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 ii 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.

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.

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.

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.

No description available.

The effects of oxidation by sodium hypochlorite (0, 0.8, 2, and 5%, NaOCl), the presence of endogenous proteins, and amylose content on waxy and common rice flours (WF, CF) and starches (WS, CS) were investigated in terms of in vitro starch digestibility, morphology and surface properties, and thermal and rheological characteristics. The concentration of NaOCl had an effect on all the samples including WF, CF, WS, and CS. The carbonyl and carboxyl group contents increased up to 25 and 10 folds (P < 0.05) of oxidized starches (WS, CS), respectively. Only mild oxidation (P < 0.05) occurred in flours (WF, WS). In addition, endogenous proteins were oxidized according to amino acid analysis and SDS–PAGE results. Glu+Gln, Gly, His, Arg, Tyr, and Lys were more sensitive to NaOCl oxidation. Disulfide bonds, hydrophobic force, and hydrogen bonds were involved in protein polymerization after NaOCl oxidative modification. In granular state, the in vitro starch digestibility of WF, WS, and CS decreased by 5% NaOCl oxidation. After gelatinization, only 2 and 5% oxidized WS had lower digestibility. Scanning electron microscopy and confocal laser scanning microscopy further demonstrated that protein existed on the surface of starch granules and had aggregation by oxidation. X-ray diffraction patterns showed the crystallinity of 5% oxidized flours and starches was reduced compared with all their non-oxidized samples. Thermal and rheological properties were analyzed by differential scanning calorimetry and rheometer, respectively. Starch gelatinization peak temperature of flours (WF, RF) was increased by 3 °C, but starches (WS, CS) had a significantly decrease by 8 °C. Viscoelastic patterns were dramatically changed by oxidation. Oxidized WF and CF had increased in both viscosity and elasticity by oxidation, whereas both WS and CS had significantly lower viscoelasticity after oxidative modification.

O acúmulo de açúcares solúveis observados em banana madura é resultado da ação de diversas enzimas que atuam sob o amido acumulado durante o seu desenvolvimento. Este processo pode ocorrer fora da planta e, em um período relativamente curto conhecido como amadurecimento. A rápida mobilização associado à escassez de trabalhos que enfoquem o processo de degradação de amido em órgãos que armazenam amido temporariamente, como bananas, o objetivo desse estudo foi localizar cultivares que se comportem diferente do modelo Nanicão, que é objeto de estudo do laboratório há muito tempo, sendo por isso, a cultivar mais conhecida e estudada. Bananas pertencentes a diferentes grupos genômicos foram selecionadas e analisadas, sendo elas: Nanicão (AAA), Terra (AAB), Mysore (AAB), Pacovan (AAB) e Figo (ABB). Quanto aos parâmetros de amadurecimento analisados, as cultivares Nanicão, Terra e Pacovan tiveram um comportamento climatérico típico. A sacarose foi o açúcar predominante em todas as cultivares seguida pela glicose e frutose que mantiveram uma proporção 1: 1. A cultivar Nanicão teve maiores teores de açúcares solúveis seguida pela Figo, Pacovan, Terra e Mysore. Em extrato bruto de banana, os maiores valores de atividade da &#946;-amilase foi verificada nos estágios finais do amadurecimento, enquanto que a da &#945;-amilase foi, praticamente, constante durante o período analisado, com exceção da Figo. Para detectar a presença das amido-fosforilases foi realizado ensaio, em extrato bruto de banana, de eletroforese em condições nãodesnaturantes (PAGE-nativo) e, duas bandas com atividade foram visualizadas nas cultivares, com exceção da Mysore. O inicio do processo de degradação de amido ocorreu anterior a produção autocatalítica de etileno e, as cultivares tiveram diferentes percentuais de degradação de amido, sendo os maiores obtidos na Pacovan seguida pela Mysore, Nanicão, Figo e Terra. Os grânulos de amido das cultivares Terra e Figo mostraram-se resistentes a hidrólise enzimática. Todas as cultivares tiveram atividade de &#945; e &#946;-amilase associadas aos grânulos presentes desde as fases iniciais do amadurecimento. Somente na cultivar Mysore a atividade ligada da &#945;-amilase foi superior ao da &#946;-amilase. O conteúdo de amilose e a distribuição de tamanhos de cadeias da amilopectina não tiveram grandes variações ao longo do amadurecimento. As micrografias revelaram que com o avanço do amadurecimento as superfícies dos grânulos de amido sofrem a exposição de estruturas, os anéis de crescimento, decorrentes da ação de enzimas. As imagens de microscopia de força atômica relevaram a presença de estruturas semelhantes ao modelo dos bloquetes na superfície dos grânulos de amido em banana. / The soluble sugars accumulation in mature banana is consequence of several enzymes action on accumulate starch obtained during its development processo The starch degradation can occur outside the plant in a relatively short time called ripening. Associate at the faster starch mobilization, few works are available focusing this process in organs that store starch temporarily like banana. The aim of this study was identified cultivars that show different pattern of starch degradation when compared with Nanicão model. Bananas of different genomic groups were selected and analyzed, Nanicão (AAA), Terra (AAB), Mysore (AAB), Pacovan (AAB) and Figo (ABB). The Nanicão, Terra and Pacovan cultivars had a typical c1imacteric behavior. Sucrose was the predominant sugar followed by glucose and fructose that maintained 1:1 ratio, in all cultivars. Nanicão had the higher leveis of soluble sugars followed by Figo, Pacovan, Terra and Mysore. The onset of starch degradation seems to be independent of ethylene. The higher activities of &#946;-amylase, in banana pulp, was obtained in the last ripening stages, while &#945;-amylase activities was constant and low at Nanicão and Pacovan. Two bands with activities were visualized in native PAGE which corresponded to cytosolic and plastidial forms of starchphosphorylases, with exception of Mysore. All cultivars had different perceptual of starch degradation and the higher one was observed at Pacovan, followed by Mysore, Nanicão, Figo and Terra. Starches granules of Terra and Figo showed to be resistant to enzymatic hydrolysis. All cultivars had &#945; and &#946;-amylase activities associated to granule throughout the ripening process. Only Mysore showed aamylase activity associated to granule higher than &#946;-amylase. No significant change along the ripening was observed in amylose content and amylopectin chain length distribution. The micrographs revealed that granule surface suffer changes along the ripening, exposing structures called growth rings. The Atomic Force Microscopy revealed the presence of blockets at banana granule surface.

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.