY18 overexpressors supported a biological difference between the At and Aa homodimers in mediating the targets regulation, thus contributing to the difference in disease responses. Overall, our findings suggested that the rapid differential alleles expression and altered protein-protein or protein-DNA interactions of WRKY transcription factors could contribute to the improved defense in the allotetraploids, providing a molecular basis of for heterotic phenotype development in hybrids.
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Cavaliere, Chiara. "Studies of plant proteomics and metabolomics by means of multidimensional analytical techniques". Doctoral thesis, La Sapienza, 2007. http://hdl.handle.net/11573/916872.
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Lau, Edward y 劉家明. "Combinatorial use of SCX and RP-RP separation for iTRAQ-based quantitative proteomics profiling". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44923120.
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Tan, Yew-Foon. "Metal-protein interactome in plant mitochondria". University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, 2009. http://theses.library.uwa.edu.au/adt-WU2009.0162.
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[Truncated abstract] Transition metals in the plant mitochondrion have dual roles in regulating the function of the organelle. While metals participate in mitochondrial respiratory metabolism as ligands in bioenergetic, detoxifying, and various other metabolic enzymes, a breakdown in metal homeostasis during oxidative stress can perpetuate the cycling of ROS by redox active metal ions. Large-scale studies into the duplicitous roles of metal ions in biological systems has been lacking and in this thesis, a combination of metallomics, database annotations, membrane proteomics, metal-protein interactomics, structural biology, functional assays and mass spectrometry were all used to gain a clearer insight into the involvement of metal ions in affecting plant mitochondrial function. The Arabidopsis mitochondrion was shown to contain the transition metals cobalt, copper, iron, manganese, molybdenum, and zinc. Interestingly, the redox active copper and iron represented 75% of the mitochondrial metallome and these metal species were revealed to be highly labile during oxidative stress suggesting a possible contribution of metal-catalysed oxidation (MCO) in the damage of biological macromolecules. Bioinformatic analysis of metalloproteins predicted and experimentally determined to be mitochondrially localised revealed that metal ion transporters are poorly characterised. An in-depth proteomic analysis of the membrane proteome was conducted on mitochondria isolated from unstressed and stressed cell cultures resulted in the identification of stress-responsive as well as potential metal ion transporters. Also, many of the annotated metalloproteins predicted to be mitochondrial lack experimental evidence for subcellular localisation. ... However, based on evidence in the literature, it was hypothesised that metal-interacting sites may be the targets for MCO due to their affinity for metal ions. Attempts were made to identify the site specificity of MCO on mitochondrial proteins but no carbonyl sites could be found owing to technical problems associated with non-specific binding of proteins to the enrichment resin and low abundance of the labelled protein carbonyls. The use of the model protein BSA showed that protein oxidation occurs in clusters and the use of model peptides demonstrated that the ability of amino acid residues to complex metal ions is important in dictating susceptibility to MCO. Further experimental verification for the site specificity of MCO is required to determine the consequences of MCO on mitochondrial protein function. Overall, this thesis provided a large-scale analysis of the contributions of metal ions to mitochondrial respiratory metabolism with an emphasis on metal ion induced toxicity. Using multi-facetted approaches, an insight into the dynamic nature of mitochondrial metal homeostasis, stress responsive transporters, the interactions of metal ions with mitochondrial proteins and the possible mechanism in which proteins are specifically oxidised by MCO has been uncovered paving the way for future focused studies characterising the consequences of oxidative stress on specific proteins and their function.
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Tan, Hooi Sin. "Proteomics analysis of somatic embryogenesis in tissue culture of oil palm (Elaeis guineensis Jacq)". Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33755/.
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Oil palm is an important commercial crop in Malaysia where Malaysia is the second largest producer and exporter of palm oilin the world. In order to meet the increasing demand for palm oil, elite oil palm planting materials with higher palm oil yield are the desirable planting materials. Hence, the oil palm plantation companies have incorporated in vitro micropropagation technique through somatic embryogenesis in producing elite oil palm. However, low embryogenesis rate has hampered large production of elite oil palm ramets. In this study, proteomic technology was deployed to compare protein expression and identify differential expressed protein between high and low proliferated embryogenic lines of oil palm tissue culture. From the study, total protein of oil palm young and old leaves was extracted using an optimized trichloroacetic acid/acetone precipitation protocol followed by polyethylene glycol (PEG) fractionation to isolate low abundance proteins. Then, the extracted proteins were separated on two-dimensional (2D) gel electrophoresis and protein profiles between the high and low proliferated embryogenic lines were compared. Total of 40 differentially expressed protein spots were isolated from the 2D gel for mass spectrophotometry (MS/MS) identification. However, only 26 out of 40 protein spots were identified and just 8 of the identified protein spots were isolated from young leaves. Quantitative real-time PCR were conducted on 17 proteins candidates to study on the relationship between the protein and mRNA expression level. There was 29% of the 17 proteins’ expression showed linear correlation with their mRNA expression. These proteins candidates were highlighted for further validation in the future.
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Nqumla, Ntombekhaya. "Evaluation of various proteomic techniques to identify proteins involved in cereal stress responses to aphid infestation". Thesis, University of Fort Hare, 2012. http://hdl.handle.net/10353/d1004572.
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All plants are exposed to abiotic and biotic stresses and have developed intricate signalling responses to survive. They respond to cell-structure disruption caused by herbivore probing and feeding by the formation of callose. Callose is a linear homopolymer made up of β-1,3-linked glucose residues with some β-1,6-branches. Plant responses to abiotic or biotic stress share events such as phosphorylation, membrane depolarization, calcium influx and the release of reactive oxygen species such as hydrogen peroxide. These events lead to the up-regulation of several pathways leading to biosynthesis of signalling molecules such as salicylic acid, jasmonate, abscisic acid and ethylene pathways. The aim of this study was to determine the most suitable proteomic approach for identifying proteins and signalling pathways involved in cereal response to aphid infestation. An in silico approach was first evaluated in which the 5ʹ upstream regulatory region of proteins belonging to the family of callose synthases was scanned for cis-regulatory elements in order to identify which callose synthases are possibly expressed in plants during biotic or abiotic stresses. Bioinformatics tools were used in the identification of twelve Arabidopsis and ten rice callose synthase coding regions. Genome sequences for rice and Arabidopsis were scanned for the 2000 bp 5ʹ region upstream of the start codon of each callose synthase coding region. PlantCare, PLACE and Athena software were used to identify putative cis-regulatory elements present in the 2000 bp 5ʹ upstream sequences. The majority of cis-acting elements identified were involved in drought and high temperature responses and only one cis-acting element was involved in wound stress. These results therefore indicated a probable role for plant callose synthases in drought stress responses rather than in biotic stress responses. Genevestigator analysis of Arabidopsis results of micro-array experiments indicated that AtGSL10 is highly up-regulated, with AtGSL1, 3, 5, 6, 7, 8, 11 and 12 showing medium up-regulation and AtGSL2, 4 and 9 no up-regulation during aphid infestation of Arabidopsis plants, implicating a possible role for AtGSL10 in the plant response to aphid infestation. An LC/MS/MS approach was used to identify specific signalling pathways involved in wheat resistance or stress response to aphid infestation. Eight proteins were identified as being up-regulated during aphid feeding in wheat, and 11 proteins were identified as possibly involved in the wheat resistance mechanism against aphid infestation. Several proteins were also identified as constitutively expressed proteins, during normal conditions and aphid infestation. Most pathways identified with proteins up-regulated in the resistance mechanisms of TugelaDN plants, were related to energy metabolism and located in the chloroplast. Evaluation of two dimensional gel electrophoresis to identify phosphoproteins differentially regulated in wheat during aphid infestation, revealed the up-regulation of three proteins namely photosystem II oxygen-evolving complex protein 2, HVUNKNOWN from Hordeum vulgare subsp vulgare and HSKERAT9 NID from Homo sapiens. Additional 57 proteins were partially identified as involved in the stress response but due to low protein levels, the percentage of matching peptides to these proteins was below the required confidence level. Although these protein identifications were below the confidence level, it is interesting to note that several of the proteins are known stress response proteins, and therefore could serve as potential targets for future investigations. In conclusion, the down and up-regulation of wheat proteins after aphid feeding reported in this study suggest that several signalling pathways are involved in the cereal stress response to aphid feeding. In silico approaches require knowledge or identification of potential proteins whereas 2D and LC/MS can identify numerous proteins still unknown to be involved in specific stress responses. The 2D approach is also limited in that the proteins of interest may be in low abundance and therefore not detected in the gels due to the presence of high abundant proteins. Therefore the best approach to identify proteins and signalling pathways involved in the stress response of wheat to aphid infestation, is the LC/MS/MS approach, as this proved to be the most sensitive and robust, identifying the most proteins with a high degree of confidence.
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Schenck, Craig A. "Using Quantitative Proteomics to Study the Early Events of Gravitropism". Ohio University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1338380163.
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Spadoni, Sara y SARA SPADONI. "The role of pectins in the regulation of plant defence responses against pathogens". Doctoral thesis, La Sapienza, 2006. http://hdl.handle.net/11573/917331.
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Xie, Zhengzhi. "Investigation of Plant Specialized Metabolism (Secondary Metabolism) Using Metabolomic and Proteomic Approaches". Diss., The University of Arizona, 2007. http://hdl.handle.net/10150/195218.
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Specialized metabolism (secondary metabolism) in glandular trichomes of sweet basil (Ocimum basilicum L.) and accumulation of specialized metabolites (secondary metabolites) in rhizomes of turmeric (Curcuma longa L.) was investigated using proteomic and metabolomic approaches, respectively. In an effort to further clarify the regulation of metabolism in the glandular trichomes of sweet basil, we utilized a proteomics-based approach that applied MudPIT (multidimensional protein identification technology) and GeLC-MS/MS (gel enhanced LC-MS/MS) to protein samples from isolated trichomes of four different basil lines: MC, SW, SD, and EMX-1. Phosphorylation, ubiquitination and methylation of proteins in these samples were detected using X!tandem. Significant differences in distribution of the 755 non-redundant protein entries demonstrated that the proteomes of the glandular trichomes of the four basil lines were quite distinct. Correspondence between proteomic, EST, and metabolic profiling data demonstrated that both transcriptional regulation and post-transcriptional regulation contribute to the chemical diversity. One very interesting finding was that precursors for different classes of terpenoids, including mono- and sesquiterpenoids, appear to be almost exclusively supplied by the MEP (2-C-methyl-D-erythritol 4- phosphate) pathway, but not the mevolonate pathway, in basil glandular trichomes. Our results suggest that carbon flow can be readily redirected between the phenylpropanoid and terpenoid pathways in this specific cell type. To investigate the impact of genetic, developmental and environmental factors on the accumulation of phytochemicals in rhizomes of turmeric, we performed metabolomic analysis in a 2x2x4 full factorial design experiment using GC-MS, LC-MS, and LC-PDA. Our results showed that growth stage had the largest effect on levels of the three major curcuminoids. Co-regulated metabolite modules were detected, which provided valuable information for identification of phytochemicals and investigation of their biosynthesis. Based on LC-MS/MS data, 4 new diarylheptanoids were tentatively identified in turmeric rhizomes using Tandem-MSASC, a home-made software tool that automatically recognizes spectra of unknown compounds using three approaches. Based on our metabolomic results, we proposed two new strategies, “metabolomics-guided discovery” and “correlation bioassay”, to identify bioactive constituents from plant extracts based on information provided by metabolomic investigation.
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Lee, Alex Chun Pong. "Dynamics of the plant mitochondrial proteome : towards the understanding of metabolic networks". University of Western Australia. School of Biomedical and Chemical Sciences, 2009. http://theses.library.uwa.edu.au/adt-WU2009.0181.
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[Truncated abstract] The mitochondrion is the energy powerhouse that provide energy to many metabolic functions in the form of ATP. Mitochondria in plants are also known to carry out a variety of other important biochemical processes within the cell, including the anaplerotic function of tricarboxylic acid (TCA) cycle, one-carbon metabolism and portions of photorespiration. Dynamics of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles under different developmental and environmental conditions. A quantitative comparative proteomic approach was carried out to analyze mitochondria isolated from non-photosynthetic models, cell culture and root, and compared them to mitochondria isolated from photosynthetic shoots. The glycinedependent respiration rate and the protein abundance of the photorespiratory apparatus was found to be higher in shoot than cell culture and root mitochondria. Also, there were major differences in the abundance and/or activities of enzymes in the TCA cycle between the three systems examined. The metabolic pathways that relied on the supply of intermediates from TCA cycle and photorespiration were also altered, namely cysteine, formate and one-carbon metabolism, as well as amino acid metabolism focused on 2-oxoglutarate generation, and branched-chain amino acids degradation. To further provide insight into the extent of mitochondrial heterogeneity in plants, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, stem and silique were analyzed. Of the 251 protein spots on a 2D-gel of the mitochondrial soluble/matrix fraction, the abundance of 213 spots were significantly varied between different samples. Identification of these spots revealed a non-redundant set of 79 proteins which were differentially expressed between organ/cell types. ... Importantly, posttranslational modifications played a significant role in the dynamics of the leaf mitochondrial proteome during the diurnal cycle. Overall, these findings indicated that the mitochondrial proteome is dynamic in order to fulfil different functional and physiological requirements in response to organspecific growth and changes in the external environments. These results also indicated that the majority of the changes in the mitochondrial proteome occurred in the matrix and suggested differences in substrate choice/availability in various plant organs and during the diurnal cycle. Further, these analyses demonstrate that, while mitochondrial proteins are regulated transcriptionally by the nucleus, post-transcriptional regulation and/or post-translational modifications play a vital role in modulating the activation state and/or regulation of proteins in key biochemical pathways in plant mitochondria. The integration of proteomics data with respiratory measurements, enzyme assays and transcript datasets will allow the identification of organ-enhanced and/or light/darkresponsive metabolic pathways as well as providing potential targets for reverse genetic approaches for further functional analysis of plant mitochondria.
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Mabiya, Thembeka. "Development of a plum chromosome doubling method and proteomics and biochemical characterization". University of the Western Cape, 2015. http://hdl.handle.net/11394/4875.
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>Magister Scientiae - MScChromosome doubling has become an important tool in breeding programmes as it offers the ability of introducing novel traits into existing plants. Doubled haploid plants are highly valued by both consumers and breeders as these plants usually show larger flower, leaves and fruit, thus making them more marketable. Marianna open pollinated plum rootstocks’ adaptability to different soil types and moisture conditions has been favoured in polyploidy studies as parental material in breeding programmes. The potential of the microtubule depolymerizing herbicide (oryzalin) for in vitro chromosome doubling were investigated by optimizing the concentration and incubation time of plant shoots to the antimitotic agent. Meristem tissues were treated for two time intervals (24 and 48 h) with five different concentrations of oryzalin (50, 75, 100, 150 or 200 μM) in liquid Murashige and Skoog (MS) medium. After treatment, plants were allowed to grow under a 16/8 h light/dark photoperiod at 24±2˚C for 4 weeks. One and two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) was used to separate, visualise and identify differently expressed proteins. Furthermore, changes in ROS accumulation, photosynthetic pigmentation, lipid peroxidation and antioxidant enzyme activity (SOD, APX and GR) were investigated. Flow cytometry results revealed that treatment of plants with oryzalin concentrations ranging from 75 to 150 μM induced ploidy after 24 h exposure whereas, 200 μM produced mixoploids containing both tetraploid and octoploids plants after 24 h exposure. Longer incubations of 48 h were detrimental to plant tissues as complete mortality was observed in the higher concentration (100 to 200 μM) treatments. Mass spectrometry analysis identified 14 differentially expressed protein spots that were characterized into different functional categories. ROS accumulation, the extent of lipid peroxidation and antioxidant capacity were differentially regulated in response to oryzalin treatment whereas photosynthetic pigments were significantly enhanced. The results suggests that oryzalin-induced proteins may act as potential biomarkers to improve fruit characteristics in future breeding programs whereas antioxidant enzymes play an important role in scavenging ROS in plants to enhance their adaptability to different environmental conditions.
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Kröber, Eileen. "Environmental genomics and proteomics of plant-associated microbial dimethylsulfide degradation in a coastal salt marsh". Thesis, University of Warwick, 2016. http://wrap.warwick.ac.uk/81391/.
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The methylated sulfur compound dimethylsulfide (DMS) plays a major role in the biogeochemical sulfur cycle and atmospheric chemistry. Bacteria are a main sink for DMS in the global sulfur cycle and can utilise DMS as a sole carbon and energy source. This study investigated the diversity and activity of bacteria capable of DMS degradation and associated with the salt marsh plant Spartina anglica known to be a producer of the DMS precursor dimethylsulfoniopropionate (DMSP). Initially, it was shown that S. anglica is rich in DMSP throughout the entire seasonal cycle in the Stiffkey salt marsh providing a likely hotspot for DMSP- and DMS-degrading bacteria. DMS uptake experiments demonstrated that DMS degradation takes place in the phyllosphere and rhizosphere of S. anglica and denaturing gradient gel electrophoresis (DGGE) and high-throughput amplicon sequencing of the 16S rRNA revealed the dominance of bacteria related to α - and γ- Proteobacteria, as well as Flavobacteria in the phyllosphere of S. anglica, whereas the rhizosphere was mainly colonised by members of the classes γ-, δ-, α-, and ε-Proteobacteria and Bacteroidia. The diversity of DMS-degrading bacteria associated with S. anglica was first assessed by enrichment culture. DGGE analysis and high-throughput sequencing diversity of DMS enriched samples using the 16S rRNA gene as a marker suggested the dominance of Piscirickettsiaceae, Methylophaga and Methylophaga-like bacteria in DMS-enrichments of phyllosphere and rhizosphere samples of S. anglica. A functional gene marker analyses was carried out using the gene encoding methanethiol oxidase (mtoX), a key enzyme in DMS degradation and the gene encoding a DMSP lyase (dddP) to determine the diversity of bacteria degrading DMS and DMSP, respectively, in the phyllosphere and rhizosphere of S. anglica. The analysis for mtoX showed a great diversity of this gene in phyllosphere and rhizosphere of S. anglica and that major clades of mtoX clustered together with Sedimenticola, Methylohalobius, Methylophaga and other mtoX clones previously detected in surface sediments of the same salt marsh. The results for the functional marker gene analysis for the dddP gene suggested the dominance of Ruegeria-like species and Roseobacter-like bacteria but also of unidentified Ddd+ bacteria in the phyllosphere and rhizosphere of S. anglica. In order to identify the active DMS degraders in the phyllosphere and rhizosphere of S. anglica stable-isotope probing (SIP) combined with DGGE and highthroughput sequencing of the 16S rRNA gene was carried out. The SIP experiments revealed the dominance of Piscirickettsiaceae, Methylophaga and Methylophaga-like microorganisms in the rhizosphere of S. anglica. However, the DMS-degrading microbial community in the phyllosphere seemed more diverse than in the rhizosphere and microorganisms like Halothiobacillus, Xanthomonadaceae, Rhodanobacter but also Piscirickettsiaceae seemed to be involved. A comparative proteomic and transcriptomic experiment of Methylophaga thiooxydans, a microorganism found in phyllosphere and rhizosphere of S. anglica, revealed the general pathways involved in methanol but especially DMS degradation. During DMS cycling the protein and the protein-encoding gene for the methanethiol oxidase (MtoX/mtoX) was highly expressed. A metaproteogenomic experiment provided an insight into the taxonomy and functional diversity of the microbial community associated with the Spartina anglica phyllosphere. Analysis of the metagenome provided evidence that the microbial community associated with S. anglica is dominated by γ-Proteobacteria such as Halomonadales, Alteromonadales, Oceanospirillales, and Thiotrichales and the alphaproteobacterial order Rhodobacterales and showed therefore a major difference to the bacterial community composition in the phyllosphere of for instance A. thaliana, clover, soybean and rice. The detection of DMSP lyase encoding genes and genes encoding proteins for DMS degradation confirmed the genetic potential for the observed DMSP and DMS degradation activity previously measured in the phyllosphere of S. anglica. The metaproteomic experiment allowed a first insight into the proteins expressed in the phyllosphere of S. anglica which also suggested that mainly γ-Proteobacteria and α-Proteobacteria are dominant populations occurring in this habitat. New insights were gained into the activity and diversity of DMS-degrading microbial communities associated with a salt marsh plant that represents a significant component of salt marsh plant communities world wide. Not only was the taxonomic and functional diversity of DMS-degrading microorganisms associated with S. anglica greater then previously realised, the observation of considerable potential of above-ground plant-associated DMS degradation in the phyllosphere demonstrates a previously unrealised sink in the DMS cycle in coastal ecosystems, which is clearly more complex than previously appreciated.
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Maltman, Daniel James. "Characterization of endoplasmic reticulum from castor bean and the cloning of a plant phosphatase : a basis for comprehensive plant organelle proteomics research". Thesis, Durham University, 2000. http://etheses.dur.ac.uk/4958/.
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The plant endoplasmic reticulum is the location of storage oil and membrane lipid assembly, and for fatty acid modifying reactions (desaturation, elongation, hydroxylation). It therefore represents a source of enzymes involved in these processes. Many of these defy traditional purification strategies. In this study, ER membranes have been isolated biochemically pure and in milligram quanties from the endosperm of developing and germinating castor bean. One-dimensional SDS- PAGE, used to routinely assess sample integrity, showed resolution limitations. Two-dimensional gel electrophoresis was optimized regarding sample preparation and solubilization, and reproducible profiles confirmed its suitability as a sound basis for analysis of stage-specific ER components. In large format 2-D experiments, preparative loadings were reproducibly resolved. MALDI TOP mass spectrometry was evaluated for high throughput peptide signature generation with individual ER components. Resolution problems were again highlighted with 1-D separations, although some functional assignments were made. Subsequently analysis of selected spots from a preparative 2-D gel of germinating ER was used to establish the limitations of the procedure. Database matching of a single component at very low levels of mass error tolerance also demonstrated the power and accuracy of the technology. Membranes were subfractionated to simplify protein patterns. It is proposed that an organellar approach, including subfractionation, provides enrichment of specific subsets of cellular components. A putative plant phosphatidic acid phosphatase gene has been investigated following identification from the EST database. The aim of this research is the identification of proteins involved in storage lipid synthesis in castor bean in reactions specific to the endoplasmic reticulum.
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Ozgazi, Nese. "Proteome Analysis Of Blumeria Graminis F. Sp. Hordei Inoculated Barley". Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/3/12611152/index.pdf.
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Blumeria graminis f. sp. hordei is a biotroph pathogen that causes powdery mildew disease in barley. In this study, Pallas01 and Pallas03 barley lines having Mla1, Ml (Al2) and Mla6, Mla14 R-genes were inoculated with Bgh103(64/01) race of the Blumeria graminis f. sp. hordei having avirulence and virulence to Pallas01 and Pallas03, respectively.
The proteins were isolated from the three biological replicates of 12, 24, and 48 hpi samples following the method in Rampitsch et al., 2006. These there biological replicates of three time points together with the mock inoculated plant proteins were separated on 2D-PAGE using IPG strips of 4-7 pH values as three technical replicates, resulting 108 gels.
The gels were analyzed using PdQuest (Bio Rad) in order to assess up- or down-regulated protein spots by comparing against controls and the samples having resistance or susceptible responses with each other. According to the analysis, 36 proteins were found to be differentiated and among them 18 proteins were found up-regulated and 8 proteins were found down-regulated. The spots were manually
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excised and subjected to the nano-LC-ESI-MS/MS analysis (Proteome Factory, Germany). The MASCOT algorithm was used for identification of the possible proteins. The experimental pI and MW values were used for selecting the differentiated proteins from the mass results.
The relative abundance of each of the 38 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be carbohydrate metabolism related. The relative distribution of the proteins into four main functional categories was taken into consideration.
Statistical tests (Students‟
T-test) were carried among the identified proteins in order to reveal statistically significant proteins throughout the study. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, most of the proteins were found to be located in cytoplasm or chloroplast.
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Whitehill, Justin G. A. "INVESTIGATIONS INTO MECHANISMS OF ASH RESISTANCE TO THE EMERALD ASH BORER". The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306863052.
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Greenham, Trevor. "Pointillism in Plant Systems Biology: I. Proteomic Analysis of Plant Exosome-like Particles II. Amyloplast-binding Puroindoline Fusion Proteins for Recombinant Protein Expression". Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39647.
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Expanding upon our understanding of plant defense is critical, particularly with the perilous threats of climate change and overpopulation to our food security, health and well-being. In this study, we focused on plant defense using two distinct approaches. First, we performed a proteomic analysis of plant exosome-like nanoparticles in order to elucidate their defense related protein cargo. Secondly, we used a wheat antimicrobial protein, puroindoline, as a fusion partner for the expression of recombinant proteins in rice endosperm.
Plant exosome-like nanoparticles (ELP) were isolated from fresh tomato and subjected to mass spectrometry (MS) analysis. The ELPs were compared to fresh pressed tomato juice, and the proteins that were significantly upregulated in the ELPs were analyzed for their defensive properties. Bioinformatic analysis identified 30 proteins upregulated in the ELPs, with a majority of these being involved in plant defense.
Puroindoline is a protein found in soft wheat varieties. A unique feature of this protein is the presence of a tryptophan-rich domain, which causes it to localize and tether onto starch granule surfaces; a property we are seeking to exploit for recombinant protein isolation. We hypothesized that when expressed in a pin-null crop, such as rice, puroindoline along with its fusion partner will localize and adhere to starch granule surfaces. PIN fusions were expressed in rice, and their subcellular localization was determined by immunolocalization. It was observed that PIN localizes to rice starch
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granules in vitro and in planta, and retains its starch granule binding abilities as a fusion partner.
To identify other possible starch granule binding fusion partners, an anhydrous cleavage method was developed that can scan dry biological materials for associated proteins, in this case the starch granule surface. Incubation of our cleavage reagent with isolated rice starch granules yielded several cleavage products as determined through SDS-PAGE. These cleavage products were compared with previous proteomic data of trypsin digested rice starch granules.
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Mackay, Stephen. "Identification of the genes encoding enzymes involved in the synthesis of the biopolymer paramylon from Euglena gracilis". Thesis, Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/5420.
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Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010.Includes bibliography.
Title page: Dept. of Genetics, Faculty of Science
ENGLISH ABSTRACT: Recent advances in medical pharmacology have identified the immune-potentiating effects of β-1,3-glucans on mammalian immune systems. Extensive research has identified and described the mechanisms of action and receptor binding specificity of different β-1,3-glucans as well as their structural and functional relationships. Molecular mass, solubility, structural order, degree of branching as well as chemical modification all determine the effectiveness of the β-1,3-glucan immune-modulating activities, which typically include; macrophage activation, antibody adjuvant activities, reduction of LDL-cholesterol, leukocyte mitogenic activities, cytokine and chemokine production as well as antiviral and antitumor activities. Currently β-1,3-glucans have been sold commercially under the name β-glucan, mostly in the form of Betafectin, a genetically modified yeast derived β-1,3-glucan. Recent studies of different β-1,3-glucans have identified the pharmacological activities of paramylon, a Euglena derived β-1,3-glucan. Although paramylon has relatively low immune-stimulating activities, chemical modification of the paramylon granule increased immune-potentiation with specific antimicrobial and anti-HIV activities. Due to these specific immune-potentiating activities, paramylon is novel in terms of both structure as well as functional activity. In terms of biotechnological application, paramylon is greatly favoured as it is synthesized as an insoluble membrane bound granule in the cytosol of Euglena where most plant and fungal β-1,3-glucan synthases are cell membrane bound highly regulated multifunctional complexes, synthesizing β-1,3-glucan as cell wall components. Due to the novel granular nature of paramylon, expression in other systems with genetic modification could potentially further increase immuno-potentiating activities. In this study, different approaches were attempted in order to identify the genes involved in paramylon synthesis including; constructing and screening a Euglena gracilis cDNA library, sequence analysis of the purified proteins as well as transcription analysis of the sequenced transcriptome and genome of E. gracilis. Putative candidates that encode subunits of the paramylon synthase complex have been identified.
AFRIKAANSE OPSOMMING: Onlangse vordering in mediese farmakologie het die immuun-stimulerende effek van β-1,3-glukane op die soogdier immuunsisteem geïdentifiseer. Intensiewe navorsing het die meganisme van die werking en reseptor bindingspesifisiteit van verskillende β-1,3-glukane, asook hulle struktuur en funksionele verhoudings, geïdentifiseer. Die molekulêre massa, oplosbaarheid, strukturele orientasie, mate van vertakking asook chemiese modifikasies bepaal almal die effektiwiteit van die β-1,3-glukaan immuun-modulerende aktiwiteite. Tipiese immuno-moduleringsaktiwiteite sluit makrofaag aktivering, teenliggaampie adjuvant aktiwiteite, verlaging van LDL-cholesterol, leukosiet mitogeniese aktiwiteite, sitokien en chemokien produksie asook anti-virale en antitumor aktiwiteite in. Huidiglik word β-1,3-glukane onder die naam β-glukaan verkoop meestal in die vorm van Betafectin, ‘n geneties gemodifiseerde gis wat van β-1,3-glukaan afkomstig is. Onlangse studies van verskillende β-1,3-glukane het die farmakologiese aktiwiteit van paramylon, ‘n Euglena afkomstige β-1,3-glukaan geïdentifiseer. Alhoewel paramylon relatiewe lae immuun-stimulerende aktiweite toon, verhoog chemiese modifikasies van die paramylon granules immuun-stimulering, spesifiek die anti-mikrobiese en anti-MIV aktiwiteite. Weens hierdie spesifieke immuun-stimulerende aktiweite, word paramylon as nuut beskou veral in terme van beide struktuur asook funksionele aktiwiteit. In terme van biotegnologiese toepassing, verkry paramylon voorkeur aangesien dit as ‘n onoplosbare membraangebonde granule in die sitosol van Euglena gesintetiseer word terwyl meeste plant en fungus β-1,3-glukaan sintases hoogs gereguleerde multifunksionele selmembraan gebonde komplekse is wat β-1,3-glukaan asook ander selwand komponente sintetiseer. Weens die unieke granulêre natuur van paramylon, sal uitdrukking in ander sisteme ‘n moontlike industrie skep waar deur die transgeniese uitdrukking van granulêre paramylon verdere verbetering van die immuun-stimulerings aktiwiteite kan lei. In hierdie studie is verskillende benaderings aangewend om die gene wat by paramylon sintese betrokke is te identifiseer, dit sluit in die konstruksie en sifting van ‘n E. gracilis cDNS biblioteek, aminosuur volgorde analise van gesuiwerde proteiene asook die transkripsionele analise van die volgorde van die transkriptoom en genoom van E. gracilis. Moontelike kandidate wat vir die subeenhede van die paramylon syntase kompleks kodeer is geïdentifiseer.
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Wu, Ming Jie. "Identification and characterisation of polymorphic proteins in wheat grain: a proteomic and immunological approach". Thesis, The University of Sydney, 2007. https://hdl.handle.net/2123/28092.
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The development of wheat cultivars with unique quality traits is of major importance to the Australian wheat grower. Varietal diversity and flour quality of wheat are determined by genes and their products - proteins. Therefore, characterisation of the grain proteins will lead to discovery of potential protein markers for varietal identification and desirable quality attributes such as hardness, dough strength and extensibility. In order to screen the identified
protein markers among thousands of breeder’s lines, specific high throughput detection methods are needed. In this study, a proteomics approach was applied to search for polymorphic grain proteins, and specific and high
throughput antibody—based immunoassays were then developed. In order to increase resolution of inter-varietal polymorphic proteins, sub-fractionation (referred to as “de-complexing”) on the basis of protein solubility and multiple
comparative analyses of hundreds of wheat varieties were carried out. A series of protein analytical techniques were used including native and denaturing one-dimensional polyacrylamide gel electrophoresis (PAGE), twodimensional
gel electrophoresis (2-DE) and electrospray ionisation tandem mass spectrometry (ESl-MS/MS).
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Strandberg, Linnéa. "Isolation of the native chloroplast proteome from plant for identification of protein-metabolite interactions". Thesis, KTH, Proteinvetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-301783.
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För att kunna livnära en växande population behöver avkastningen på skördar öka. En lösning på dettaär att optimera plantornas fotosyntes, vilket innefattar förbättrad koldioxidfixering. För att lyckas meddet krävs kunskap i hur reglering av nyckelproteiner i kloroplasten går till. Syftet med detta projekt är identifiera möjliga reglerande protein-metabolitinteraktioner i Arabidopsis thaliana. Målproteinerna ärde 11 enzymerna i Calvin-Benson-Basshamcykeln. Metaboliterna som testas är 3PGA, ATP, FBP, GAP, vilka är mellan produkter eller kofaktorer i cykeln; 2PG, som är en produkt av en konkurrerande reaktion i cykeln; och slutligen G6P, citrat och sackaros, vilka är centrala metaboliter i andra viktiga reaktioner i cellen. Före experimenten med Arabidopsis testades protokollen med spenat. Som ett första steg isolerades kloroplasterna från blad. När intakta kloroplaster verifierats extraherades proteinerna. Inter-aktioner mellan metaboliterna och proteinerna analyserades med en metod kallad limited proteolysis-small molecule mapping. Denna teknik, vilken kombinerar begränsad proteolys med masspektrometri, detekterade flertalet protein-metabolit interaktioner. I Arabidopsis uppvisade alla enzym förutom FB-Pase, PPE och TIM minst en interaktion. I spenat sågs interaktioner med FBA, GAPDH, PGK, PRK, RuBisCO, TIM och TK. Resultaten visar möjliga reglerande interaktioner, vilka skulle kunna användasför att identifiera flaskhalsar i kolfixeringen. Denna kunskap kan i sin tur utnyttjas för att öka flödet i Calvin-Benson-Basshamcykeln och därigenom förbättra växters koldioxidfixering.In order to feed a growing population, the crop yield needs to be increased. One way to do this is to optimise the photosynthetic activity in the plant, which includes improvement of carbon fixation. To succeed with this, knowledge of the regulation of key proteins in the chloroplast is required. The aim of this project is to identify possible regulatory protein-metabolite interactions in chloroplasts from Arabidopsis thaliana. The target proteins are the 11 enzymes of the Calvin-Benson-Bassham cycle. The metabolites of interest are 3PGA, ATP, FBP, GAP, which are intermediates or co-factors of the cycle;2PG, which is a product of a competing reaction in the cycle; and finally G6P, citrate and sucrose, which are central metabolites in other vital reactions in the cell. Before the experiments with Arabidopsis, spinach was used as a test organism to evaluate the proposed protocols. First, chloroplasts were isolatedfrom leaves. When the integrity of the chloroplasts had been validated, the proteins were extracted. Metabolic interactions with the extracted proteins were analyzed with limited proteolysis-small molecule mapping. This method, which combines limited proteolysis with mass spectrometry, detected severalprotein-metabolite interactions. In Arabidopsis, all enzymes except for FBPase, PPE and TIM had atleast one interaction. In spinach, interactions were seen with FBA, GAPDH, PGK, PRK, RuBisCO,TIM and TK. The results highlight potential regulatory events, which could be used to target bottlenecks in carbon fixation. This could provide a pathway to increase the flux in the Calvin-Benson-Bassham cycle, and thereby improve carbon fixation in plants.
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Valadares, Rafael Borges da Silva. "Identification of genes and proteins involved in the regulation of orchid mycorrhiza". Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/11/11140/tde-24032014-133439/.
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Orchids are characterized by producing minute endosperm-lacking seeds, which depend on mycorrhizal fungi for germination and embryo development. Some aclorophyllous orchids remain dependent on the mycorrhizal association for carbon acquisition during their whole life history, whereasother orchids develop photosynthesis. Despite the biological significance of orchid mycorrhiza, gene expression studies are lacking. We have used different highthroughput approaches in order to understanding the mechanisms regulating orchid mycorrhiza development and functioning. Firstly, we have used a 2D-LC-MS/MS approach coupled to isobaric tagging for relative and absolute quantification (iTRAQ) to identify proteins with differential accumulation in Oncidium sphacelatum at different stages of mycorrhizal protocorm development (achlorophyllous and green protocorms) after seed inoculation with a Ceratobasidium sp. isolate. Quantitative analysis showed that the expected changes in carbon metabolism in green protocorms were accompanied by enhanced accumulation of proteins involved in the modulation of reactive oxygen species homeostasis, defense related responses, phytoalexins and carotenoid biosynthesis, suggesting that orchid protocorms undergo profound metabolic changes during the switch from the fully mycoheterotrophic to the photosynthethic stage. Secondly, three different proteomic techniques were carried out in independent experiments aiming to identify changes in protein accumulation in mycorrhizal roots of the terrestrial orchid Oeceoclades maculata.Finally, O. maculatamycorrhizal roots were used for transcriptome analyses. The data revealed a strong increase in general stress responses, accompanied by changes in signaling pathways possibly related to fungal recognition and establishment of a compatible interaction. Some of the upregulated genes may be involved in the reorganization of cell structure, likely related to accommodation of the fungal symbiont in the plant roots. We have also observed in mycorrhizal roots up-regulation of genes involved in carbon metabolism, including glycolysis/gluconeogenesis and amino sugars metabolism, as well as genes involved innitrogen assimilation. The down-regulation of genes involved in the jasmonate and ABA transduction pathways, and key genes encoding anti-fungal proteins, such as chitinase and a mannose-specific binding lectin, strongly suggests an alleviation of plant defense responses in O. maculata mycorrhizal roots. In general, our data suggest that the physiology of an orchid mycorrhiza is more similar to a compatible interaction than to an arm-race between plant and fungi. Overall orchid mycorrhiza have proved to be a promising model for investigating plantfungal interactions and further studies should now address the specific roles of the genes showing differential regulation in this study.As orquídeas são caracterizadas por produzirem sementes diminutas, que não possuem endosperma. Necessitam, portanto, da interação com fungos micorrízicos para germinação e desenvolvimento do embrião. Algumas orquídeas aclorofiladas se mantêm dependentes dos fungos micorrízicos para a aquisição de carbono, enquanto outras desenvolvem a maquinaria fotossintética. Apesar do significado biológico das micorrizas de orquídeas, alterações na expressão gênica e no acúmulo de proteínas foram altamente negligenciads nos últimos anos. Neste trabalho, foram utilizadas diferentes técnicas sequenciamento e identificação de genes e proteínas em larga-escala para acessar as alterações moleculares responsáveis pela regulação das micorrizas de orquídeas. Uma abordagem baseada em 2D-LC MS/MS acoplada a técnica de quantificação absoluta e relativa iTRAQ, foiutilizada para identificar proteínas com acúmulo diferencial em Oncidium sphacelatum em diferentes estágios do desenvolvimento do protocormo (protocormos aclorofilados versus protocormos fotossintetizantes), após inoculação com um fungo do gênero Ceratobasidium. As análises mostraram que, as alterações esperadas no metabolismo do carbono foram acompanhadas de um acúmulo aumentado de proteínas envolvidas na modulação de espécies reativas de oxigênio, respostas de defesa, biossíntese de fitoalexinas e carotenóides, sugerindo que os protocormos de orquídeas passam por profundas alterações metabolicas durante a transição do metabolismo micoheterotrófico para o fotossintético. Posteriormente foram utilizadas três diferentes técnicas de proteômica quantitativa para explorar alterações fisiológicas em raízes micorrizadas e não-micorrizadas de Oeceoclades maculata.Este estudo foi ampliado, pela utilização de uma abordagem transcritômica ao mesmo modelo biológico. Em conjunto, os dados revelaram um forte aumento em respostas relacionadas ao estresse, acompanhadas de alterações em vias de transdução de sinal possivelmente relacionadas ao reconhecimento do simbionte fúngico e estabelecimento de uma interação compatível. Alguns genes com expressão aumentada devem estar envolvidos na reorganização celular, provavelmente ligada a acomodação do simbionte fúngico nas raízes das plantas. Também foi observado o aumento de genes envolvidos no metabolismo do carbono e de açúcares aminados, juntamente a genes relacionados a assimilação de nitrogênio em raízes micorrizadas. A expressão diminuída de genes envolvidas nas vias do jasmonato e ácido abscícico, juntamente a genes-chave que codificam para proteínas anti-fúngicas sugerem fortemente uma atenuação das respostas de defesa da planta em raízes micorrizadas de Oeceoclades maculata. No geral, parece que as micorrizas de orquídeas são fisiológicamente mais próximas de uma simbiose compatível do que de uma interação unilateral em favor da planta. Sobretudo, este sistema biológico provou ser promissor para investigação de interações planta-fungo e, próximas pesquisas devem agora ser focadas em funções específicas dos genes que mostraram regulação diferencial neste estudo.
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Grosse-Holz, Friederike. "Proteases and inhibitors in the interaction between Nicotiana benthamiana and Agrobacterium tumefaciens : systematic analysis and emerging solutions for molecular farming". Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:6146918c-3749-4604-88fa-01d426e4a817.
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Nicotiana benthamiana is now an established platform for molecular farming, the production of biopharmaceuticals in plants. Infiltration with Agrobacterium tumefaciens (agroinfiltration) is commonly used to transiently express one or multiple transgenes in N. benthamiana leaves. Agroinfiltrated N. benthamiana is a flexible and scalable recombinant protein (RP) production platform, but is impeded by low RP yields. Plant proteases can degrade RPs and thus limit RP accumulation. To inform, design and implement strategies for enhancing RP accumulation, I present four papers about proteases and protease inhibitors in agroinfiltrated N. benthamiana. First, I investigated the transcriptome, extracellular proteome and active secretome to understand the plant response to agroinfiltration and investigate the expressed proteases. I show that an extracellular immune response is mounted at the expense of photosynthesis. Comprehensive annotation and monitoring uncover a large, diverse repertoire of proteases in agroinfiltrated leaves, indicating that broad-range depletion of protease activity may be required to enhance RP accumulation. Second, I reviewed the literature on multifunctional plant protease inhibitors (PIs) and grouped them into three types of multifunctional PIs that evolved independently. Third, I screened candidate PIs and discovered that three new, unrelated PIs enhance RP accumulation. I present universal elements of the RP degradation machinery, uncovering new questions on our understanding of the protease network that degrades RPs. Fourth, I identified targets of SlCYS8, a PI that enhances RP accumulation. The target proteases of SlCYS8 are implicated in RP degradation and the high specificity of SlCYS8 can be used to study their role in other processes. By elucidating the immune response to agroinfiltration, by uncovering the N. benthamiana protease repertoire and by providing new tools to deplete the activity of specific proteases, this thesis makes a relevant contribution to both basic plant research and molecular farming.
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Jereissati, Camila Barbosa Pinheiro. "Análise proteômica de plastídeos do endosperma de sementes em desenvolvimento de pinhão manso (Jatropha curcas L.)". reponame:Repositório Institucional da UFC, 2015. http://www.repositorio.ufc.br/handle/riufc/19405.
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JEREISSATI, Camila Barbosa Pinheiro. Análise proteômica de plastídeos do endosperma de sementes em desenvolvimento de pinhão manso (Jatropha curcas L.). 2015. 127 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2015.Submitted by Vitor Campos (vitband@gmail.com) on 2016-09-01T22:55:24Z No. of bitstreams: 1 2015_teses_cbpjereissati.pdf: 1568499 bytes, checksum: 2f41598abfda8cf6bcab51817b1b742a (MD5)
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Jatropha curcas L. is a plant native to America and belongs to the Euphorbiaceae family. Currently it is gaining economical interest mainly because it is an oilseed crop with potential to produce biodiesel. However, presence of phorbol esters (a class of diterpenes) that are the major toxic constituents of the seeds, limits a better usage of the plant, by making the use of the residue, obtained after the oil extraction from the seeds, unfeasible for animal feed, due to its pro-carcinogenic activity and inflammatory action. Proteomic analysis of the plastids isolated from developing seeds of Jatropha is important because the synthesis of fatty acid as well as phorbol esters, the two most attractive compounds in the study of Jatropha curcas, occur in plastids. Proteomic analysis of this organelle is crucial to better understand and explore not only the biosynthetic pathway of these two compounds but other metabolic pathways , and addtionaly providing foundation for researchs that aimed to develope genotypes with more suitable characteristics for industrial applications. In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled online to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in three different plastids proteins databases i.e. PPDB, SUBA and PlProt, while homologues for 13 proteins were not found in any of these three databases but were marked as plastidial by at least one of the three prediction programs used (TargetP, ChloroP and PlantMPloc). Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, this study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level.
O pinhão manso (Jatropha curcas L.) é uma planta nativa da América, pertencente à família Euphorbiaceae. Atualmente, ela desperta interesse econômico principalmente por se tratar de uma oleaginosa com potencial para a produção de biodiesel. Entretanto, a presença de ésteres de forbol (uma classe de diterpeno), que são os principais constituintes tóxicos das sementes, limita uma melhor utilização dessa planta, por inviabilizar o uso do resíduo de extração do óleo das sementes na alimentação animal, bem como, por apresentar atividade pró-carcinogênica e ação inflamatória. A análise proteômica de plastídeos, isolados de sementes em desenvolvimento de pinhão manso, é uma importante vertente de estudo, pois tanto a síntese de ácidos graxos como dos ésteres de forbol, os dois compostos mais atrativos no estudo de Jatropha curcas, ocorrem nos plastídeos. O estudo proteômico dessa organela torna-se crucial para melhor compreender e explorar não somente as vias biossintéticas desses dois compostos, como de outras vias metabólicas, além de proporcionar um conjunto de dados que pode ser utilizado em pesquisas voltadas para o desenvolvimento de genótipos com características mais adequadas para aplicações industriais. No presente trabalho, realizou-se uma análise proteômica de plastídeos isolados do endosperma de sementes em desenvolvimento do pinhão manso, que estavam nos estágios iniciais de deposição de lipídios e proteínas de reserva (25-30DAA), confirmados por meio de análises histológica e histoquímica. As proteínas extraídas dos plastídeos foram digeridas com tripsina e os peptídeos foram aplicados no sistema de nano-LC EASYII acoplado online ao espectrômetro de massa nano ESI LTQ-Orbitrap velos, o que resultou na identificação 1103 proteínas, representando 804 grupos de proteínas, dos quais 923 foram consideradas identificações verdadeiras. Isso expandiu consideravelmente o repertório de proteínas do pinhão manso até agora identificas. Dentre as proteínas identificadas, apenas 5 são codificadas pelo genoma plastidial, e nenhuma delas está envolvida na fotossíntese, o que evidencia a natureza não fotossintética dos plastídeos isolados. Homólogos de 824, dentre as 923 proteínas identificadas, estavam presentes nos bancos de dados PPDB, SUBA e PlProt, enquanto homólogos para 13 proteínas não foram encontrados em nenhum dos três bancos de dados plastidiais, mas foram detectados como plastidiais por pelo menos um dos três programas de predição de localização subcelular utilizados (TargetP, ChloroP, PlantMPloc). A classificação funcional mostrou que a maioria das proteínas identificadas pertencia ao metabolismo dos aminoácidos, seguido dos metabolismos dos carboidratos, energético e dos lipídios. As subunidades maiores e menores da Rubisco foram identificadas, e sua presença nos plastídeos foi considerada uma característica adaptativa para contrabalancear a perda de um terço do carbono na forma de CO2 como um resultado da conversão de carboidratos em óleo através da glicólise. Apesar de enzimas envolvidas na biossíntese de diversos precursores dos diterpenóides terem sido identificadas, não foi detectado nenhuma terpeno sintase/ciclase, o que sugere que os plastídeos isolados do endosperma de sementes em desenvolvimento não sintetizam ésteres de forbol, apesar de uma grande quantidade desse composto ser encontrada neste tecido. Como conclusão, o presente trabalho proporciona insights sobre as principais vias biossíntéticas, e sobre características peculiares dos plastideos isolados do endosperma de sementes em desenvolvimento.
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Koziol, Adam. "Application of Direct-sequencing Peptide Proteomics to the Characterization of Antagonistic (Endogenous and Exogenous) Proteins in Cereal Grains". Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23853.
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The cereal seed plays a crucial role in society – both in the “food as medicine” paradigm, but also in food security. It is the starch and proteins present in the seed that lend it importance in these dissimilar anthropomorphic activities. This thesis investigation first characterized the post-translational processing of the potential diabetogen, wheat globulin-3. Globulin-3-like peptides were observed primarily in the embryo. These peptides varied significantly in their molecular masses and isoelectric points, as determined by two dimensional electrophoresis and immunoblotting. Five major polypeptide spots were sequenced by mass spectrometry, allowing for the development of a model of the post-translational events contributing to the globulin-3 processing profile. Three separate investigations of starch granules from different cereal species were performed. In the first series of experiments, pathogen-susceptible maize kernels were injected with either conidia of the fungal pathogen Fusarium graminearum or sterile water controls. Proteins in the desiccated fungal remnants on the surface of the kernels as well as in the endosperm and embryo tissues of the control and infected kernels were isolated and these proteomes were sequenced using tandem mass spectrometry. Approximately 250 maize proteins were identified. These proteins were classified into functional categories. There was an increased representation of defense proteins in the both the embryo and endosperm tissues of infected maize samples. The proteome of the fungal remnants was composed of 18 proteins. Several of these proteins were categorized as being involved in the metabolism of plant-sourced molecules, or in stress response. The second series of experiments detail the investigation of commercially prepared rice and maize starches using tandem mass spectrometry. The majority of identified proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. Markers for seed maturity and for starch mobilization were also documented. Finally, the third series of experiments investigated the non-host proteomes present in commercially-prepared starches. Non-host proteins from a variety of species, including Homarus americanus were found in the starch samples. This documentation of H. americanus proteins in these starch samples may have food safety implications with regards to shellfish allergies.
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Jiang, Nan. "Characterization of TaXPol-1, a Xylan Synthase Complex from Wheat". Ohio University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1437153132.
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Dias, Leonardo Lucas Carnevalli. "Aspectos fisiológicos, bioquímicos e análise proteômica comparativa durante a maturação, germinação e conversão em plantas de embriões de Ocotea catharinensis Mez. (Lauraceae)". Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-10082009-172459/.
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A semelhança entre os eventos da embriogênese zigótica e somática permite que sejam estabelecidos parâmetros para o acompanhamento destes dois processos. Neste trabalho foi estudada a embriogênese somática em Ocotea catharinensis, nas fases de maturação e germinação, além do processo germinativo da semente zigótica, avaliando-se parâmetros bioquímicos, e a expressão diferencial de proteínas. O tratamento com ABA + PEG em culturas de embriões somáticos apresentou variações semelhantes a apresentadas por embriões zigóticos no estádio de maturação. Não se observou a germinação de embriões somáticos, contudo verificou-se que a desidratação prévia promoveu importantes alterações bioquímicas. Durante a germinação, não se observou à síntese de novas proteínas no embrião zigótico. Os estudos proteômicos no desenvolvimento da semente permitiram a seleção de polipeptídios, marcadores estádio-específicos. Os resultados obtidos possibilitam a otimização e melhor entendimento das etapas da embriogênese somática, em espécies com sementes recalcitrantes, como O. catharinensis.The great similarity among the events of zygotic and somatic embryogenesis allows the establishment of parameters for accompaniment of these processes. In the present work it was studied the somatic embryogenesis in Ocotea catharinensis, in the maturation and germination phases, and the zygotic embryogenesis. The biochemical parameters and differential expression of proteins were evaluated: The treatment with ABA + PEG presented similar variations for zygotic embryos in the maturation stage. The germination was not observed for somatic embryos; however, it was verified that the previous dehydration promoted important biochemical alterations. With relation to the zygotic embryo, throughout the germination process, the synthesis of new proteins was not observed. The proteomic studies carried out throughout seed development, allowed the selection of polypeptides with differential expression. The results obtained open perspectives for the methodology optimization of the somatic embryogenesis, for species with recalcitrant seeds, like O. catharinensis.
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Basu, Proma. "Proteomic Analysis of Arabidopsis Seedlings Germinated in Microgravity to Identify Candidate Genes for Gravity Signal Transduction". Ohio University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1565216423464876.
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Jereissati, Camila Barbosa Pinheiro. "Proteomic analysis of plastids the endosperm of developing seeds of Jatropha (Jatropha curcas L.)". Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=13786.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorJatropha curcas L. is a plant native to America and belongs to the Euphorbiaceae family. Currently it is gaining economical interest mainly because it is an oilseed crop with potential to produce biodiesel. However, presence of phorbol esters (a class of diterpenes) that are the major toxic constituents of the seeds, limits a better usage of the plant, by making the use of the residue, obtained after the oil extraction from the seeds, unfeasible for animal feed, due to its pro-carcinogenic activity and inflammatory action. Proteomic analysis of the plastids isolated from developing seeds of Jatropha is important because the synthesis of fatty acid as well as phorbol esters, the two most attractive compounds in the study of Jatropha curcas, occur in plastids. Proteomic analysis of this organelle is crucial to better understand and explore not only the biosynthetic pathway of these two compounds but other metabolic pathways , and addtionaly providing foundation for researchs that aimed to develope genotypes with more suitable characteristics for industrial applications. In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled online to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in three different plastids proteins databases i.e. PPDB, SUBA and PlProt, while homologues for 13 proteins were not found in any of these three databases but were marked as plastidial by at least one of the three prediction programs used (TargetP, ChloroP and PlantMPloc). Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, this study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level.
O pinhÃo manso (Jatropha curcas L.) à uma planta nativa da AmÃrica, pertencente à famÃlia Euphorbiaceae. Atualmente, ela desperta interesse econÃmico principalmente por se tratar de uma oleaginosa com potencial para a produÃÃo de biodiesel. Entretanto, a presenÃa de Ãsteres de forbol (uma classe de diterpeno), que sÃo os principais constituintes tÃxicos das sementes, limita uma melhor utilizaÃÃo dessa planta, por inviabilizar o uso do resÃduo de extraÃÃo do Ãleo das sementes na alimentaÃÃo animal, bem como, por apresentar atividade prÃ-carcinogÃnica e aÃÃo inflamatÃria. A anÃlise proteÃmica de plastÃdeos, isolados de sementes em desenvolvimento de pinhÃo manso, à uma importante vertente de estudo, pois tanto a sÃntese de Ãcidos graxos como dos Ãsteres de forbol, os dois compostos mais atrativos no estudo de Jatropha curcas, ocorrem nos plastÃdeos. O estudo proteÃmico dessa organela torna-se crucial para melhor compreender e explorar nÃo somente as vias biossintÃticas desses dois compostos, como de outras vias metabÃlicas, alÃm de proporcionar um conjunto de dados que pode ser utilizado em pesquisas voltadas para o desenvolvimento de genÃtipos com caracterÃsticas mais adequadas para aplicaÃÃes industriais. No presente trabalho, realizou-se uma anÃlise proteÃmica de plastÃdeos isolados do endosperma de sementes em desenvolvimento do pinhÃo manso, que estavam nos estÃgios iniciais de deposiÃÃo de lipÃdios e proteÃnas de reserva (25-30DAA), confirmados por meio de anÃlises histolÃgica e histoquÃmica. As proteÃnas extraÃdas dos plastÃdeos foram digeridas com tripsina e os peptÃdeos foram aplicados no sistema de nano-LC EASYII acoplado online ao espectrÃmetro de massa nano ESI LTQ-Orbitrap velos, o que resultou na identificaÃÃo 1103 proteÃnas, representando 804 grupos de proteÃnas, dos quais 923 foram consideradas identificaÃÃes verdadeiras. Isso expandiu consideravelmente o repertÃrio de proteÃnas do pinhÃo manso atà agora identificas. Dentre as proteÃnas identificadas, apenas 5 sÃo codificadas pelo genoma plastidial, e nenhuma delas està envolvida na fotossÃntese, o que evidencia a natureza nÃo fotossintÃtica dos plastÃdeos isolados. HomÃlogos de 824, dentre as 923 proteÃnas identificadas, estavam presentes nos bancos de dados PPDB, SUBA e PlProt, enquanto homÃlogos para 13 proteÃnas nÃo foram encontrados em nenhum dos trÃs bancos de dados plastidiais, mas foram detectados como plastidiais por pelo menos um dos trÃs programas de prediÃÃo de localizaÃÃo subcelular utilizados (TargetP, ChloroP, PlantMPloc). A classificaÃÃo funcional mostrou que a maioria das proteÃnas identificadas pertencia ao metabolismo dos aminoÃcidos, seguido dos metabolismos dos carboidratos, energÃtico e dos lipÃdios. As subunidades maiores e menores da Rubisco foram identificadas, e sua presenÃa nos plastÃdeos foi considerada uma caracterÃstica adaptativa para contrabalancear a perda de um terÃo do carbono na forma de CO2 como um resultado da conversÃo de carboidratos em Ãleo atravÃs da glicÃlise. Apesar de enzimas envolvidas na biossÃntese de diversos precursores dos diterpenÃides terem sido identificadas, nÃo foi detectado nenhuma terpeno sintase/ciclase, o que sugere que os plastÃdeos isolados do endosperma de sementes em desenvolvimento nÃo sintetizam Ãsteres de forbol, apesar de uma grande quantidade desse composto ser encontrada neste tecido. Como conclusÃo, o presente trabalho proporciona insights sobre as principais vias biossÃntÃticas, e sobre caracterÃsticas peculiares dos plastideos isolados do endosperma de sementes em desenvolvimento.
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Kierul, Kinga. "Comprehensive proteomic study of Bacillus amyloliquefaciens strain FZB42 and its response to plant root exudates". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16805.
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Bacillus amyloliquefaciens FZB42 ist ein frei lebendes Bakterium, das Pflanzenwurzeln besiedelt und das Pflanzenwachstum durch viele verschiedene Wirkmechanismen anregt. In dieser Arbeit wurden die molekularen Grundlagen dieser positiven Wirkungen, die dieses „Pflanzenwachstum fördernde Rhizobakterium“ (PGPR) auf seine Wirte ausübt, untersucht. Um den gegenseitigen Austausch von B. amyloliquefaciens und seinen Wirtspflanzen zu entschlüsseln, wurden umfangreiche Proteomstudien durchgeführt. Es wurden Referenzkarten der extrazellulären und zytosolischen Proteinfraktionen erstellt. Die größte Anzahl an ausgeschiedenen Proteinen konnte während der stationären Phase beobachtet werden. Die identifizierten extrazellulären Proteine gehören verschiedenen Funktionsklassen an, wobei die prominentesten Klassen am Kohlenhydrat-Abbau und den Transport von Molekülen durch die Zellwand beteiligt sind. Die zytosolischen Extrakte von Kulturen, die in 1C-Medium bzw. Mineralmedium angezogen wurden, und in der zweidimensionalen Gelelektrophorese (2 DE) aufgetrennt wurden, ergaben 461 und 245 verschiedene Protein-Einträge. Die erstellten Referenz-Karten wurden anschließend verwendet, um Proteine und Prozesse, in an der Interaktion mit Pflanzen beteiligt sind, zu identifizieren. Dafür wurden die Bakterien Wurzelexudaten von Mais (Zea mays L.) ausgesetzt. Die Proteine aus zwei Stämmen, denen die globalen Transkriptionsregulatoren (Degu, AbrB) und vier Sigma-Faktoren (SigB, SigM, SigV, und SigX) fehlen, wurden ebenfalls untersucht, um ihre Beteiligung an den bakteriellen Reaktionen auf die Wurzelausscheidungen zu analysieren. Zusammenfassend ist dies die erste Studie, die umfangreiche Proteomdaten von Gram-positiven PGPR präsentiert, wobei gleichzeitig die Veränderung der Expression von extrazellulären und zytoplasmatischen Proteinen, nach Zugabe von Wurzelexudaten, ausgewertet wurde.Bacillus amyloliquefaciens strain FZB42 is a free-living bacterium that competitively colonizes plant roots and stimulates plant growth by many different modes of action. The molecular basis of singular beneficial effects that this Plant Growth-Promoting Rhizobacteria (PGPR) exert on their hosts have been studied. To decipher the molecular cross-talk of B. amyloliquefaciens and its’ host plants as a whole system, an extensive proteomic approach was performed. Reference maps of the extracellular and cytosolic protein fractions were established. The highest number of secreted proteins was observed during stationary growth phase. Identified extracellular proteins belong to different functional classes, with the most prominent classes involved in carbohydrate degradation and transportation of molecules across the cell wall. Cytosolic extracts obtained from cultures grown in 1C and minimal media subjected to the 2 Dimensional Electrophoresis (2 DE), revealed 461 and 245 different protein entries, respectively. Created reference maps were subsequently used to identify proteins and processes involved in the interaction with plants, prior to exposure of bacteria to maize (Zea mays L.) root exudates. The proteomics of two strains lacking expression of genes coding for global transcriptional regulators (degU, abrB) and four sigma factors (sigB, sigM, sigV, and sigX) were also inves-tigated, in order to analyse their involvement in bacterial responses to root exudates. In summary, this is the first study presenting comprehensive proteomics of Gram-positive PGPR, evaluating at the same time changes in protein expression caused by addition of root exudates at the extracellular and cytosolic level.
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Bernardo, Letizia. "IDENTIFICATION AND CHARACTERIZATION OF PROTEINS INVOLVED IN BIOTIC STRESS RESISTANCE OF CEREALS". Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3426966.
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Leaf rust is one of the most important diseases of barley (Hordeum vulgare) and is caused by the biotrophic fungal pathogen Puccinia hordei. The rust fungi penetrate barley leaves through stomata and colonize cells of the mesophyll, then growing systemically through the leaf vascular tissue.
The leaf rust resistance gene Rph15 is of outstanding interest for resistance breeding because it confers resistance to over 350 Puccinia hordei isolates collected from around the world (Weerasena et al. 2004).
Plant-pathogen interactions activate many cellular signalling processes and, most likely, changes on protein accumulation and phosphorylation pattern of proteins play a pivotal role in plant responses to biotic stress. In this work, a proteomic approach was undertaken to study changes in total proteins accumulation and protein phosphorylation pattern in response to the leaf rust pathogen infection in two barley near isogenic lines, Bowman and Rph15, which differ for the introgression of the leaf rust resistance gene Rph15. Two infection time points, 24 hours and four days, were considered for the analysis.
No statistically significant differences were identified at the early time point, 24 hours post infection, for total and phosphorylated proteins.
At four days after inoculation, total protein analysis led to the identification of twenty-one protein spots significantly up or down regulated with a fold-change equal or higher than two following pathogen infection. Most of down-regulated proteins were found in the Rph15 near-isogenic line while no significantly differential protein abundance was recovered in the susceptible line. Nineteen out of 21 protein spots were characterized by LC-MS/MS analysis and found to be involved in photosynthesis, sugar metabolism, energy balance and defence.
Phosphoproteomics analysis was performed at four day after inoculation. A phosphoprotein enrichment methodology based on MOAC (metal oxide affinity chromatography) was optimized for subsequent 2DE analyses.La ruggine fogliare è una delle malattie più importanti della coltura dell'orzo (Hordeum vulgare) ed è causata dal patogeno fungino biotrofo Puccinia hordei. Il fungo penetra attraverso gli stomi delle foglie dell’orzo e colonizza le cellule del mesofillo, crescendo poi per via sistemica nei tessuti vascolari della foglia. Il gene Rph15 di orzo è di considerevole importanza per il miglioramento genetico della resistenza in quanto conferisce resistenza a più di 350 isolati di P. hordei provenienti da tutto il mondo (Weerasena et al. 2004). L’interazione pianta-patogeno attiva numerosi processi di signalling cellulare e, molto probabilmente, l’accumulo delle proteine e i cambiamenti nel pattern di fosforilazione delle proteine giocano un ruolo centrale nella risposta della pianta in seguito a stress biotico. In questo lavoro, un approccio di tipo proteomico è stato intrapreso per studiare i cambiamenti nei pattern proteici totali e delle proteine fosforilate in seguito a risposta alla ruggine fogliare in due linee quasi isogeniche di orzo, Bowman e la linea Rph15, che differiscono per l’ introgressione del gene Rph15. Due tempi di infezione, 24 ore e quattro giorni, sono stati presi in considerazione per le analisi. Nessuna differenza statisticamente significativa è stata individuate nel primo tempo di infezione precoce, a 24 ore dopo l’inoculo, sia per quanto riguarda le proteine totali che per le proteine fosforilate. A 4 giorni dall’infezione, l’ analisi delle proteine totali ha consentito di identificare ventuno spot proteici significativamente up o down regolati in risposta all’ infezione con un fold-change almeno di 2. La maggior parte delle proteine down-regolate sono state trovate nel campione infettato della linea isogenica contenente il gene di resistenza Rph15, mentre non è stata riscontrata alcuna differenza statisticamente significativa nel pattern proteico della linea isogenica suscettibile. Diciannove dei 21 spot proteici sono stati caratterizzati mediante analisi LC-MS/MS e identificati essere implicati in processi come fotosintesi, metabolismo degli zuccheri, bilancio energetico e risposte di difesa. L’analisi del fosfoproteoma è stata condotta a quattro giorni dopo l’inoculo. Una tecnica di arricchimento in fosfoproteine basata su MOAC (cromatografia di affinità mediante ossidi metallici) che è stata ottimizzata per la successiva analisi 2DE.
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MURATORE, CHIARA. "CHARACTERIZATION OF PROTEOMIC CHANGES IN CROPS DURING METABOLIC ADAPTATION TO DIFFERENT NITROGEN INPUTS". Doctoral thesis, Università degli Studi di Milano, 2023. https://hdl.handle.net/2434/951273.
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Nitrogen availability is one of the major factors that influence plant growth, morphology, and metabolism and, hence, crop productivity. In agricultural soils, nitrogen is present in different forms, both inorganic (nitrate and ammonium) and organic (amino acids, short peptides and urea), with variable and heterogeneous distribution. Nowadays, improving knowledge about the nitrogen nutrition in plants is crucial to address the urgent need for a more sustainable agricultural production. In the last years, the “-omics” approaches provided a holistic perspective of the molecular mechanisms underlying plant metabolic adaptations to different nitrogen inputs. Among these, proteomics was largely and successfully applied to analyze various aspects, including the role of post-translational modifications and enzyme isoforms. The aim of this PhD project was to obtain new insights about the biochemical events during sensing and adaptation to different availabilities of nitrogen forms in crops, through an approach based on the integration of physiological, metabolic and proteomic evaluations.
The first research activity consisted in an extensive literature revision about plant nitrogen nutrition and plant proteomics, which led to the publication of a review article. This activity highlighted that the majority of the information derives from studies conducted in Arabidopsis and in model crops, such as maize, rice and tomato. Nevertheless, analogies and peculiarities remain to be verified in other crop species. For instance, nitrogen nutrition has been only slightly investigated in perennial plants, and proteomics has been rarely applied in this context. At the same time, although in herbaceous species great progress has been made in understanding plant metabolic responses to inorganic nitrogen forms, several aspects await a clear elucidation. A representative case consists in the fact that a clear overview of the role of specific subcellular compartments during nitrate and/or ammonium nutrition is still fragmentary. Moreover, the literature revision pointed out an increased interest in understanding the relevance of organic nitrogen forms as nutrients influencing plant growth and development. Although it is recognised that plants are able to take up organic forms of nitrogen, such as amino acids, their actual contribution to plant nitrogen nutrition is currently unknown. Starting from these considerations, during the PhD three studies have been designed and conducted, leading to the publication of an article and the preparation of two manuscripts.
The first study was devoted to the analysis of nitrogen metabolism in a perennial woody species. In particular, it was aimed at investigating the biochemical and proteomic responses to nitrate in a grapevine rootstock genotype. Indeed, even though grapevine has been adopted as a model perennial species, little is known about the biochemical roles played by roots in nitrogen acquisition. Moreover, this topic has never been addressed through a proteomic approach.
The aim of the second study was to deepen the knowledge about the responses to nitrate, ammonium or their co-provision in maize seedlings, adopting a novel proteomic approach. Specifically, key proteins involved in these biochemical mechanisms are localized in different cellular compartments and their functionality is deeply affected by the kind of inorganic source. Hence, a subcellular proteomic approach was applied to obtain new insights about the roles of distinct organelles in the ability of roots to manage different inorganic nitrogen availabilities.
Finally, the third study aimed to investigate whether maize plants are able of using and metabolizing amino acids as nitrogen source, when they were externally supplied as a mixture that mimics soil conditions. Considering the complexity of the amino acid metabolism in plants, the application of a proteomic approach was chosen as a useful holistic strategy to obtain a comprehensive overview and to gain new information. In particular, the physiological, biochemical and proteomic changes in roots and leaves were compared to those associated with nitrate availability as a reference inorganic nitrogen source.
Overall, these studies provide novel information about how plants perceive and adapt to different nitrogen availabilities. According with the literature, nitrate influenced several aspects acting as either a nutrient, an osmolyte and a signal molecule. In addition, it has been possible to highlight that, in comparison to herbaceous species, specific responses to nitrate occurred in grapevine. Moreover, the subcellular proteomic investigation allowed to appreciate how nitrate and ammonium have different, and sometimes contrasting, effects on the cell organelles functionalities, especially with regard to mitochondria and vacuoles. Finally, new hints about the metabolic pathways involved in amino acid-based nutrition were provided. In particular, the provision of amino acids to the maize plants impacted on the carbon and energy metabolism in roots and influenced the translocation of amino acids to shoot.
In conclusion, these studies confirm that the different nitrogen sources have distinct and significant effects on plant growth and physiology, and also put in evidence some interesting peculiarities related to plant species and developmental stages. Moreover, they underline the key role of roots in response to nitrogen forms, providing new evidence that amino acid metabolism represents a key point in the carbon/nitrogen balance in plants.
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Katam, Ramesh. "MOLECULAR AND BIOCHEMICAL ANALYSIS OF WATER STRESS-INDUCED RESPONSES IN GRAPE". MSSTATE, 2008. http://sun.library.msstate.edu/ETD-db/theses/available/etd-11052008-162021/.
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Water stress affects vine productivity, disease tolerance, and enological characteristics of grape. Florida Hybrid Bunch grape are developed through hybridization of local grape spp with Vitis vinifera. These cultivars are mostly grown in southeast region of United States. Water deficit conditions resulted due to failure of rains in the region has developed concern among Florida grape growers to increase water use efficiency of grape. The goal of this research is to identify genes and proteins differentially expressed in response to water stress and to correlate these changes with enological characteristics. Investigating transcripts and proteins will allow us to correlate them and confirm the involvement of specific genes responding to stress. Florida hybrid bunch Suwannee grape plants were maintained under green house conditions. Water stress was induced by withholding irrigation. The leaf samples were collected from both irrigated and stressed plants at 5, 10, 15 and 20 day interval. We generated over 200 Subtractive Hybridization PCR products from control and water stressed leaf tissues. Cloning, sequencing and transcript analysis revealed that, 54 genes related to drought and defense regulated pathways out of 125 characterized transcripts. Proteins were extracted from leaf tissue with trichloroacetic acid /acetone and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The proteins were sequenced in LC/Mass Spectrophotometer. The most important differentially expressed genes include sucrose synthase, actin, isoprene synthase, ABF3, SNF1 related protein kinase, WRKY type transcription factors, AP2, ASR2, glyoxalase I and, cytochrome b which play significant role in cell permeability, transportation, photosynthesis and, maintenance in osmotic stress. We have found that ribulose bisphosphate carboxylase and phosphoribulokinase, which play major role in photosynthesis, were suppressed in response to water stress in Florida hybrid bunch. The results suggested that water stress affects expression of cDNAs associated with defense and drought regulated functions. Such profiling studies will be used to explicate specific pathways disconcerted by water deficit treatments, and in the identification of varietal differences.
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Teplitski, Max I. "Quorum sensing in Sinorhizobium meliloti and effect of plant signals on bacterial quorum sensing". The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1029777185.
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Nogueira, FÃbio CÃsar Sousa. "AnÃlise ProteÃmica da DeposiÃÃo de ProteÃnas em Sementes em Desenvolvimento e SuspensÃes Celulares EmbriogÃnicas de FeijÃo-de-Corda [Vigna unguiculata (L.) Walp.]". Universidade Federal do CearÃ, 2007. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=848.
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CoordenaÃÃo de AperfeiÃoamento de NÃvel SuperiorO feijÃo-de-corda (Vigna unguiculata) Ã uma leguminosa bastante utilizada na alimentaÃÃo de famÃlias de baixa renda da regiÃo nordeste do Brasil. Embora suas sementes sejam ricas em proteÃnas, estas sÃo deficientes em aminoÃcidos sulfurados na sua composiÃÃo. Dessa forma, um aumento na qualidade nutricional dessas sementes tem sido um dos principais objetivos dos programas de melhoramento genÃtico para essa espÃcie. AlÃm de determinar o padrÃo de deposiÃÃo de proteÃnas durante o desenvolvimento de embriÃes zigÃticos e somÃticos, nÃs pretendemos identificar genes com padrÃes especÃficos de expressÃo durante a embriogÃnese com a finalidade de utilizÃ-los em programas de melhoramento genÃtico. Utilizando a tÃcnica de eletroforese bidimensional (2D-PAGE) foi comparado o padrÃo protÃico de sementes em desenvolvimento com 10 dias apÃs a antese (DAA), sementes maduras e de suspensÃes celulares embriogÃnicas (SCE) obtidas de calos embriogÃnicos friÃveis (CEF) de feijÃo-de-corda. Para cada estÃgio de desenvolvimento da semente e para as SCE foram obtidos mapas de referÃncia proteÃmicos altamente reprodutÃveis numa faixa de pH de 3-10 e 4-7. VÃrios âspotsâ foram selecionados baseando-se na quantidade ou volume relativo de cada âspotâ e na taxa de expressÃo. Cerca de 800 (para sementes em desenvolvimento) e 130 (para SCE) âspotsâ protÃicos regulados para cima, para baixo, que se mantÃm constantes ou que sÃo especÃficos durante o desenvolvimento foram retirados dos gÃis 2D para anÃlise por espectrometria de massa. Algumas estratÃgias foram utilizadas para a identificaÃÃo das proteÃnas, como: PMF (peptide mass fingerprinting) e busca atravÃs de dados nÃo processados (MS/MS ion search). Dessa forma, cerca de 400 proteÃnas foram identificadas em sementes e cerca de 70 proteÃnas foram identificadas para SCE. A maioria das proteÃnas fram classificadas como proteÃnas do metabolismo primÃrio, energÃtico, proteÃnas de destinaÃÃo/proteÃnas de reserva e proteÃnas de defesa ou relacionadas a algum tipo de estresse.
Cowpea (Vigna unguiculata) is a leguminous plant highly utilized by low-income earners of Northeastern Brazil. However, proteins found in the crop are highly deficient in sulfur-containing amino acids. In line with this, improvement of nutritional quality of cowpea seeds has been one of the major goals of breeding programs of the crop. A starting point in this respect is a concerted effort to study the basic biochemical and physiological aspects of the development of cowpea seed. Besides determining the protein deposition pattern during the development of zygotic embryos and somatic embryos of the species, we set out to identify genes with specific pattern of expression during the two processes which will be of immense importance in cowpea breeding. Using the technique of two-dimensional electrophoresis (2D-PAGE), we compared the protein deposition pattern of developing cowpea seeds with 10 days after anthesis (DAA) and mature seeds and embryogenic cell suspension (ECS). From each developmental stage and for ECS, we obtained proteomic reference maps that were highly reproducible within the pH of 3-10 and 4-7. Various spots were selected based on quantity or relative volume and rate of expression. About 800 (for seeds development) and 130 (for ECS) proteins spots up- or down-regulated, remained constantly expressed and were specific for each developmental stage. The spots were removed from the 2-D gels, processed, and subjected to mass spectrometry. Some strategies like peptide mass fingerprinting (PMF) and non-processed data search (MS/MS ion search) were employed for protein identification. Over 400 proteins in seeds and over 70 proteins in ECS were identified. A large number of these proteins were found to be primary metabolism proteins, energy, protein destination and storage and defense proteins and others related to stress.
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Budzinski, Ilara Gabriela Frasson. "Avaliação do metabolismo primário da região cambial e casca de Eucalyptus grandis". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-02012013-172036/.
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O gênero Eucalyptus é uma das principais espécies arbóreas comercialmente plantadas em todo o mundo. Apresenta inúmeras características favoráveis para a produção e comercialização de sua madeira, tais como o rápido crescimento, rotação de ciclo curto e produção de biomassa renovável, com potencial para geração de biocombustível. Apesar de sua grande importância econômica, pouco é conhecido sobre os processos moleculares que envolvem a formação da madeira e casca, principalmente no que diz respeito ao metabolismo primário. Ademais, não se têm muitas informações sobre mudanças moleculares que ocorrem durante a formação desses tecidos em resposta a variações sazonais. Sabe-se que a região cambial das árvores apresenta maior atividade metabólica durante o verão, quando comparada ao inverno. Deste modo, visando compreender mudanças dinâmicas ao nível transcricional, protéico e metabólico, principalmente em relação ao metabolismo primário, o presente trabalho teve por objetivo comparar os tecidos da região cambial e casca, coletados em diferentes períodos climáticos (verão e inverno). A expressão do padrão transcricional foi analisada por PCR em tempo real, seguido de normalização e análise estatística, usando os programas NormFinder e Rest, respectivamente. O perfil protéico foi obtido por eletroforese bidimensional (2D-PAGE), seguido de análise por espectrometria de massas associada a cromatografia líquida (LC-MS/MS) e posterior identificação das proteínas pelo programa Mascot Daemon. Já o perfil metabólico foi obtido por espectrometria de massas associada à cromatografia gasosa (GC/MS), com posterior análise dos picos identificados pelo programa MatLab. Em seguida, as análises estatísticas foram geradas pelo programa SIMCA P+ e \"R\". Para os transcritos, foi possível observar tanto para a região cambial quanto para a casca padrão diferencial na expressão dos genes analisados, principalmente aqueles atuantes na glicólise, durante os dois períodos sazonais. Quanto ao perfil protéico, foram identificadas um total de 77 e 75 proteínas estatisticamente significativas na região cambial e casca, respectivamente. Proteínas pertencentes ao metabolismo primário foram identificadas em ambos os tecidos. Metabólitos relacionados ao metabolismo de açúcares também foram encontrados.Eucalyptus genus is the most widely planted hardwood crop in the world because of its superior growth, broad adaptability and multipurpose wood properties. In today\'s \"new carbon economy\", eucalypts are receiving attention as fast-growing, short-rotation, renewable biomass crop for energy production. In spite of its economical importance, little information is available about the molecular changes that occur in primary metabolism in the wood and bark forming tissues. Furthermore, there is less information about molecular changes that occur during wood and bark formation in response to seasonal variation. It\'s known that Eucalyptus cambial region presents higher metabolic activity in summer than in winter. Thus, in order to observe the dynamic changes in transcript, protein and metabolite levels, mainly related to primary metabolism, we compared cambial tissue and bark collected in two different seasons (summer high temp + high rainfall) and winter (lower temp and little rainfall). Transcript expression patterns were analyzed by Real-Time PCR, normalization chosen by NormFinder and statistical analysis carried out using REST. The protein profile was obtained by bidimensional electrophoresis (2D-PAGE) followed by liquid chromatography associated with mass spectrometry (LC-MS/MS) and analyzed by Mascot Daemon. Metabolite profile was obtained by GC-TOF/MS, peaks were analyzed in MatLab and statistical analyses were done using SIMCA and \"R\". The results obtained with transcripts indicate differential gene expression in the cambial region and bark during summer and winter. A total of 77 and 75 proteins in cambium and bark, respectively, presented statistically significant alterations and were identified and classified into functional categories. We identified many proteins from primary metabolism. Metabolites from carbohydrate metabolism were also identified.
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Juárez, Ortega Paloma. "Production of recombinant Immunoglobulin A in plants for passive immunotherapy". Doctoral thesis, Universitat Politècnica de València, 2014. http://hdl.handle.net/10251/37015.
Texto completoResumen
Mucosal passive immunization is the transfer of active antibodies from one organism
to the mucosal surfaces of another organism for preventing or treating infectious diseases.
Mucosal passive immunization has a great potential for the prevention and treatment of
enteric infections like Rotavirus, which causes more than 114 million episodes of diarrhoea
annually with a death toll of more than 450.000 per year. However, the high cost of
recombinant antibodies with the current manufacturing systems based on mammalian cells
hampers the production of the high antibody quantities required for passive immunization
strategies. Alternative expression platforms such as plants could provide higher scalability and
reduced costs. Moreover, the use of edible plant organs, which are Generally¿Regarded¿As¿
Safe (GRAS), could reduce manufacturing costs even further by easing the requirements for
antibody purification. We analyze here the feasibility of utilizing fruits as inexpensive
biofactories of human antibodies that can be orally delivered as crude extracts or partially
purified formulations in mucosal passive immunization strategies.
In the first section of this thesis, the construction of tomato plants producing a model
human Immunoglobulin A (IgA) against rotavirus in their fruits is described. As a result, an elite
homozygous line was obtained whose fruits produced on average 41 ¿g of IgA per gram of
fresh weigh, equivalent to 0.69 mg IgA per gram of dry tomato powder. Minimally processed
products derived from IgA¿expressing tomatoes were shown to strongly inhibit virus infection
in an in vitro neutralization assay. Moreover, in order to make IgA¿expressing tomatoes easily
distinguishable from wild¿type tomatoes, they were sexually crossed with a transgenic tomato
line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors,
which confer purple colour to the fruit. The resulting transgenically¿labelled purple tomatoes
contained not only high levels of recombinant neutralizing human IgA but also increased
amounts of anthocyanins.
In the second section of the thesis the composition of IgA¿expressing tomatoes was
analyzed in search of possible unintended effects that could compromise the GRAS status of
the final product. To this end, transgenic IgA¿tomatoes were compared with wild type
tomatoes and also commercial tomato varieties using proteomic and metabolomic
approaches. 2D¿DIGE gels coupled with LC¿MSMS for protein identification showed that all the
uptrend differential proteins detected corresponded only to immunoglobulin chains or
antibody fragments. On the other hand, non¿targeted metabolite data obtained by UPLC¿MSJuárez Ortega, P. (2014). Production of recombinant Immunoglobulin A in plants for passive immunotherapy [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37015
TESIS
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Cipriano, Aline Kelly de Aquino Lima. "The interaction of cashew (Anacardium occidentale L.) with the fungus Lasiodiplodia theobromae reprograms the expression of proteins in the stem, the site of pathogen infection". Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=12618.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorNo Brasil, a indÃstria do caju à uma das principais fontes de renda e trabalho no campo e representa a maior parcela da economia na regiÃo nordeste, que concentra 94% da produÃÃo, destacando-se os estados do CearÃ, Piauà e Rio Grande do Norte, respectivamente. Neste contexto, para otimizar o setor produtivo, estratÃgias de melhoramento genÃtico de cultivares tÃm focado na seleÃÃo e desenvolvimento de clones anÃos. Entretanto, essa prÃtica tem contribuÃdo para diminuiÃÃo da variabilidade genÃtica e, consequentemente, para maior vulnerabilidade ao ataque de patÃgenos. A resinose, causada pelo fungo Lasiodiplodia theobromae (Pat.) Griff & Maubl. à considerada a principal doenÃa do cajueiro nas condiÃÃes semiÃridas. MÃtodos eficientes de controle da doenÃa ainda nÃo foram estabelecidos. Baseado na ausÃncia de dados publicados com relaÃÃo Ãs respostas bioquÃmicas e fisiolÃgicas do cajueiro infectado com L. theobromae, associado ao fato de que as plantas, para se defenderem do ataque de patÃgenos, acionam/alteram vias metabÃlicas controladas por diversas proteÃnas, o estudo da identidade dessas proteÃnas fornecem informaÃÃes sobre os mecanismos relacionados à interaÃÃo de compatibilidade/incompatibilidade entre o cajueiro e L. theobromae. Dessa forma, nesse estudo, foi realizada anÃlise proteÃmica diferencial de caules de cajueiro (Anacardium occidentale), clone BRS 226 (resistente), mantido em condiÃÃes controladas, em tempos iniciais pÃs-infecÃÃo com o L. theobromae, assim como, de plantas de cajueiro resultantes da polinizaÃÃo aberta do BRS 226, classificadas como resistentes e suscetÃveis à resinose, em condiÃÃes de campo, onde hà alta pressÃo do patÃgeno. ProteÃnas diferencialmente expressas foram identificadas por eletroforese bidimensional (2D-PAGE) combinada com espectrometria de massas ESI-Q-TOF MS/MS. Plantas de cajueiro infectadas com L. theobromae acionam respostas fisiolÃgicas associadas à reprogramaÃÃo da expressÃo de um total de 73 proteÃnas, nos cenÃrios investigados. Destas, 36 foram identificadas no clone BRS 226, artificialmente inoculado, e 37 nas plantas de cajueiro, cultivadas em condiÃÃes de campo. Portanto, um nÃmero equivalente de proteÃnas à responsivo dentro dos cenÃrios analisados e compartilham funÃÃes celulares em rotas metabÃlicas e de produÃÃo de energia, estresse/defesa, sinalizaÃÃo celular e enovelamento/metabolismo de proteÃnas. ProteÃnas responsivas a hormÃnios e envolvidas com estrutura celular foram diferencialmente expressas somente em plantas crescidas em condiÃÃo de campo, enquanto proteÃnas de transporte foram reprogramadas no clone BRS 226. Plantas de cajueiro, cultivadas em campo, e inoculadas do clone BRS 226 compartilharam a expressÃo reprogramada de 6 proteÃnas idÃnticas, dentre as quais, proteÃnas 14-3-3 e anexinas, envolvidas com a sinalizaÃÃo celular, foram influenciadas ao mesmo tempo, aparentemente, pelos estresses abiÃtico e biÃtico. A reprogramaÃÃo de funÃÃes celulares comuns somado a alteraÃÃo na expressÃo de 6 proteÃnas idÃnticas, nas condiÃÃes estudadas, mostrou sobreposiÃÃo de respostas, que parece ser indicativo da infecÃÃo pelo L. theobromae no tecido caulinar. Essas observaÃÃes revelam proteÃnas que sÃo alvos dos mecanismos celulares acionados em plantas de cajueiro desafiadas com L. theobromae e constituem uma base inicial de resultados que podem ser, futuramente, integrados a programas de melhoramento genÃtico do cajueiro, visando resistÃncia, particularmente, à resinose.
Recent studies demonstrated the efficacy of mangrove retaining nutrients and, in particular, the ability of the mineral component of the buffer in its high soil phosphorus levels. In general, the mangroves have been considered as important sinks of nutrients due to its high capacity purification of effluents. However, depending on the geochemical conditions exist, these soils can act as a source of phosphorus to other environments and / or coastal waters. Given that the geochemical behavior of phosphorus and its role in eutrophication of water bodies, is best measured by the behavior of its different fractions, this paper aims at a fractionation of the different forms of phosphorus in wetlands impacted by different effluent. The objective of this project is to study three areas in order to assess how activities impacting interfere in the process of nutrient cycling (with special emphasis on the dynamics of phosphorus forms) and, as the marsh supports the stress caused by these activities. Also, if you evaluate the potential for eutrophication of each human activities. Taking into consideration the impact that these environments suffer as a result of separate activities, were established the following areas of study: a marsh impacted by effluents from shrimp, a marsh impacted by effluents, and a control area located in a preserved area that still finds is little affected by human impacts. The samples were determined pH, Eh, salinity, grain size and the total content of C and P. In addition, extraction was performed sequentially phosphorus which allows differentiation of fractions 7: P exchangeable (NaClP), P associated with iron oxides (Fe-P); organic P (AH-P), the bound phosphorus hydroxides Al (Al-P), P associated with compounds of calcium (Ca-P); phosphorus associated refracting matter (P-RES) and unreacted phosphorus (P-NR). The results indicate that the discharge of effluents in mangrove increases the amount of phosphorus in these environments, especially in organic form, the phosphorus bound to carbonate is the major inorganic fraction in these environments.
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Tahmasian, Arineh. "Lupin: Prospective superfood or potential allergen?" Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2023. https://ro.ecu.edu.au/theses/2655.
Texto completoResumen
The demand for plant-based protein sources is on the rise. Lupins, important members of the legume family, are one of the richest natural sources of protein and fibre and can positively contribute to global food and nutritional security. Despite their strong potential, lupins remain under-utilised as a human food and are predominantly grown as green manure and livestock feed. One constraint to the widespread adoption of lupins in the food industry is allergenicity, which has led to its inclusion in the list of food allergens subjected to mandatory labelling in many countries.
The research presented herein focused on the study of proteins in lupin seeds, aiming to incentivise exploitation of this under-utilised legume by enhancing the available proteome-level knowledge and addressing the allergenicity challenges attributed to this legume. Firstly, the efficiency of four solvents (IPA-DTT, Tris-HCl, Urea and IPA→Urea) in extracting proteins from three narrow-leafed lupin genotypes (Tanjil, Unicrop and P27255) were evaluated through global discovery and quantitative proteomic approaches. The integration of complementary solvent systems enabled identification of 2,760 proteins from these genotypes. In addition, the proteome-wide relative quantitative analysis highlighted differences in the protein profiles of the wild and domesticated lupin genotypes and demonstrated the substantial influence of the protein extraction method on the proteome coverage and downstream biological interpretation of the data.
The diversity of the major lupin seed storage proteins, known as conglutins, were assessed across a panel of 46 genetically diverse narrow-leafed lupin genotypes. The differentiation and relative quantitation of the 16 conglutin sub-families, belonging to the four major α-, β-, γ-, and δ- families, was achieved by monitoring a set of maker peptides specific to each protein sub-family. Whilst this comparative evaluation determined distinct differences in the conglutin profiles of the lines under investigation, the major variability was observed for the β-, and δ-conglutin sub-families, wherein, the allergenic β-conglutin proteins were found at considerably lower levels in a subset of Australian and Polish domesticated varieties. These narrow-leafed lupin cultivars can serve as potential hypoallergenic varieties and be implemented in breeding programs for further enhancing the lupin grain as a human food ingredient.
Finally, a combination of discovery and targeted quantitative proteomic approaches were applied to examine the changes driven by solid-state fermentation (induced by the starter culture Rhizopus oligosporus) in the white lupin allergenic protein profiles. The comparative proteomics study of the allergen derived peptides across the pre-fermented and fermented samples revealed a significant decrease in the levels of ~94% of the monitored peptides as result of fermentation. This effect was more prominent across the β-conglutin peptides, for which a decrease > 50% was observed for ~70% of the monitored peptides. These observations suggest good efficiency of solid-state fermentation for the degradation of the allergenic proteins and development of innovative lupin-based food products with reduced allergenicity.
The findings of these proteomic studies contribute to advancing the proteome-level knowledge available for lupin seeds, thereby providing opportunities to now enhance lupin seed protein composition and stimulate the broader application of this grain legume as a food ingredient.
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Marques, Felipe Garbelini. "Análise de metabólitos e proteínas totais em folhas de Eucalyptus grandis durante a infecção por Puccinia psidii". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-07062016-184853/.
Texto completoResumen
Os mecanismos moleculares envolvidos na resistência de plantas contra patógenos são um tema bastante discutido no meio acadêmico, sendo o objetivo maior dos estudos a diminuição das perdas de produtividade provocadas por doenças em plantações do mundo todo. Muitos modelos de interação patógeno-hospedeiro foram propostos e desenvolvidos priorizando plantas e culturas de rápido desenvolvimento com ciclo de vida curto. Espécies de ciclo longo, porém, devem lidar durante anos - ao menos até a idade reprodutiva - contra o ataque de bactérias, fungos e vírus, sem contar, nesse meio tempo, com recombinações genéticas e mutações que tornariam possível o escape contra as moléstias causadas por microrganismos. Assim, como alternativa aos modelos usuais, o presente trabalho estudou um diferente par de antagonistas: Eucalyptus grandis e Puccinia psidii. Apesar da contribuição de programas de melhoramento genético, o patossistema E. grandis X P. psidii ainda é pouco descrito no nível molecular, havendo poucos estudos sobre os processos e as moléculas que agem de forma a conferir resistência às plantas. Assim, buscando o melhor entendimento da relação entre E. grandis X P. psidii, o presente trabalho estudou a mudança dos perfis de proteínas e metabólitos secundários ocorrida nos tecidos foliares de plantas resistentes e susceptíveis durante a infecção pelo patógeno, com o auxílio da técnica de cromatografia líquida acoplada à espectrometria de massas. Os resultados obtidos indicam que as plantas resistentes percebem a presença do patógeno logo nas primeiras horas pós-infecção, produzindo proteínas ligadas à imunidade (HSP90, ILITYHIA, LRR Kinase, NB-ARC disease resistance protein). Essa percepção desencadeia a produção de proteínas de parede celular e de resposta oxidativa, além de modificar o metabolismo primário e secundário. As plantas susceptíveis, por outro lado, têm o metabolismo subvertido, produzindo proteínas responsáveis pelo afrouxamento da parede celular, beneficiando a absorção de nutrientes, crescimento e propagação de P. psidii. No trabalho também são propostos metabólitos biomarcadores de resistência, moléculas biomarcadoras de resposta imune e sinais da infecção por patógeno em E. grandis.The molecular mechanisms involved in the plant resistance against pathogens is a well-discussed theme in the academy, overall objecting to diminish worldwide plantation yield losses caused by diseases. Many pathogen-host models were proposed and developed prioritizing model plants and fast growing crops with short life cycles. However, long life cycle species need to cope with the attack of bacteria, fungi and virus throughout many years, or at least until its reproduction period, being unable, meanwhile, to escape the attack of these microorganisms through genetic recombination and mutation. Therefore, as an alternative to the usual models, the present work studied a different pair of antagonists: Eucalyptus grandis and Puccinia psidii. Despite the contribution of plant breeding programs, the E. grandis X P. psidii pathosystem is still poorly described in the molecular level, with few studies about processes and molecules that confer resistance to the plants. Thus, in order to better understand the E. grandis X P. psidii relationship, this project aimed to study the proteome and metabolome changes that occur on leaf tissues of resistant and susceptible plants infected by the pathogen, with the aid of the liquid chromatography coupled to mass spectrometry technique. The results show that the resistant plants notice the presence of the pathogen shortly after being infected, producing immunity related proteins such as HSP90, ILITYHIA, LRR Kinase, NB-ARC disease resistance protein. This perception triggers the production of cell wall and oxidative burst proteins, also changing the primary and secondary metabolism. On the other hand, susceptible plants have its metabolism subverted, producing proteins responsible for the cell wall loosening, favoring P. psidii nutrient uptake, growth and spread. Metabolite biomarkers, Immune response biomarker molecules and infection signals triggered by P. psidii on E. grandis are also proposed on this work.
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Huet, Joëlle. "Etude structurale relative au protéome présent dans les cellules laticifères de Carica papaya". Doctoral thesis, Universite Libre de Bruxelles, 2010. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210052.
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Le latex de Carica papaya est un milieu riche en cystéine protéinases. Celles-ci ont été régulièrement utilisées en cosmétique ou pour l’attendrissement de la viande. Mais ces protéines ont aussi un intérêt pharmaceutique. En effet, le latex est bien connu pour posséder une activité antifongique mais aussi une activité anthelminthique. Ces effets sont régulièrement attribués aux cystéine protéinases qui se trouvent en concentration importante dans le latex. Malgré ces concentrations importantes en protéinases, d’autres protéines restent actives dans ce milieu. C’est le cas de la glutamine cyclase, qui a été extraite intacte de ce milieu et cristallisée. Sa structure nous a révélé une architecture particulière en ‘’&61538;-propeller‘’ à cinq pales avec double fermeture. Cette structure lui confère sa très grande stabilité. Les industries pharmaceutiques sont aussi à la recherche de protéines très stables et résistantes aux protéinases endogènes. Nous avons donc entrepris l’étude du protéome de Carica papaya afin de mettre en évidence d’autres protéines minoritaires relativement stables pouvant conférer au latex son activité anthelminthique. Cette analyse a permis la mise en évidence de différentes protéines appartenant à diverses familles des « pathogenesis related protéins » (PR-proteins): une &61538;-1,3 glucosidase, une analogue à la barwin, une thaumatine et deux chitinases.
Nous nous sommes particulièrement intéressés à ces deux dernières au cours de cette thèse. Une caractérisation de ces deux protéines a permis de montrer que celles-ci étaient bien deux protéines distinctes, identifiées comme chitinases majeure et mineure selon leur abondance dans le latex. Elles sont relativement stables et résistantes à la protéolyse. Une analyse de la séquence de la chitinase majeure a montré que celle-ci était homologue à la chitinase issue de l’orge et une analyse de sa structure révèle la présence d’une grande concentration en prolines localisées principalement dans les neuf boucles de sa structure. Cela pourrait expliquer sa grande résistance vis à vis des cystéine protéinases.
La cristallisation de cette même chitinase en présence de N-acétyl-glucosamine comme additif, a conduit à une structure contenant trois molécules de GlcNac, deux dans le centre actif de notre protéine et une participant au réseau cristallin. Aucune structure de chitinase n’avait encore pu être obtenue en co-cristallisation avec un substrat. A partir des deux GlcNac observés dans le centre actif, nous avons reconstruit un complexe chitinase/(GlcNac)4. L’analyse de ce complexe a permis de mettre en évidence de nouvelles interactions entre (GlcNac)4 et les acides aminés du centre actif ainsi que de confirmer le mécanisme de la famille GH 19.
Des tests préliminaires sur nématodes ont finalement confirmé l’activité anthelminthique du latex et montré que la chitinase pouvait aussi être un bon nématocide
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Dawit, Abigail Ngugi. "Improvement of Helicoverpa armigera resistance in pigeonpea (Cajanus cajan) through 'omics and breeding". Thesis, Queensland University of Technology, 2021. https://eprints.qut.edu.au/226045/1/Abigail_Dawit_Thesis.pdf.
Texto completoResumen
Pigeonpea (Cajanus cajan) is a sub-tropical and tropical pulse rich in plant-based protein, carbohydrates, minerals, and vitamins. Helicoverpa armigera is the most devastating insect pest in pigeonpea. This study focussed on deciphering the molecular host plant resistance (HPR) mechanisms applied by Cajanus scarabaeoides a wild pigeonpea against insect using transcriptomic and proteomic studies. These HPR mechanisms were transferred to the cultivated pigeonpea via interspecific hybridisation, and they are stable at F2 generation. The study outcome provides a unique insight into the insect resistance mechanisms employed by C. scarabaeoides and lays the foundation for further studies and applications.
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Sengupta, Sameer. "The influence of BRCA1's ubiquitin ligase activity on cell motility". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:b14af4ae-1f95-4d0c-85a8-faaf4fa950c4.
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Breast cancer type 1 susceptibility protein (BRCA1) has been established as an important tumour suppressor protein and its loss of function is associated with hereditary breast and ovarian cancer. An emerging body of work suggests that BRCA1 is involved in sporadic cases of breast and ovarian cancers and may also have a role in other cancers, indicating a more global role in tumourigenesis. BRCA1-mutated cancers can be early-onset and are characterised by being highly aggressive with a propensity to metastasize, thus from a clinical perspective there is a requirement to understand the molecular mechanisms in order to be able to tailor treatments and develop therapeutics. BRCA1 has numerous cellular functions, many ascribed to its role in maintenance of genome integrity, transcription and checkpoint control. More recently, a number of extra-nuclear roles have been established. An interesting novel function is the role of the E3 ubiquitin ligase activity on cell motility. Abrogation of the ubiquitin ligase activity of BRCA1 results in cells exhibiting a hypermotile, invasive phenotype which may help to account for the metastatic nature of BRCA1-mutated tumours. Our aim was to further elucidate BRCA1’s role in cell motility, starting with the identification of relevant candidate ubiquitin ligase substrates. To date, there has yet to be a systematic approach to identify BRCA1’s ubiquitin ligase substrates. Thus we undertook an unbiased proteomic approach to identify extra-nuclear candidates by comparing the profiles of ubiquitinated proteins in breast cancer epithelial cells expressing either functional BRCA1 or ubiquitin ligase-dead BRCA1. We identified 55 candidates which were differentially enriched between the two cell lines and through pathway analysis we determined a significant proportion were cytoskeletal and translation related proteins. Using an ubiquitin-remnant profiling approach, we also identified the site(s) of ubiquitination for many of the candidates. To assess the role of these candidates in cell motility initially we adopted an in silico approach. We used existing time-lapse movies from the online database (www.mitocheck.org) which systematically siRNA knocked down every single gene in the human genome. We developed a series of algorithms which track cell motility from these movies and used these to analyse 192,000 movies containing 3.5 billion cell steps. We have produced a complete database containing motility information after siRNA knockdown of every gene in the human genome, which has been annotated with gene ontologies, KEGG families, Gene Descriptions, SwissProt, Ensembl IDs and siRNA information. In addition to providing motility data of our candidates, we also carried out gene set enrichment analysis on the whole dataset to uncover structural or functional families that may be involved in up-regulating motility when knocked down by siRNA. This is the first report of a genome-wide motility database. Based on overlaps between the results from these two large-scale unbiased proteomic and in silico datasets, we selected 4 candidates, namely, ezrin, moesin, fermitin-2 and delta-catenin. Through monolayer wound healing, cell spreading and single cell motility assays, we determined that ezrin was a particularly relevant and informative candidate. The hypermotile phenotype observed in cells expressing ubiquitin ligase dead BRCA1 was rescued through siRNA knockdown of ezrin and thus we suggest that BRCA1 may regulate cell motility through effects on ezrin. This thesis has investigated candidate BRCA1’s role in cell motility, identified candidate substrates for the E3 ubiquitin ligase activity, established a genome-wide motility database and proposed a possible pathway through which BRCA1 may mediate cell motility and by extension metastasis.
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Dias, Keith. "Proteomic comparison of «Arabidopsis thaliana» under high and low nitrogen fertilization". Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110688.
Texto completoResumen
Nitrogen (N) and the levels of N in plants play a vital role in the physiology, regulating their development and metabolism. We grew Arabidopsis thaliana under agronomic conditions at low (6 mg N/L) and high (106 mg N/L) N fertilizer regimes, maintaining a constant NO3-N to NH4-N ratio (3:1). Using a shotgun mass spectrometry proteomics approach, multi-dimensional protein identification technology (MudPIT), we characterized a total of 2134 reproducibly identified proteins shared between the two N treatments. By statistical analysis in both treatments we found 37 differentially expressed proteins that satisfied both the AC test and the FDR q-value specified cutoffs, where 18 proteins were down regulated and 19 proteins were up regulated under low and high N treatments. We also found 35 differentially expressed proteins that are statistically important but did not satisfy the q- test. These differentially expressed proteins appear to have roles in glycolysis, metabolic, developmental, and signaling processes, or protein binding, transport and nucleic acid binding. The proteins associated with glycolysis indicate glutamine metabolism is of major importance in the plant N economy since it provides N to young developing tissues. Our study indicates that under varying N level treatments, proteins responsible for glutamate synthase (GOGAT), glutamine synthase (GS), and dehydrogenase activity (DH) that serve as enzymes to catalyze a link between carbohydrate and amino acid metabolism are up regulated. Thus, this study has enabled us to apply comparative shotgun proteomics to characterize A. thaliana at the proteomic level and will provide the tools necessary to provide an improved understanding of how and what up-regulates and down regulates different proteins under varying environmental conditions.La fertilisation en azote (N) et la teneur en N des plantes ont un rôle clef dans leur physiologie, régulant leur développement et métabolisme. Tout en gardant un rapport de NO3-N à NH4-N de 3:1, des plants d'Arabidopsis thaliana (L.) Heynh furent cultivés sous deux régimes de fertilisation: bas (6 mg N/L) et élevé (106 mg N/L). Utilisant une technique protéomique en vrac par spectrométrie de masse et une technologie d'identification multidimensionnelle des protéines (MudPIT), nous avons pu caractériser un total de 2134 protéines identifiées de façon récurrente comme apparaissant dans les deux traitements de fertilisation azotée. Une analyse statistique des deux traitements a indiqué la présence de 37 protéines différentiellement exprimées, satisfaisant à la fois le test Audic-Claverie (AC) et le seuil de valeur q dans l'estimation du taux d'erreur (FDR). De celles-ci, 18 protéines furent régulées à la baisse lors des traitements à haut ou bas niveau de N, et 19 furent régulées à la hausse dans les mêmes circonstances. En plus, 35 protéines différentiellement exprimées du point de vue statistique ne passèrent tout de même pas le test de la valeur q. Les protéines différentiellement exprimées semblent avoir des rôles dans les processus de glycolyse, de métabolisme, de développement, de signalisation, et de transport, ainsi que dans la liaison des protéines et des acides nucléiques. Une analyse des protéines associées à la glycolyse indique que le métabolisme de la glutamine est d'une importance majeur dans l'économie en N de la plante, puisqu'il fournit l'azote aux jeunes tissus en voie de développement. Notre étude indique que, sous différents niveaux de fertilisation en N, les protéines responsables pour l'activité glutamate synthétase (GOGAT), glutamine synthétase (GS), et déshydrogénase (DH), servant comme enzymes dans la catalyse du lien entre les voies de métabolisme des glucides et celui des acides aminés, sont régulés à la hausse. Ainsi, cette étude nous permettra d'utiliser une technique protéomique en vrac comparative afin de caractériser A. thaliana au niveau protéomique, et nous fournira les outils nécessaires à mieux comprendre quelles protéines sont régulées à la hausse ou à la baisse sous différentes conditions environnementales et comment cette régulation est mise en œuvre.
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Rugen, Nils [Verfasser]. "From single proteins to supercomplexes : a proteomic view on plant mitochondria / Nils Rugen". Hannover : Gottfried Wilhelm Leibniz Universität Hannover, 2019. http://d-nb.info/1204458707/34.
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Delaunois, Bertrand. "Protection de la vigne contre Botrytis cinerea via la stimulation des défenses : identification de marqueurs de protection et de potentialisation par approche protéomique". Thesis, Reims, 2011. http://www.theses.fr/2011REIMS037.
Texto completoResumen
La pourriture grise causée par Botrytis cinerea est l'une des principales maladies de la vigne. La principale solution pour faire face à cette maladie est l'utilisation de produits chimiques, mais la lutte chimique cause des dommages environnementaux et conduit au développement de souches de B. cinerea résistantes. Une stratégie alternative consiste à stimuler les mécanismes de défense naturelle de la vigne. Cependant, B. cinerea est connu pour contourner les défenses de la plante (El Oirdi et Bouarab, 2011) et à ce jour aucun éliciteur n'a montré d'effet protecteur probant contre B. cinerea. De plus, les résultats prometteurs observés au laboratoire se révèlent souvent décevants au vignoble et à ce jour aucun produit stimulateur de défense ne confère à la vigne une protection acceptable contre la pourriture grise. Pour développer des éliciteurs efficaces contre la pourriture grise, il s'avère donc essentiel de caractériser des marqueurs de protection qui permettraient de distinguer stimulation des défenses et protection efficace de la vigne contre B. cinerea. Pour ce faire des analyses comparatives ont été réalisées par 2D-PAGE afin de comparer au niveau protéique l'effet d'éliciteurs induisant une protection et l'effet d'éliciteur n'induisant pas une protection. Les recherches se sont concentrées sur l'apoplaste, qui est un réseau continu dans les plantes créant une interface avec l'environnement. Après la cuticule, l'apoplaste est ainsi la première barrière contre les attaques d'agents pathogènes (Agrawal et al., 2010). Afin d'obtenir un aperçu du protéome constitutif de l'apoplaste (secrétome), une méthode d'infiltration-centrifugation a été optimisée afin de recueillir le fluide apoplastique à partir de feuilles de vigne. Les profils protéiques apoplastiques ont ensuite été comparés aux profils protéiques de feuilles entières par 2D-PAGE et les protéines identifiées par spectrométrie de masse. Cette approche nous a permis d'établir une carte protéique des feuilles entières et du secrétome de la vigne. A notre connaissance, cette étude fournit la première carte protéique du secrétome de la vigne. Cette étude nous donne ainsi un aperçu des protéines les plus abondantes de l'apoplaste de feuilles de vigne. Une prédiction de la fonction des protéines nous a permis de conclure que l'apoplaste de vigne contient principalement (i) des protéines liées aux stress, (ii) des protéines impliquées dans le métabolisme de la paroi cellulaire et (iii) des protéases. Pour confirmer la qualité des extractions, l'analyse prédictionnelle de la sécrétion des protéines a révélé une forte proportion de protéines secrétées de manière classique et non classique (Leaderless Secreted Proteins, LSP). Cette approche offre un grand nombre de protéines candidates impliquées dans les diverses fonctions physiologiques de l'apoplaste. (…)Parallèlement cette étude présente les travaux préliminaires entrepris, également par 2D-PAGE, pour (i) mettre en évidence des marqueurs de potentialisation chez la vigne, (ii) mettre en évidence les bouleversements, au niveau protéique, causés par une infection de B. cinerea sur la vigne et (iii) comprendre comment un traitement avec un éliciteur protecteur pourrait limiter ces effets néfastes. La validation de ces marqueurs par différentes approches permettra d'améliorer la compréhension des mécanismes de défense des plantes et le développement d'outils pour le criblage de nouveaux éliciteurs candidats pour leurs effets protecteurs contre la pourriture grise et pour le suivi des défenses de la vigne directement au vignobleGrey mould caused by Botrytis cinerea infection is one of the main diseases affecting grapevine. The main solution to cope with this disease is the use of chemicals, but chemical control cause environmental damages and lead to the development of B. cinerea resistant strains. An alternative strategy to prevent diseases consists in stimulating plant defense mechanisms. Nevertheless B. cinerea is known to manipulate plant defenses (El Oirdi and Bouarab, 2011) and to date no elicitors have shown expected protective effect against B. cinerea even if they stimulate defense markers. Moreover despite that numerous studies showed an excellent efficacy of elicitors in laboratory conditions, in vineyard the obtained results are often disappointing.Thus it is necessary to distinguished elicitors inducing protection (protective elicitor) from elicitors inducing defense but no protection (non protective elicitors). For that it appears crucial to characterize markers which would enable to discriminate grapevine defense stimulation from effective protection against B. cinerea. To reach this goal, comparative analyses were performed by 2D-PAGE in order to compare the effect of protective elicitors from non protective elicitors against B. cinerea on grapevine apoplastic fluids. Those biomarkers could be defined as protection biomarkers.Researches were focused on the apoplast, which is a continuous network in plants and creates an interface with the environment. After the cuticle, apoplast is the first barrier against pathogen attacks (Agrawal et al., 2010). In order to obtain an overview of the constitutive apoplastic proteome (secretome), a vacuum-infiltration-centrifugation method was optimized to collect the apoplastic fluid from grapevine leaves. Apoplastic protein profiles were compared to whole-leaf protein profiles by 2D-PAGE and protein identifications were performed by tandem mass spectrometry. This approach allowed us to establish a well-defined proteomic map of whole-leaf and apoplastic leaf. To our knowledge, it is the first time that the apoplastic fluid is recovered from grapevine to characterize its protein content. This study provides a comprehensive overview of the most abundant proteins present in grapevine apoplast. Protein function prediction allowed us to conclude that the grapevine apoplast mainly contains a high proportion of (i) stress-related proteins, (ii) proteins involved in cell wall metabolism, (iii) proteases. To confirm quality of extractions, proteins secretion prediction tools revealed a high proportion of classical and non-classical secreted proteins namely Leaderless Secreted Proteins (LSP). This approach provides thus a large number of candidate proteins involved in physiological functions of the apoplast under various stresses.Differential analyses let us to highlight 7 putative markers, namely an aspartyl protease, a β-1,3 glucanase, an isoflavone reductase for induced markers and an another aspartyl protease, an another β-1,3 glucanase, a germin-like protein and a serine pyruvate amino transferase for repressed markers. This proteins could act in a concert manner to (i) regulate reactive oxygen species homeostasy and dercease programmed cell death, (ii) counteract B. cinerea virulence factors, (iii) increase plant cell wall stiffening, (iv) cause fungal membrane leakage and (v) participate in the induction of systemic defence responses.In addition, this study provides preliminary research performed to highlight (i) priming biomarkers in grapevine, (ii) damages caused by B. cinerea infection on grapevine proteome and (iii) how a protective elicitor treatment could limit those damages.Further functional analyses of these proteins will improve the understanding of plant defense mechanisms and tools development for large scale screening of new elicitors regarding their protective effects against grey mould disease and for following grapevine defenses in vineyard
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Winck, Flavia Vischi. "Nuclear proteomics and transcription factor profiling in Chlamydomonas reinhardtii". Phd thesis, Universität Potsdam, 2011. http://opus.kobv.de/ubp/volltexte/2011/5390/.
Texto completoResumen
The transcriptional regulation of the cellular mechanisms involves many different components and different levels of control which together contribute to fine tune the response of cells to different environmental stimuli. In some responses, diverse signaling pathways can be controlled simultaneously. One of the most important cellular processes that seem to possess multiple levels of regulation is photosynthesis. A model organism for studying photosynthesis-related processes is the unicellular green algae Chlamydomonas reinhardtii, due to advantages related to culturing, genetic manipulation and availability of genome sequence. In the present study, we were interested in understanding the regulatory mechanisms underlying photosynthesis-related processes. To achieve this goal different molecular approaches were followed. In order to indentify protein transcriptional regulators we optimized a method for isolation of nuclei and performed nuclear proteome analysis using shotgun proteomics. This analysis permitted us to improve the genome annotation previously published and to discover conserved and enriched protein motifs among the nuclear proteins. In another approach, a quantitative RT-PCR platform was established for the analysis of gene expression of predicted transcription factor (TF) and other transcriptional regulator (TR) coding genes by transcript profiling. The gene expression profiles for more than one hundred genes were monitored in time series experiments under conditions of changes in light intensity (200 µE m-2 s-1 to 700 µE m-2 s-1), and changes in concentration of carbon dioxide (5% CO2 to 0.04% CO2). The results indicate that many TF and TR genes are regulated in both environmental conditions and groups of co-regulated genes were found. Our findings also suggest that some genes can be common intermediates of light and carbon responsive regulatory pathways. These approaches together gave us new insights about the regulation of photosynthesis and revealed new candidate regulatory genes, helping to decipher the gene regulatory networks in Chlamydomonas. Further experimental studies are necessary to clarify the function of the candidate regulatory genes and to elucidate how cells coordinately regulate the assimilation of carbon and light responses.Pflanzen nutzen das Sonnenlicht um Substanzen, sogenannte Kohlenhydrate, zu synthetisieren. Diese können anschließend als Energielieferant für das eigene Wachstum genutzt werden. Der aufbauende Prozess wird als Photosynthese bezeichnet. Ein wichtiges Anliegen ist deshalb zu verstehen, wie Pflanzen äußere Einflüsse wahrnehmen und die Photosynthese dementsprechend regulieren. Ihre Zellen tragen diese Informationen in den Genen. Die Pflanzen nutzen aber in der Regel nicht alle ihre Gene gleichzeitig, die sie zur Anpassung an Umwelteinflüsse besitzen. Zu meist wird nur eine Teilfraktion der gesamten Information benötigt. Wir wollten der Frage nachgehen, welche Gene die Zellen für welche Situation regulieren. Im Zellkern gibt es Proteine, sogenannte Transkriptionsfaktoren, die spezifische Gene finden können und deren Transkription modulieren. Wenn ein Gen gebraucht wird, wird seine Information in andere Moleküle übersetzt (transkribiert), sogenannte Transkripte. Die Information dieser Transkripte wird benutzt um Proteine, Makromoleküle aus Aminsäuren, zu synthetisieren. Aus der Transkription eines Gens kann eine große Zahl des Transkripts entstehen. Es ist wahrscheinlich, dass ein Gen, dass gerade gebraucht wird, mehr Transkriptmoleküle hat als andere Gene. Da die Transkriptionsfaktoren mit der Transkription der Gene interferieren können, entwickelten wir in der vorliegenden Arbeit Strategien zur Identifikation dieser im Zellkern zu findenden Proteine mittels eines „Proteomics“-Ansatzes. Wir entwickelten weiterhin eine Strategie zur Identififikation von Transkripten Transkriptionsfaktor-codierender Gene in der Zelle und in welche Menge sie vorkommen. Dieser Ansatz wird als „Transcript-Profiling“ bezeichnet. Wir fanden Zellkern-lokalisierte Proteine, die als Signalmoleküle funktionieren könnten und Transkripte, die bei unterschiedlichen Umweltbedingungen in der Zelle vorhanden waren. Wir benutzten, die oben genannten Ansätze um die einzellige Grünalge Chlamydomonas zu untersuchen. Die Informationen, die wir erhielten, halfen zu verstehen welche Transkriptionsfaktoren notwendig sind, damit Chlamydomonas bei unterschiedlichen Umweltbedingungen, wie z.B. unterschiedliche Lichtintensitäten und unterschiedlicher Konzentration von Kohlenstoffdioxid, überlebt.
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Nkomo, Mbukeni Andrew. "The role of p-coumaric acid on physiological and biochemical response of chia seedling under salt stress". University of the Western Cape, 2020. http://hdl.handle.net/11394/7954.
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Philosophiae Doctor - PhDThe role of phenolic acids in mitigating salt stress tolerance have been well documented. However, there are contradicting reports on the effect of exogenously applied phenolic acids on the growth and development of various plants species. A general trend was observed where phenolic acids were shown to inhibit plant growth and development, with the exception of a few documented cases. One of these such cases is presented in this thesis. This study investigates the role of exogenously applied p-coumaric acid (p-CA) on physio-biochemical and molecular responses of chia seedlings under salt stress. This study is divided into three parts. Part one (Chapter 3) focuses on the impact of exogenous p-coumaric acid on the growth and development of chia seedlings. In this section, chia seedlings were supplemented with exogenous p-CA and the various biochemical and plant growth parameters were measured. The results showed that exogenous p-CA enhanced the growth of chia seedlings. An increase in chlorophyll, proline and superoxide oxide contents were also observed in the p-CA treatment relative to the control. We suggested that the increase in chia seedling growth could possibly be via the activation of reactive oxygen species-signalling pathway involving O2− under the control of proline accumulation (Chapter 3). Given the allopathy, nature of p-coumaric acid it is noteworthy that the response observed in this study may be species dependent, as contrasting responses have been reported in other plant species. Part two (Chapter 4) of this study investigates the influence of piperonylic acid (an inhibitor of endogenous p-coumaric acid) on the growth and development of chia seedlings. In trying to illustrate whether p-CA does play a regulatory role in enhancing pseudocereal plant growth, we treated chia seedlings with the irreversible inhibitor of C4H enzyme, to inhibit the biosynthesis of endogenous p-CA. In this section, chia seedlings were treated with piperonylic acid and changes in plant growth, ROS-induced oxidative damage, p-CA content and antioxidant capacity was monitored. Inhibition of endogenous p-CA restricted chia seedling growth by enhancing ROS-induced oxidative damage as seen for increased levels of superoxide, hydrogen peroxide and the extent of lipid peroxidation. Although an increase in antioxidant activity was observed in response to piperonylic acid, this increase was not sufficient to scavenge the ROS molecules to prevent oxidative damage and ultimate cellular death manifested as reduced plant growth. The results presented in this section support our hypothesis that p-CA play an important regulatory role in enhancing chia seedling growth and development as shown in Chapter 3. Part three (Chapter 5) seeks to identify and functionally characterise p-coumaric acid induced putative protein biomarkers under salt stress conditions in chia seedlings. Previous studies have shown that p-CA reversing the negative effect caused by NaCl-induced salt stress. While these studies were able to demonstrate the involvement of p-CA in promoting plant growth under salt stress conditions, they focussed primarily on the physiological aspect, which lacks in-depth biochemical and molecular analysis (ionomic and proteomic data) which could help in detecting the genes/proteins involved in salt stress tolerance mechanisms. A comparative ionomics and proteomic study was conducted, with the aim of elucidating the pivotal roles of essential macro elements and/or key protein markers involved in p-CA induced salt stress tolerance in chia seedlings. With the exception of Na, all the other macro elements were decreased in the salt treatment. Contrary to what was observed for the salt treatment most of the macro elements were increased in the p-CA treatment. However, the addition of exogenous p-CA to salt stressed seedlings showed an increase in essential macro elements such as Mg and Ca which have been shown to play a key role in plant growth and development. In the proteomic analysis we identified 907 proteins associated with shoots across all treatments. Interestingly, only eight proteins were conserved amongst all treatments. A total of 79 proteins were unique to the p-CA, 26 to the combination treatment (NaCl + p-CA) and only two proteins were unique to the salt stress treatment. The unique proteins identified in each of the treatments were functionally characterised to various subcellular compartments and biological processes. Most of the positively identified proteins were localised to the chloroplast and plays key roles in photosynthesis, transportation, stress responses and signal transduction pathways. Moreover, the protein biomarkers identified in this study (especially in the p-CA treatment) are putative candidates for genetic improvement of salt stress tolerance in plants.
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MILLI, Alberto. "2D-page coupled to mass spectrometry for proteomic analysis of human, microbial and plant samples". Doctoral thesis, Università degli Studi di Verona, 2009. http://hdl.handle.net/11562/337513.
Texto completoResumen
Il progetto di dottorato è stato condotto nel Laboratorio di Proteomica del Dipartimento
di Biotecnologie dell’Università di Verona. Per la stesura di tale progetto sono state
instaurate collaborazioni con alcuni laboratori, sia interni allo stesso Dipartimento sia
appartenenti ad altre Università o Istituti di Ricerca. In particolare, per quanto riguarda
lo studio su cellule di cancro colorettale trattate con un nuovo chemiofarmaco, il
Laboratorio del Prof. Zunino (Fondazione IRCCS, Istituto Nazionale dei Tumori, Milano)
ha fornito tutti i campioni biologici, mentre il gruppo del Prof. Marengo (Dipartimento di
Scienze dell’Ambiente e della Vita, Università degli Studi del Piemonte Orientale,
Alessandria) ha effettuato l’analisi statistica multivariata dei dati.
Per quanto riguarda invece la caratterizzazione microbiologica, biochimica e proteomica
del ceppo tannasi-positivo VP08 di Lactobacillus plantarum, il lavoro è stato condotto in
collaborazione con il Dottor Zapparoli (Laboratorio di Microbiologia, Dipartimento di
Biotecnologie, Università di Verona).
L’indagine proteomica su campioni di foglie di Vitis vinifera suscettibile a Plasmopara
viticola è stata realizzata in collaborazione col gruppo della Dottoressa Polverari
(Laboratorio di Biotecnologie Fitopatologiche, Dipartimento di Biotecnologie, Università
di Verona).
Infine, il gruppo del Prof. Zolla (Laboratorio di Proteomica, Dipartimento di Scienze
Ambientali, Università degli Studi della Tuscia, Viterbo) ha effettuato le analisi di
spettrometria di massa per l’identificazione delle proteine di interesse individuate nei
diversi studi. Ulteriori analisi di massa sono state svolte, in parte, presso il medesimo
Laboratorio di Proteomica del Dipartimento di Biotecnologie dell’Università di Verona e,
in parte, presso il Laboratorio di Proteomica del Dottor Vindigni (Centro Internazionale
di Ingegneria Genetica e Biotecnologia “ICGEB”, Trieste).
In questo progetto di dottorato sono stati applicati i metodi classici della proteomica
comparativa basata sull’elettroforesi bidimensionale per l’analisi di campioni umani,
microbici e vegetali legati a tre differenti problematiche, riguardanti rispettivamente:
1) la risposta di una linea cellulare di cancro al colon-retto ad un nuovo tipo di inibitore
delle istone deacetilasi
2) l’effetto dell’acido tannico sul microrganismo del vino Lactobacillus plantarum in
condizioni di carenza di nutrienti
3) i cambiamenti nel proteoma di foglia di Vitis vinifera cultivar Pinot Noir a diversi tempi
dopo infezione con Plasmopara viticolaThe proteome of a cell or an organelle provides information about the ensemble of proteins and protein isoforms expressed in that cell or organelle under specific physiological conditions and at a specific time. Proteomic approaches provide several novel possibilities to address biological questions. In fact, the large-scale screening approach of proteomics enables protein expression studies that are impossible to perform using classical molecular biology and biochemical techniques, in which the expression of only one or a few proteins is studied at a time. Instead, proteomic techniques allow for the analysis of up to thousands of proteins simultaneously, in any tissue or organelle, under any given physiological condition. Thus, proteomic applications are growing in many areas of research and proteomic approaches are nowadays widely exploited for cancer, microbial, and plant investigations. The present work was focused on three proteomics studies: 1) evaluation of the cell response to a novel histone deacetylase inhibitor in colon cancer cell 2) effect of tannic acid on lactobacillus plantarum wine strain during starvation 3) analysis of grapevine leaves after Plasmopara viticola infection The thesis work was conducted at the Proteomics Laboratory of the Biotechnology Department of the University of Verona, in collaboration with other laboratories: concerning the study on colorectal cancer cells, the Laboratory of Oncology of the IRCCS Foundation “Istituto Nazionale dei Tumori”, Milan, provided all the biological samples, whilst the multivariate analysis of protein profiles was possible thanks to the collaboration with the Department of Environmental and Life Sciences of the University of Eastern Piedmont, Alessandria. The biochemical and proteomic analysis of lactobacillus plantarum wine strain was the result of the collaboration with laboratory of Dr. Zapparoli (Department of Biotechnology of the University of Verona). Proteomic investigations on the Grapevine leaves infected by Plasmopara viticola were performed in collaboration with laboratory of Dr. Polverari (Department of Biotechnology of the University of Verona). Finally, the identification of proteins for all the proteomic analyses performed were possible thanks to the collaboration with the Proteomics laboratories of Department of Environmental Sciences, Tuscia University, Viterbo, and of International Centre for Genetic Engineering and Biotechnology, Trieste. The results thus obtained are here discussed and evaluated.
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Kamies, Rizqah. "A methodological investigation into the roots of the resurrection plant, Xerophyta viscosa, for further proteomic analyses". Master's thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/10955.
Texto completoResumen
In order to conduct proteomic analysis on the hydrated root tissues of the resurrection plant, Xerophyta viscosa Baker, aeroponically grown plant roots were subjected to various proteomic techniques. Three protein extraction methods were investigated, of which one method, was the most suited in isolating total protein from the root tissues of X. viscosa.