BACKGROUND:The opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary.RESULTS:In this study, we combined transcript profiling and proteomic analysis to better understand which genes and proteins are altered by opaque2 in the W64A inbred line. These analyses showed that the accumulation of some lysine-rich proteins, such as sorbitol dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, was increased in mature kernels and may contribute substantially to the lysine content of opaque2 endosperm. Some defense proteins such as beta-glucosidase aggregating factor were strongly down regulated and may be regulated directly by opaque2. The mutant also had altered expression of a number of starch biosynthesis genes and this was associated with a more highly crystalline starch.CONCLUSIONS:The results of these studies revealed specific target genes that can be investigated to further improve nutritional quality and agronomic performance of high lysine maize lines, particularly those based on the presence of the opaque2 mutation. Alteration of amylopectin branching patterns in opaque2 starch could contribute to generation of the soft, starchy endosperm.

Quality Protein Maize (QPM) with the mutant gene opaque-2 (o2), has higher lysine and tryptophan content and hard endosperm which is less susceptible to mechanical and biological damage. Three experiments were conducted to characterize the phenotypic and genotypic characteristics of high lysine maize. In the first experiment two separate diallels including 7 white and 9 yellow QPM inbreds were evaluated in five southern USA environments to estimate the general (GCA) and specific combining abilities (SCA) for grain yield and to identify potential heterotic relationships among them. QPM hybrids yielded less than commercial checks. GCA effects across environments were non-significant for grain yield but highly significant for secondary traits. Best yielding hybrids resulted from crosses among inbreds from different programs (CIMMYT, Mexico; University of Natal, South Africa and TAMU, USA). In the second experiment testcrosses between QPM inbreds and Tx804, were evaluated for agronomic performance, aflatoxin resistance and quality. QPM inbreds in testcrosses have similar flowering dates, plant height, ear height and test weights but lower grain yield than normal checks. Population 69 inbreds and their testcrosses were least susceptible to aflatoxin. Aflatoxin in testcrosses was positively correlated with endosperm texture (0.67) and kernel integrity (0.60) but negatively correlated with grain yield (-0.30) and silking date (-0.50). Tryptophan content was negatively correlated with endosperm modification. Amino acid levels of inbred lines were significantly correlated with those of hybrids, but with low predictive value. In the third experiment 92 high lysine maize inbreds with different origins [Stiff Stalk, Non Stiff Stalk, Pop 69, temperate (Tx802, Tx804, Tx806, B97, B104) and exotic subtropical lines (CML161, Do940y and Ko326y)] were haplotyped on a cM scale utilizing 43 mapped SSR markers to characterize genetic diversity on chromosome 7, estimate linkage disequilibrium around opaque-2 locus and determine the parental contribution in some inbreds. Dendrograms of genetic similarity showed clusters in agreement with the different origin of inbreds. A total of 200 alleles were detected with an average of 4.7 alleles/locus. Significant linkage disequilibrium was detected around opaque-2 locus. Parental contributions of haplotypes showed segments of chromosome 7 exclusively contributed by one or the other parent.

Maize genetic diversity includes an array of kernel colors (red, blue, purple) with blue concentrated in the aleurone and red primarily in the pericarp. Quality protein maize (QPM) is improved over normal maize in regards to grain concentration of the essential amino acids lysine and tryptophan but has not been widely adapted in part due to lower than conventional yields. These are minimally-utilized specialty corns when compared to the yellows and whites commonly grown. Red, blue, and purple pigments are antioxidant phytochemicals produced by the plant as secondary metabolites. Antioxidants have been linked to anti-cancer and other anti-inflammatory health benefits. QPM hybrids are desirable in developing countries where subsistent agriculture is commonly practiced and quality protein cereals are non-existent. These two diverse maize categories have been the subject of little breeding research compared to normal maize and the potential for high phenolic content as well as the characterization of these QPM hybrids has not been previously investigated. We evaluated 153 maize hybrids (84 colored, 69 QPM) across three locations. High heritability estimates were found for phenolic content (0.80), tryptophan (0.46), and endosperm opacity (0.82). It was encouraging that all three traits observed little genotype by environment (GxE) interaction across diverse environments. This proved the trait analysis procedure to be robust in detecting and separating genotypes for both total phenolic content in colored maize, and amino acids in QPM. Top combiners for phenolics were the purple maize "maize morado" and red lines, with blue, yellow and white maize performing in descending order. Within the tested hybrids, high per kernel antioxidants (measured by total phenolics) may be the answer for producing the most total phenolics, with the top hybrid yielding greater than twice the total phenolics as the top yielding yellow hybrid. The top QPM hybrid out yielded the top normal hybrid by 35 and 30% for lysine and tryptophan. Additionally, QPM endosperm opacity primarily followed an additive, mid-parent trend, with some hybrids (20%) from diverse germplasm backgrounds deviating from that trend displaying the complexity and recessive nature of multiple modifier loci. Additional agronomic and composition traits were minimally correlated with phenolics.

Quality protein maize (QPM), high protein corn (HPC) and food grade maize (FGM) were processed into tortillas and direct expanded extruded snacks. QPM had similar test weight, density and kernel size with 45% more lysine and 38% more tryptophan compared to FGM. HPC had the largest kernel with density and test weight similar to FGM. During alkaline cooking, HPC absorbed water faster than QPM and FGM. White QPM required shorter cooking time and had less dry matter losses compared to FGM. All corn varieties had excellent pericarp removal at the optimum cooking time. Tortillas from QPM had better pliability and rollability after storage compared to FGM and HPC. HPC tortillas had lower rupture force after storage. The use of QPM for tortilla production may reduce energy and sewage cost, and could produce a tortilla with longer shelf stability with improved nutritional value. Decorticated and non-decorticated QPM, FGM and HPC grain were processed into corn meal and direct expanded snacks. A modified short scale dry milling system was used to produce the corn meal. QPM produced more coarse meal with greater fat content compared to FGM. Decortication decreased fiber content and coarse meal yield. Non-decorticated meal had greater protein, fiber and fat content compared to decorticated meal. The modified short flow milling system provides reduced lost fractions for extrusion into nutritionally improved products. Extrusion was performed in a low cost friction extruder. QPM extruded faster than FGM and HPC. FGM required greater specific mechanical energy than QPM. Extrudates from FGM were the most expanded followed by QPM and HPC. Extrudates from the three corn varieties were acceptable to the panelists and decortication did not affect acceptability. The improved nutritional value of QPM, was retained during dry milling and extrusion. Current QPM varieties can be processed into tortillas with longer shelf stability and meal for extrusion into a wide variety of snacks and other foods. These may have application in specialty health foods and in developing countries where maize is a staple food.

The protein quality of maize can be improved by replacing normal Opaque2 alleles with non-functional recessive alleles opaque2 (o2). The allele o2 produces a severe phenotype with soft endosperm enhancing its protein quality but decreasing its agronomical value. Plant breeders have restored a desirable ratio of hard to soft endosperm in o2 germplasm known as Quality Protein Maize (QPM). Neither the mechanism nor the genetic components by which the modification of the endosperm in QPM lines occurs are well understood. To increase the understanding of the genetics of endosperm modification, a population of 146 recombinant inbred lines derived from a cross between the o2 inbred line B73o2 and the QPM inbred line CML161 was evaluated in two Texas locations from 2004 to 2006. Four traits related to endosperm texture were measured and showed significant effect of the inbred lines, high heritability estimates and high genetic correlations. Relative content of the essential amino acids lysine, tryptophan and methionine were measured and showed significant effects of the lines and considerable high genetic correlations and heritabilities. Negative correlation was observed between endosperm texture traits and amino acid content. Quantitative trait loci (QTL) were mapped for traits related to the modification of endosperm texture and the content of lysine, tryptophan and methionine. QTLs clusters for endosperm texture traits were detected on chromosomes 3, 5, 6 and 8 explaining 62-68% of the variation. QTLs clusters for amino acid contents were located on chromosomes 7 and 8 that explained up to 39% of the observed variation. The product of the O2 gene is a transcription factor that affects the expression of a number of endosperm genes. A group of 29 endosperm genes associated with the O2 activity were evaluated in developing endosperm of the recombinant inbred lines. Genomic regions controlling gene transcript abundance in developing endosperm were identified by expression QTL mapping. Evidence is presented of QTL hot spots that segregate in association with endosperm texture modification or amino acid contents and are associated with the regulation of the expression of a group of endosperm genes.

Quality protein maize (QPM), high protein corn (HPC) and food grade maize (FGM) were processed into tortillas and direct expanded extruded snacks. QPM had similar test weight, density and kernel size with 45% more lysine and 38% more tryptophan compared to FGM. HPC had the largest kernel with density and test weight similar to FGM. During alkaline cooking, HPC absorbed water faster than QPM and FGM. White QPM required shorter cooking time and had less dry matter losses compared to FGM. All corn varieties had excellent pericarp removal at the optimum cooking time. Tortillas from QPM had better pliability and rollability after storage compared to FGM and HPC. HPC tortillas had lower rupture force after storage. The use of QPM for tortilla production may reduce energy and sewage cost, and could produce a tortilla with longer shelf stability with improved nutritional value. Decorticated and non-decorticated QPM, FGM and HPC grain were processed into corn meal and direct expanded snacks. A modified short scale dry milling system was used to produce the corn meal. QPM produced more coarse meal with greater fat content compared to FGM. Decortication decreased fiber content and coarse meal yield. Non-decorticated meal had greater protein, fiber and fat content compared to decorticated meal. The modified short flow milling system provides reduced lost fractions for extrusion into nutritionally improved products. Extrusion was performed in a low cost friction extruder. QPM extruded faster than FGM and HPC. FGM required greater specific mechanical energy than QPM. Extrudates from FGM were the most expanded followed by QPM and HPC. Extrudates from the three corn varieties were acceptable to the panelists and decortication did not affect acceptability. The improved nutritional value of QPM, was retained during dry milling and extrusion. Current QPM varieties can be processed into tortillas with longer shelf stability and meal for extrusion into a wide variety of snacks and other foods. These may have application in specialty health foods and in developing countries where maize is a staple food.

O melhoramento genético clássico de sementes milho (Zea mays L.) permitiu desenvolver inúmeras variedades, incluindo o milho com qualidade proteica melhorada (Quality Protein Maize, QPM), que visava aumentar os teores proteicos e as propriedades nutricionais. Por outro lado, novas variedades comerciais foram obtidas por vegetais geneticamente modificados (GM), com foco em parâmetros agronômicos. Em ambos os casos, a segurança dessas variedades para uso como alimento é uma das principais preocupações dos desenvolvedores e dos órgãos de regulamentação. A Equivalência Substancial é a base do sistema de avaliação da segurança de culturas geneticamente modificadas, no entanto alterações na expressão de proteínas não são devidamente analisadas e esclarecidas. As abordagens proteômicas complementam as técnicas de avaliação de biossegurança para alimentos GM, bem como permitem investigar possíveis efeitos indesejáveis derivados do melhoramento clássico. Os objetivos do presente estudo foram caracterizar e comparar os perfis proteicos de variedades de milhos convencionais melhorados (QPM) e geneticamente modificados (GMs), contra suas respectivas linhas convencionais utilizando técnicas proteômicas como eletroforese bidimensional (2-DE) e bottom up shotgun (gel-free). Num primeiro estudo, foram utilizadas três amostras de milho, sendo duas variedades convencionais com QPM (QP1 e QP2) e uma variedade convencional normal (CN). No segundo estudo, foram analisadas duas cultivares de milho GM (GM1 e GM2) e seus respectivos convencionais genitores (CG1 e CG2). As composições químicas de todas as amostras também foram avaliadas quanto a Equivalência Substancial. O extrato bruto proteico foi submetido à análise de eletroforese unidimensional (1-DE), bidimensional (2-DE) e bottom up shotgun (gel-free). As imagens dos mapas proteicos foram analisadas pelo software Image Master 2D Platinum 7.0 (GE). Os spots diferencialmente expressos e selecionados foram sequenciados por MS. Pela composição química das principais frações das amostras de milho foi possível identificar a equivalência substancial entre as amostras convencionais e GMs, bem como QPMs e sua convencional dentro das faixas de variabilidade esperadas da espécie. Nos géis 1-DE foram observadas bandas proteicas com perfis similares entre os grupos de amostras avaliadas para ambos estudos. Nas imagens dos géis 2-DE não houveram alterações extremas entre as amostras de milhos GMs e seus respectivos convencionais genitores (CGs), mas apenas diferenças na intensidade dos spots proteicos. As variedades QPMs e CN apresentaram diferenças devido à distribuição dos spots. Os mapas proteicos das amostras CG1 x GM1 e CG2 x GM2 apresentaram maior semelhança com porcentagens de matchings superiores a 70 %, enquanto as porcentagens de matchings entre variedades diferentes (QPMs e CN) foram menores. No total foram identificadas 219 proteínas das amostras CGs x GMs e QPMs x CN, classificadas quanto aos seus processos biológicos e função molecular. Em conclusão, foram encontradas diferenças entre os cultivares GMs e CGs, indicando uma variação normal entre variedades de milho, que não comprometem a segurança alimentar das amostras estudadas. Quanto às amostras com QPM e CN as diferenças encontradas são devido à sua distância nas linhagens ou germoplasma. / The classic genetic breeding of corn seeds (Zea mays) has enabled the development of many varieties, including corn with improved protein quality (Quality Protein Maize, QPM), which aimed to increase protein levels and nutritional properties. On the other hand, new commercial varieties have been obtained out of genetically modified (GM) vegetables, with a focus in agronomic parameters. In both cases, the safety of these varieties for food use is one of the main concerns for the developers and for the regulatory agencies. Substantial Equivalence is the basis of the safety evaluation system for genetically modified crops, however, alterations in the protein expressions are not been properly analyzed and clarified. The protein approaches complement the techniques of biosafety evaluation for GM foods, as well as allow for possible undesirable effects derived from classic improvement to be investigated. The goals of the current studies were to characterize and compare the protein profiles of the different varieties of conventionally improved (QPM) and genetically modified (GM) corn, against their respective conventional lines using proteomic techniques, such as, two-dimensional electrophoresis (2-DE), bottom up shotgun (gel-free) and masses spectrometry (MS). In a first instance of the study, three samples of corn were used, two of conventional varieties with QPM (QP1 and QP2) and one conventional normal variety (CN). In a second instance of the study, two cultures of GM corn (GM1 and GM2) were analyzed and their respective conventional genitors (CG1 and CG2). The chemical compositions of all the samples were also evaluated for their Substantial Equivalence. The protein raw extract was submitted to analysis of one-dimensional (1-DE), two-dimensional (2-DE) electrophoresis, and bottom up shotgun (gel-free). The protein image maps were analyzed by the Image Master 2D Platinum 7.0 (GE) software. The spots which were expressed and selected differentially were sequenced by MS. By the chemical composition of the main fractions of the samples of corn, it was possible to identify the substantial equivalence between the conventional samples and GMs, likewise with OPMs and their conventional in the ranges of variability which were expected for the species. On the 1-DE gel, it was observed protein bands with similar profiles amongst the groups of evaluated samples for both studies. In the images of the 2-DE gel, there were no alterations between the GM corn and their respective conventional genitors (CGs), but only differences in intensity of the protein spots. The OPM and CN varieties presented differences due to the distribution of the spots. The protein maps of samples CG1 vs. GM1 and CG2 vs. GM2 presented greater similarities with the percentages of matchings superior to 70%, while the percentage of matchings among different varieties (QPMs and CN) were smaller. In total, there were 219 proteins identified in the samples CGs vs. GMs and QPMs vs. CN, classified by the biologic processes and molecular function. In conclusion, there were found differences between the cultures of GMs and CGs, indicating a normal variation among the corn varieties, which do not affect the food security of the studied samples. As per the samples with QPM and CN, the differences found were due to the line distances or germplasm.

O melhoramento genético clássico de sementes milho (Zea mays L.) permitiu desenvolver inúmeras variedades, incluindo o milho com qualidade proteica melhorada (Quality Protein Maize, QPM), que visava aumentar os teores proteicos e as propriedades nutricionais. Por outro lado, novas variedades comerciais foram obtidas por vegetais geneticamente modificados (GM), com foco em parâmetros agronômicos. Em ambos os casos, a segurança dessas variedades para uso como alimento é uma das principais preocupações dos desenvolvedores e dos órgãos de regulamentação. A Equivalência Substancial é a base do sistema de avaliação da segurança de culturas geneticamente modificadas, no entanto alterações na expressão de proteínas não são devidamente analisadas e esclarecidas. As abordagens proteômicas complementam as técnicas de avaliação de biossegurança para alimentos GM, bem como permitem investigar possíveis efeitos indesejáveis derivados do melhoramento clássico. Os objetivos do presente estudo foram caracterizar e comparar os perfis proteicos de variedades de milhos convencionais melhorados (QPM) e geneticamente modificados (GMs), contra suas respectivas linhas convencionais utilizando técnicas proteômicas como eletroforese bidimensional (2-DE) e bottom up shotgun (gel-free). Num primeiro estudo, foram utilizadas três amostras de milho, sendo duas variedades convencionais com QPM (QP1 e QP2) e uma variedade convencional normal (CN). No segundo estudo, foram analisadas duas cultivares de milho GM (GM1 e GM2) e seus respectivos convencionais genitores (CG1 e CG2). As composições químicas de todas as amostras também foram avaliadas quanto a Equivalência Substancial. O extrato bruto proteico foi submetido à análise de eletroforese unidimensional (1-DE), bidimensional (2-DE) e bottom up shotgun (gel-free). As imagens dos mapas proteicos foram analisadas pelo software Image Master 2D Platinum 7.0 (GE). Os spots diferencialmente expressos e selecionados foram sequenciados por MS. Pela composição química das principais frações das amostras de milho foi possível identificar a equivalência substancial entre as amostras convencionais e GMs, bem como QPMs e sua convencional dentro das faixas de variabilidade esperadas da espécie. Nos géis 1-DE foram observadas bandas proteicas com perfis similares entre os grupos de amostras avaliadas para ambos estudos. Nas imagens dos géis 2-DE não houveram alterações extremas entre as amostras de milhos GMs e seus respectivos convencionais genitores (CGs), mas apenas diferenças na intensidade dos spots proteicos. As variedades QPMs e CN apresentaram diferenças devido à distribuição dos spots. Os mapas proteicos das amostras CG1 x GM1 e CG2 x GM2 apresentaram maior semelhança com porcentagens de matchings superiores a 70 %, enquanto as porcentagens de matchings entre variedades diferentes (QPMs e CN) foram menores. No total foram identificadas 219 proteínas das amostras CGs x GMs e QPMs x CN, classificadas quanto aos seus processos biológicos e função molecular. Em conclusão, foram encontradas diferenças entre os cultivares GMs e CGs, indicando uma variação normal entre variedades de milho, que não comprometem a segurança alimentar das amostras estudadas. Quanto às amostras com QPM e CN as diferenças encontradas são devido à sua distância nas linhagens ou germoplasma. / The classic genetic breeding of corn seeds (Zea mays) has enabled the development of many varieties, including corn with improved protein quality (Quality Protein Maize, QPM), which aimed to increase protein levels and nutritional properties. On the other hand, new commercial varieties have been obtained out of genetically modified (GM) vegetables, with a focus in agronomic parameters. In both cases, the safety of these varieties for food use is one of the main concerns for the developers and for the regulatory agencies. Substantial Equivalence is the basis of the safety evaluation system for genetically modified crops, however, alterations in the protein expressions are not been properly analyzed and clarified. The protein approaches complement the techniques of biosafety evaluation for GM foods, as well as allow for possible undesirable effects derived from classic improvement to be investigated. The goals of the current studies were to characterize and compare the protein profiles of the different varieties of conventionally improved (QPM) and genetically modified (GM) corn, against their respective conventional lines using proteomic techniques, such as, two-dimensional electrophoresis (2-DE), bottom up shotgun (gel-free) and masses spectrometry (MS). In a first instance of the study, three samples of corn were used, two of conventional varieties with QPM (QP1 and QP2) and one conventional normal variety (CN). In a second instance of the study, two cultures of GM corn (GM1 and GM2) were analyzed and their respective conventional genitors (CG1 and CG2). The chemical compositions of all the samples were also evaluated for their Substantial Equivalence. The protein raw extract was submitted to analysis of one-dimensional (1-DE), two-dimensional (2-DE) electrophoresis, and bottom up shotgun (gel-free). The protein image maps were analyzed by the Image Master 2D Platinum 7.0 (GE) software. The spots which were expressed and selected differentially were sequenced by MS. By the chemical composition of the main fractions of the samples of corn, it was possible to identify the substantial equivalence between the conventional samples and GMs, likewise with OPMs and their conventional in the ranges of variability which were expected for the species. On the 1-DE gel, it was observed protein bands with similar profiles amongst the groups of evaluated samples for both studies. In the images of the 2-DE gel, there were no alterations between the GM corn and their respective conventional genitors (CGs), but only differences in intensity of the protein spots. The OPM and CN varieties presented differences due to the distribution of the spots. The protein maps of samples CG1 vs. GM1 and CG2 vs. GM2 presented greater similarities with the percentages of matchings superior to 70%, while the percentage of matchings among different varieties (QPMs and CN) were smaller. In total, there were 219 proteins identified in the samples CGs vs. GMs and QPMs vs. CN, classified by the biologic processes and molecular function. In conclusion, there were found differences between the cultures of GMs and CGs, indicating a normal variation among the corn varieties, which do not affect the food security of the studied samples. As per the samples with QPM and CN, the differences found were due to the line distances or germplasm.