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Cotyledonal- or transverse-cracking (TVC) in certain cultivars of snapbeans, Phaseolus vulgaris ~. seeds, clearly evident during germination, seriously places affected seedlings at a competitive disadvantage. TVC is an inherited trait and occurs across cell walls of cotyledons rather than along cell walls. Thus, it might be hypothesized that internal pressure resulting from swelling of storage proteins during imbibition might account for cellular rupture. To further elucidate this possibility, experiments were designed to compare electrophoretic patterns of storage proteins from seeds of snapbeans resistant and susceptible to TVC, and to correlate the different patterns of polyacrylamide gel el ectrophoretograrns of these proteins to TVC. One hundred seeds were selected randomly from a bulk sample of 225 g from each of 17 seed lots representing 15 cultivars, seed coats removed and cotyledons finely ground (60 mesh). Seed flours were defatted twi ce with hexane (50 ml / g) at 4°C and defatted flours reground with a mortar and pestle. Seed proteins were extracted in 0.5 M NaCl (solvent to four ratio of 10:1) at pH 7.5 for 1 h at 4oc with centrifugation at 10,000 g for 30 min. Separated proteins were subjected to electrophoresis under denaturing and non-denaturing conditions and molecular weight of different protein brands determined. Different protein banding patterns were identified and correlated to the TVC phenomenon. The data showed visual differences between banding patterns of resistant and susceptible cultivars. While the electrophoretic technique shows observable differences in cultivars expressing differential TVC, it is not clear which protein bands are associated with the TVC phenomenon. For plant breeders to employ this tool in screening for TVC resistant snapbean cultivars, further requirements are needed.

A lisina é um dos aminoácidos essenciais e um dos fatores limitantes ao uso de cereais como o milho na alimentação, pois, sem suplementação, não permite a obtenção de uma dieta balanceada. A fim de melhorar a qualidade nutricional dos cereais, várias tentativas têm sido realizadas baseadas em resultados obtidos sobre as rotas de metabolismo da lisina em plantas e o acúmulo de proteínas de reserva no endosperma. Ambrozevicius (2010) com o objetivo de produzir plantas de milho transgênicas com alto teor de lisina utilizou a estratégia de expressão de proteínas de reserva de outras espécies vegetais ricas em lisina, ou seja, através da expressão de uma proteína heteróloga: a zeolina. O presente trabalho teve como objetivo estudar os 6 eventos transformados expressando a zeolina na geração F3, caracterizando as proteínas de reserva, o perfil de aminoácidos e as enzimas envolvidas no metabolismo de lisina em milho geneticamente modificado, para compreender quais as possíveis alterações bioquímicas podem ter sido geradas pela transformação, e que podem ter levado ao incremento dos aminoácidos essenciais neste material transgênico. O perfil de proteínas de reserva dos eventos transformados exibiu redução na proporção das zeínas II e glutelinas em relação ao controle HiII, e ainda aumentos muito discretos da fração globulina, porém não para todos os eventos transformados. Na composição de aminoácidos solúveis totais foram observados incrementos nos teores dos aminoácidos que fazem parte da via metabólica do ácido aspártico: lisina, metionina, treonina e isoleucina. Para os aminoácidos incorporados em proteínas, foram observados incrementos nos teores de lisina nos eventos transformados da fração globulina e da fração glutelina, ambos em relação ao controle HiII. Já a fração zeína I teve o maior conteúdo total de aminoácidos em todos os eventos transformados. A análise das enzimas envolvidas no metabolismo de lisina revelou que ocorreram alterações em duas enzimas, a primeira enzima envolvida na síntese de lisina, aspartato quinase (AK) e a segunda envolvida na degradação de lisina, lisina cetoglutarato redutase (LOR). Embora as outras enzimas envolvidas na síntese e na degradação de lisina também tenham sido alteradas, os resultados foram variáveis para os diferentes eventos. Este trabalho mostrou que a expressão da proteína heteróloga zeolina causou alterações na composição das frações protéicas, no teor dos aminoácidos solúveis totais e aminoácidos incorporados em proteína em consequencia das alterações das enzimas envolvidas na síntese e degradação da lisina. Os resultados obtidos sugerem que a expressão da proteína heteróloga zeolina, que tem a necessidade de incorporação de lisina em sua estrutura, pode ter alterado a via metabólica do ácido aspártico para suprir a nova demanda de lisina. Essas alterações podem incluir o aumento na atividade da enzima AK, que é a primeira enzima da via que leva a síntese deste aminoácido e também uma redução na atividade da enzima de degradação LOR, pois o excesso de lisina livre que seria degradada é incorporado à nova proteína. / Lysine is an essential amino acid and one of the limiting factors for the use in cereals such as corn feed therefore, without supplementation; it does not allow obtaining a balanced diet. In order to improve the nutritional quality of cereals, several attempts have been made based on the results about routes of metabolism of lysine in plants and accumulation of storage proteins in endosperm. Ambrozevicius (2010) with the objective of producing transgenic corn plants with high content of lysine used a strategy of expression of storage proteins from other plant species which are rich in lysine, it means, through the expression of a heterologous protein: zeolin. This work aimed to study the 6 events processed expressing zeolina in F3 generation, featuring the storage proteins, the profile of amino acids and enzymes involved in the metabolism of lysine in genetically modified corn, in order to understand the possible biochemical changes which may have been generated by the transformation, and that may have led to the increase of essential amino acids in transgenic material The storage protein profile of transformed events exhibited reduction in the proportion of zein II and glutelins compared to control HiII, and yet very discrete increments of globulin, but not for all events processed. In the composition of soluble amino acids, it was observed increments in concentration of amino acids forming part of the metabolic pathway of aspartic acid: Lysine, methionine, threonine and isoleucine. For the amino acids incorporated into proteins, it was observed increments in the levels of lysine in the transformed events of globulin and glutelin fraction, both in relation to the control HiII. On the other hand, zein fraction I had the highest total amino acid content in all transformed events. The analysis of the enzymes involved in the metabolism of lysine revealed that changes occurred in two enzymes, the first enzyme involved in the synthesis of lysine, aspartate kinase (AK) and the second involved in the degradation of lysine, lysine ketoglutarate reductase (LOR). Although other enzymes involved in the synthesis and degradation of lysine have also been changed, the results were variable for different events. This work showed that the expression of heterologous protein zeolina caused changes in the composition of protein fractions, in the content of soluble amino acids and amino acids incorporated in consequence of changes in enzymes involved in the synthesis and degradation of lysine. The results suggest that the expression of heterologous protein zeolina, which has the need for incorporation of lysine in its structure, may have changed the aspartic acid pathway to meet the new demand for lysine. These changes may include the increase in AK enzyme activity, which is the first enzyme of the pathway leading to the synthesis of this amino acid and also a reduction in the activity of the enzyme degradation LOR, since the excess free lysine would be degraded is incorporated into the new protein.

A lisina é um dos aminoácidos essenciais e um dos fatores limitantes ao uso de cereais como o milho na alimentação, pois, sem suplementação, não permite a obtenção de uma dieta balanceada. A fim de melhorar a qualidade nutricional dos cereais, várias tentativas têm sido realizadas baseadas em resultados obtidos sobre as rotas de metabolismo da lisina em plantas e o acúmulo de proteínas de reserva no endosperma. Ambrozevicius (2010) com o objetivo de produzir plantas de milho transgênicas com alto teor de lisina utilizou a estratégia de expressão de proteínas de reserva de outras espécies vegetais ricas em lisina, ou seja, através da expressão de uma proteína heteróloga: a zeolina. O presente trabalho teve como objetivo estudar os 6 eventos transformados expressando a zeolina na geração F3, caracterizando as proteínas de reserva, o perfil de aminoácidos e as enzimas envolvidas no metabolismo de lisina em milho geneticamente modificado, para compreender quais as possíveis alterações bioquímicas podem ter sido geradas pela transformação, e que podem ter levado ao incremento dos aminoácidos essenciais neste material transgênico. O perfil de proteínas de reserva dos eventos transformados exibiu redução na proporção das zeínas II e glutelinas em relação ao controle HiII, e ainda aumentos muito discretos da fração globulina, porém não para todos os eventos transformados. Na composição de aminoácidos solúveis totais foram observados incrementos nos teores dos aminoácidos que fazem parte da via metabólica do ácido aspártico: lisina, metionina, treonina e isoleucina. Para os aminoácidos incorporados em proteínas, foram observados incrementos nos teores de lisina nos eventos transformados da fração globulina e da fração glutelina, ambos em relação ao controle HiII. Já a fração zeína I teve o maior conteúdo total de aminoácidos em todos os eventos transformados. A análise das enzimas envolvidas no metabolismo de lisina revelou que ocorreram alterações em duas enzimas, a primeira enzima envolvida na síntese de lisina, aspartato quinase (AK) e a segunda envolvida na degradação de lisina, lisina cetoglutarato redutase (LOR). Embora as outras enzimas envolvidas na síntese e na degradação de lisina também tenham sido alteradas, os resultados foram variáveis para os diferentes eventos. Este trabalho mostrou que a expressão da proteína heteróloga zeolina causou alterações na composição das frações protéicas, no teor dos aminoácidos solúveis totais e aminoácidos incorporados em proteína em consequencia das alterações das enzimas envolvidas na síntese e degradação da lisina. Os resultados obtidos sugerem que a expressão da proteína heteróloga zeolina, que tem a necessidade de incorporação de lisina em sua estrutura, pode ter alterado a via metabólica do ácido aspártico para suprir a nova demanda de lisina. Essas alterações podem incluir o aumento na atividade da enzima AK, que é a primeira enzima da via que leva a síntese deste aminoácido e também uma redução na atividade da enzima de degradação LOR, pois o excesso de lisina livre que seria degradada é incorporado à nova proteína. / Lysine is an essential amino acid and one of the limiting factors for the use in cereals such as corn feed therefore, without supplementation; it does not allow obtaining a balanced diet. In order to improve the nutritional quality of cereals, several attempts have been made based on the results about routes of metabolism of lysine in plants and accumulation of storage proteins in endosperm. Ambrozevicius (2010) with the objective of producing transgenic corn plants with high content of lysine used a strategy of expression of storage proteins from other plant species which are rich in lysine, it means, through the expression of a heterologous protein: zeolin. This work aimed to study the 6 events processed expressing zeolina in F3 generation, featuring the storage proteins, the profile of amino acids and enzymes involved in the metabolism of lysine in genetically modified corn, in order to understand the possible biochemical changes which may have been generated by the transformation, and that may have led to the increase of essential amino acids in transgenic material The storage protein profile of transformed events exhibited reduction in the proportion of zein II and glutelins compared to control HiII, and yet very discrete increments of globulin, but not for all events processed. In the composition of soluble amino acids, it was observed increments in concentration of amino acids forming part of the metabolic pathway of aspartic acid: Lysine, methionine, threonine and isoleucine. For the amino acids incorporated into proteins, it was observed increments in the levels of lysine in the transformed events of globulin and glutelin fraction, both in relation to the control HiII. On the other hand, zein fraction I had the highest total amino acid content in all transformed events. The analysis of the enzymes involved in the metabolism of lysine revealed that changes occurred in two enzymes, the first enzyme involved in the synthesis of lysine, aspartate kinase (AK) and the second involved in the degradation of lysine, lysine ketoglutarate reductase (LOR). Although other enzymes involved in the synthesis and degradation of lysine have also been changed, the results were variable for different events. This work showed that the expression of heterologous protein zeolina caused changes in the composition of protein fractions, in the content of soluble amino acids and amino acids incorporated in consequence of changes in enzymes involved in the synthesis and degradation of lysine. The results suggest that the expression of heterologous protein zeolina, which has the need for incorporation of lysine in its structure, may have changed the aspartic acid pathway to meet the new demand for lysine. These changes may include the increase in AK enzyme activity, which is the first enzyme of the pathway leading to the synthesis of this amino acid and also a reduction in the activity of the enzyme degradation LOR, since the excess free lysine would be degraded is incorporated into the new protein.

Doctor of Philosophy / Department of Biochemistry / Gerald R. Reeck / Prolamins are the alcohol soluble storage proteins found in the endosperm of seeds from cereals and related grasses. The physical and biochemical properties of prolamins vary between species; and due to their relative abundance can greatly affect the properties and healthfulness of foods from those sources. In this work I investigate peptides from the high molecular weight glutenin of wheat, which is linked to dough elasticity and finished product quality. Using 2D NMR I determined the three-dimensional structure for the repeat peptide Ac- GQQPGQG-Am, which makes up ~50% of the 700 residue central domain. The structure was found to be a flexible β-hairpin with a type II β-turn across residues QPGQ. The NMR structure was later compared to 33 proteins with known three-dimensional structure carrying the exact sequence (backbone RMSD=0.802Å). This finding provides useful insight into the structure of high molecular weight glutenin and the molecular nature gluten elasticity. Alternatively, I studied the kafirin storage prolamins from sorghum, which do not have important physical properties, but are poorly digestible by humans and livestock. Improving digestibility of sorghum could significantly impact human health and nutrition in countries where sorghum is a dietary staple. In this work I devised a unique protocol to isolate kafirins under both non-reducing and reducing conditions. I studied kafirin extracts using SDS-PAGE, HPLC and MALDI-TOF MS, then purified β-kafirin, for the first ever characterization of this single protein. Past studies implicate β-kafirin as a source of poor digestibility due to extensive intermolecular disulfide cross-linking. Contrary to this claim I found more than 50% of β-kafirin was extractable without reducing agents. I used chymotrypsin to digest pure β-kafirin and map 10 cysteine residues to 5 intra-molecular disulfide bonds. Precise pairings have yet to be determined although the protein is largely intact after 12 hours of digestion. This work challenges us to think about sorghum protein body formation and the mechanism that leads to disulfide cross-linking during seed desiccation at maturity.

Os cereais representam importantes fontes de proteína para alimentação humana e animal. Entretanto, são caracterizados pela baixa qualidade nutricional de suas proteínas devido à composição desbalanceada de aminoácidos, causada pelo excesso dos aminoácidos prolina e glutamina e deficiência de lisina, treonina e triptofano. As proteínas de reserva prolaminas constituem 50% do conteúdo total de proteínas no endosperma e são as principais responsáveis por tais características nos cereais. As informações sobre o metabolismo de lisina e o acúmulo de proteínas de reserva no endosperma vêm sendo utilizadas para desenvolver e aplicar estratégias em programas de melhoramento de plantas que visam suprir a deficiência de lisina encontrada nos cereais. Lange e colaboradores (2007) relataram a produção de linhagens transgênicas de cevada com padrão de proteínas de reserva alterado e que apresentaram incremento no teor de lisina e outros aminoácidos essenciais. O presente trabalho teve como objetivo identificar os mecanismos responsáveis pelas alterações observadas. Para tanto, avaliou-se a atividade das enzimas envolvidas na síntese e degradação de lisina, além da caracterização das proteínas de reserva e sua composição de aminoácidos. Observou-se redução na fração protéica das prolaminas (5,91 a 18,34%) e incrementos compensatórios na fração protéica das glutelinas (2,16 a 6,52%). As demais frações apresentaram respostas variáveis dependendo do evento avaliado. Além disso, a composição de aminoácidos foi alterada nas diferentes frações protéicas. As prolaminas exibiram incrementos nos teores de lisina (1,79 a 49,13%), treonina (5,04 a 22,60%) e metionina (13,57 a 45,38%), enquanto que as globulinas aumentaram principalmente o conteúdo de metionina (32,30 a 142,56%). Para os aminoácidos solúveis, foram observados incrementos na ordem de duas a três vezes de histidina, lisina, fenilalanina e metionina. A análise das enzimas envolvidas no metabolismo de lisina revelou que ocorreram alterações nas três principais enzimas da via do ácido aspártico. A enzima aspartato quinase (AK) apresentou aumentos na atividade (4,44 a 47,27%), entretanto, foi mais sensível a inibição causada por lisina. A enzima dihidrodipicolinato sintase (DHDPS) também apresentou incremento na atividade (1,50 a 66,32%), mas diferente da AK, foi menos sensível à inibição causada por lisina. A enzima homoserina desidrogenase (HSDH), a qual compete o substrato ASA com a enzima DHDPS, exibiu redução na atividade (3,36% a 28,80%) (exceto um evento de transformação) e foi menos sensível a inibição causada por treonina. Embora as enzimas envolvidas na degradação de lisina também foram alteradas, os resultados foram variáveis para os diferentes eventos. Para aqueles que foram observados redução na atividade da enzima lisina cetoglutarato redutase (LOR), foi também verificado para enzima sacaropina desidrogenase (SDH), mas na ordem de duas vezes, sendo válido para aqueles que apresentaram incremento. Este trabalho mostrou que a alteração no padrão de proteínas de reserva ocasionou mudanças no metabolismo de aminoácidos, neste caso a lisina, para suprir a demanda necessária para incorporação em proteínas de reserva / Cereals represent an important source of protein to human food and animal feed. However, they are characterized by low nutritional quality of proteins due to the unbalanced composition of amino acids, caused by the excess of the amino acids proline and glutamine and deficiency of lysine, threonine and tryptophan. The prolamin storage proteins constitute 50% of the total protein content in the endosperm and is primarily responsible for these characteristics in cereals. Information on the metabolism of lysine and accumulation of storage proteins in endosperm have been used to develop and implement strategies in plant breeding programs that aim to address the deficiencies found in cereals. Lange and coworkers (2007) reported the production of transgenic lines of barley with a pattern of storage proteins that showed altered and increase in the levels of lysine and other amino acids. This study aimed to identify what were the mechanisms responsible for observed changes. For this, we evaluated the activity of enzymes involved in synthesis and degradation of lysine, besides the characterization of storage proteins and their amino acid composition. There was a reduction in the prolamin protein fraction (5.91 to 18.34%) and compensatory increases in the glutelin fractions (2.16 to 6.52%). The other fractions had variable responses depending on the event evaluated. Moreover, the amino acid composition was changed in the different protein fractions. Prolamins exhibited increases in levels of lysine (1.79 to 49.13%), threonine (5.04 to 22.60%) and methionine (13.57 to 45.38%), whereas increases were mainly globulins content of methionine (32.30 to 142.56%). With respect to soluble amino acids, increases were observed in the order of 2-3 fold of histidine, lysine, phenylalanine and methionine. Analysis of enzymes involved in lysine metabolism showed that changes in three key enzymes of the pathway of aspartic acid. The enzyme aspartate kinase (AK) showed increase in activity (4.44 to 47.27%), however, was more sensitive to inhibition by lysine. The enzyme dihydrodipicolinate synthase (DHDPS) also showed increased activity (from 1.50 to 66.32%), but unlike the AK, was less sensitive to inhibition by lysine. The enzyme homoserine dehydrogenase (HSDH) that competes for the substrate ASA with the DHDPS, exhibited reduced activity (3.36% to 28.80%) (an exception one transgenic line) and was less sensitive to inhibition by threonine. The enzymes involved in degradation of lysine were also changed, though the results varied for different events. Those who observed decreased activity of the enzyme lysine ketoglutarate reductase (LOR) was also found for enzyme saccharopine dehydrogenase (SDH), but the order of twice, which was valid for those who had increased. This study showed that the change in the pattern of storage proteins produced changes in amino acid metabolism, in this case lysine, to supply the demand needed for incorporation into storage proteins.