Wheat-derived products provide the basic nutrition for more than a billion of people in the world (about 40% of humankind). Humans consume more proteins from wheat than from any other source. However, the nutritional quality of wheat proteins is limited by the low content of lysine, one of the essential amino acids that we should incorporate through the diet. As part of this thesis work, we obtained transgenic wheat lines expressing the CI-2 gene from barley under the control of a promoter that is specific for endosperm. This gene has a high content of lysine, an essential amino acid in which wheat proteins are naturally deficient. These transgenic wheat lines have an improved nutritional quality because of the higher content of lysine of their proteins. Bread-making quality of wheat is determined by the number and type of high molecular weight (HMW) subunits of glutenins. The higher the number of HMW subunits of glutenins of a particular wheat variety, the better its bread-making quality. The HMW subunits of glutenins are very polymorphic (there are many different alleles or alternative forms of genes) and some types of subunits are associated with good bread-making quality while others confer a poor quality. The two HMW subunits of glutenins associated with the best bread-making quality in wheat are 1Dx5 and 1Ax1. HMW subunits of glutenins confer elasticity to the dough because of their particular spring shape.Dough with high elasticity are ideal to make bread. Using genetic engineering, we transformed (genetically modified) a commercially available variety of wheat to increase the number of HMW subunits of glutenins from 5 to 6 by introducing an extra HMW subunit known as 1Ax1, which caused an increase of 21% in the content of glutenin compared to the controls. In addition, we over-expressed 1Ax1 in a variety of wheat that already had this subunit by introducing extra gene copies of 1Ax1, which increased the content of 1Ax1 by 44% in this transgenic wheat variety compared to non-transgenic wheat control. Likewise, we over-expressed the HMW subunit of glutenin 1Dx5 causing an increase of 140% in the level of this subunit and a 20% increase in the total content of glutenin. Interestingly, the over-expression of the HMW subunits of glutenins 1Ax1 or 1Dx5 in some of the transgenic wheat lines induced the inactivation of few or all of the endogenous genes that codify for HMW subunits. This phenomenon is part of a mechanism that plants use to inactivate alien genes as a protection against viruses. As expected, these silenced transgenic wheat lines had very low levels of HMW subunit of glutenin and a very poor bread-making quality. However, these lines showed exceptional good quality for making other products such as cookies, crackers, and pizza.

The present study investigates effects of elevated atmospheric carbon dioxide concentration, different nitrogen fertilization levels, drought and UV radiation on protein content of wheat gluten fractions glutenins and gliadins. Winter wheat cultivar Bohemia was grown under ambient CO2 concentration (AC; 400 mol CO2.mol-1) and elevated CO2 concentration (EC; 700 mol CO2.mol-1). Half of the samples was fertilized with 200 kg N.ha-1 (N+) and the other part stayed unfertilized (N–). Other environmental factors were UV radiation exclusion (UV–, UV+) and drought (DRY, WET). Gliadins were separated by A-PAGE, glutenins by SDS-PAGE. Proteins were quantified by computer densitometry. Nitrogen fertilization caused an significat increase of gliadins and glutenins. While some gliadins subfractions were significantly lowered by drought, HMW glutenin subunits showed significant increase. UV radiation exclusion resulted in significant decrease of some gliadin subfractions and glutenin subunits. CO2 enrichment caused significant increase of glutenin subfractions HMW 1 and 2, while gliadin subfractions -5 1 and 1 were significantly decreased by elevated CO2 concentration.

This diploma thesis deals with an influence of high temperature and water shortage on the protein content of gliadin and glutenin fractions in four varieties of winter wheat: Bohemia, Tobak, Pannonia and var. Syria with designation S46 (IG142780). The crop was grown at 26, 29, 32, 35, 38 and 41 °C during anthesis under control irrigation treatment (with soil moisture higher than 70 %) or under drought stress (with soil moisture lower than 30 %). To separate gliadins, the A-PAGE method was used, and glutenins were separated by SDS-PAGE method. Proteins were quantified by computer densitometry. Significant influence of genotype on the gluten proteins was found. Variety Pannonia has high content of -, 5-gliadins, LMW and HMW glutenins, but low content of other gliadin fractions. It was the opposite in the other varieties. Due to temperature, as well as drought, there was an increase in the content of all gluten fractions, especially of HMW glutenins, 1,2-gliadins and total gliadins. The largest increase in the gluten fractions due to drought was observed in Syria variety. In other varieties simultaneous exposure to drought and heat caused decrease in gliadin content, but increase in glutenin content. Drought at high temperatures reduced gliadin-to-glutenin ratio, mostly in Bohemia variety. This ratio has increased due to the temperature, especially in Tobak variety. For Syria variety, no effect of stress conditions was found on gliadin-to-glutenin ratio.

In this diploma thesis the impacts of both nitrogen fertilization and drought on the content of proteins of the gliadin fraction in case of a winter wheat variety Tiguan were observed. Selected samples were cultivated at two locations in a total duration of two years. Two different conditions were selected: one without the nitrogen fertilization (0 kg N/ ha) and the other with the nitrogen fertilization in 140 kg N/ ha concentration. Second observed factor was the drought which was ensured by roofs above the crops and their cultivation in common climatic environment. Gliadin fraction was separated by the A-PAGE method and the protein quantification carried out by a computer densitometry. Such nitrogen fertilization caused a significant increase of gliadins, especially -1,2 and -5 fractions. Stress induced by the drought caused an increase of gliadin content compared to the control sample, particularly in case of - and - fractions. The most important factor influencing the gliadin content in grains was therefore the nitrogen fertilization in a dry environment. The observed increase of gliadin content was lower in case of the sample which was fertilized in a humid environment than in case of the sample which was fertilized in the dry one.

Made available in DSpace on 2015-03-26T13:42:15Z (GMT). No. of bitstreams: 1 texto completo.pdf: 503594 bytes, checksum: 9404a8d4266a856855a02a401fdb8a03 (MD5) Previous issue date: 2010-07-06 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Wheat is one of the principal cereals in the world and is processed into a wide range of products. In recent decades researchers have shown concern over acquisition of cultivars with good baking quality, a characteristic which initially refers to the protein composition of the endosperm, composed principally of the proteins making up gluten. Various methodologies have been employed in the evaluation of this characteristic, including SDS-PAGE (polyacrylamide gel electrophoresis), SE-HPLC (size exclusion high performance liquid chromatography), baking tests and employment of genetic markers. In an attempt to compare the different existing methodologies and propose a strategy for wheat genetic improvement programs based on industrial quality, four different techniques were tests with 45 national cultivars. Six allele-specific markers were used for verification of the presence of the HMW subunits: Glu-D1-1d (5+10) and Glu-B1-2a (7+8), one LMW subunit: Glu-A3d, the presence or absence of 1B/1R translocation: by the presence of Glu-B1 (long arm of the wheat chromosome) or ω- secalin (gene of the short arm of rye) and puroindoline (Pinb-D1b). Also employed where SDS-PAGE, SE-HPLC and baking test methods. As a result of applying the markers, the presence in the genotypes observed was: 71% for subunit Glu-D1-1d (5+10), 16% for Glu-B1-2a (7+8), 60% for Glu-B1 (absence of translocation) and 8% for Pinb-D1b and Glu-A3d. The results of SDS-PAGE suggested a prevalence of the subunits 2* and 1 in chromosome 1A; 7+8, 7+9 and 17+18 in 1B; and 5+10 in 1D. The average values of SE-HPLC were 35.99% and 44.99% for the polymeric protein in protein (PPP) and unextractable polymeric protein (UPP), respectively, and 1.29 for the ratio between gliadins and glutenins (GLI/GLU), with significant variation among the genotypes (p≤0.05). The baking test also showed a significant difference (p≤0.05) between the cultivars under the same conditions. The UPP data were independent of the PPP percentage data, signifying that a portion of the unextractable polymeric protein (UPP) does not depend on the greater percentage of total polymeric proteins. However there was a positive correlation between the score of HMW subunits, evaluated by SDSPAGE, and the UPP values; this indicates that scoring has apparently been efficient in classifying the genotypes of greatest quality. The cultivars without translocation with rye also showed better results for UPP, PPP and GLI/GLU in relation to those possessing translocation. The GLI/LGU ratio is correlated with the specific volume of the bread, but apparently does not guarantee quality of the qualitative characteristics, which may indicate the action of gliadins in extensibility, but with a lack of structure in the gluten network. These results corroborate for the selection of HMW subunits 5+10, cultivars without translocation with rye, with high values of UPP and an appropriate GLI/GLU ratio with the objective of obtaining greater wheat baking quality. / O trigo é um dos principais cereais em todo o mundo, sendo processado para uma gama de produtos. Surgiu nas últimas décadas a preocupação dos pesquisadores com a obtenção de cultivares com boa qualidade de panificação, característica que refere-se primordialmente a constituição protéica do endosperma, composta principalmente pelas proteínas formadoras de glúten. Várias metodologias vêm sendo empregadas na avaliação dessa característica, dentre as quais, estão o SDS-PAGE (eletroforese em gel de poliacrilamida), SE-HPLC (cromatografia líquida de alta eficiência por exclusão molecular), teste de panificação e o emprego de marcadores genéticos. Buscando comparar as diferentes metodologias existentes e propor uma estratégia a programas de melhoramento de trigo voltados à qualidade industrial, testaram-se quatro diferentes técnicas em 45 cultivares nacionais. Usaram-se seis marcadores alelo-específicos direcionados à verificação da presença das subunidades de HMW: Glu-D1-1d (5+10) e Glu-B1-2a (7+8), uma subunidade de LMW: Glu-A3d, a presença ou ausência de translocação 1B/1R: pela presença de Glu-B1 (braço longo do cromossomo de trigo) ou de ω-secalin (gene do braço curto do centeio) e puroindolina (Pinb-D1b). Também foram empregadas as metodologias de SDS-PAGE, SE-HPLC e teste de panificação. Como resultado da aplicação de marcadores obteve-se presença nos genótipos de: 71% para subunidade Glu-D1-1d (5+10), 16% para Glu-B1-2a (7+8), 60% para Glu-B1 (ausência de translocação) e 8% para Pinb-D1b e Glu-A3d. Os resultados de SDS-PAGE apontam uma prevalência das subunidades 2* e 1 no cromossomo 1A; 7+8, 7+9 e 17+18 no 1B; e 5+10 no 1D. Enquanto, os valores médios de SE-HPLC foram de 35,99% e 44,99% para as frações de proteínas poliméricas totais (PPP) e não-extraíveis (UPP), respectivamente, e 1,29 para a relação entre gliadinas e gluteninas (GLI/GLU), com variação significativa entre os genótipos (p≤0,05). O teste de panificação também apontou diferença significativa (p≤0,05) entre as cultivares nas mesmas condições. Os dados de UPP foram independentes dos dados da proporção de PPP, significando que a porção de proteínas poliméricas não-extraíveis (UPP) não depende de maior proporção de proteínas poliméricas totais. Enquanto houve correlação positiva entre o escore dado às subunidades de HMW, avaliadas por SDS-PAGE, e os valores de UPP; isto indica que a pontuação aparentemente vem sendo eficaz em classificar os genótipos de melhor qualidade. As cultivares sem translocação com centeio também obtiveram melhores resultados para UPP, PPP e GLI/GLU em relação as que a possuem. A relação GLI/GLU está correlacionada ao volume específico dos pães, mas aparentemente não garantiu boa qualidade nas características qualitativas, o que pode indicar a ação de gliadinas na extensibilidade, mas com falha na estruturação da rede de glúten. Esses resultados corroboram para uma seleção a favor das subunidades 5+10 de HMW, de cultivares sem translocação com centeio, com alto valor de UPP e razão apropriada de GLI/GLU, quando se objetiva melhorar a qualidade de panificação do trigo.