Although work on the prolamins of pearl millet (Pennisetum glaucum) has revealed partial amino acid sequences for several alcohol-soluble storage proteins (Marcellino et al. 2002) the genes encoding them have not yet been isolated. We constructed a cDNA library from developing P. glaucum seed tissue and screened it using maize zein gene probes to isolate several α-prolamin-like gene sequences. The proteins encoded by these genes generally fall into two size classes: 20.6kD and 27.1kD, which we call the 21kD and 27kD pennisetins. Both proteins are similar in composition and sequence to α-prolamins from maize, sorghum and Coix. Protein bodies that appear as occlusions within the rough ER of P. glaucum endosperm cells are also very similar in size and shape to maize and sorghum protein bodies. The SDS-PAGE gel of the alcohol soluble protein fraction shows two distinct bands in the region corresponding to the 19kD and 22kD of maize α-zein. Both classes of pennisetins appear to be more similar to the 19kD α-zein of maize than to the 22-kD α-zein judging from sequence homology and maize antibody binding. Phylogenetic reconstruction suggests that P. glaucum may have branched from maize prior to the gene duplication which created the 19kD and 22kD α-zein families.

Variable mortality of Pyrenophora semeniperda--infected Bromus tectorum seeds has been referred to as a "race for survival", stating that seeds that germinate quickly are more likely to escape pathogen-caused mortality. Dormancy status is not the only variable determining outcomes within the Bromus-Pyrenophora pathosystem. Varying temperature and exposure to water may strongly influence germination outcomes of B. tectorum when in the presence of P. semeniperda. Low water potentials characteristic of semi-arid soils are often over-looked in the context of seed pathogens, and are ecologically relevant- especially for plant species that inhabit intermittently dry environments. To adequately characterize the Bromus tectorum-Pyrenophora semeniperda pathosystem, four studies were conducted to address the following questions: (1) do temperature, water potential, and dormancy status influence germination outcomes in the Bromus-Pyrenophora pathosystem, (2) do repeated wetting-drying scenarios influence germination outcomes of infected B. tectorum seeds following dehydration at low water potentials similar to those found in the field (i.e., -4 through -150 MPa), (3) can we accurately characterize the asexual life cycle of P. semeniperda on a dormant B. tectorum seed, determining when infection takes place, and what occurs during disease development in continuously hydrated conditions, and (4) how does disease development of P. semeniperda influence the B. tectorum seed embryo and endosperm. All studies were conducted using dormant and/or non-dormant B. tectorum seeds and an intermediate strain of P. semeniperda. Study one used varying temperatures (5-20°C), and five water potentials (0, -0.5, -1, -1.5, -2 MPa) (achieved using PEG 8000). Inoculated seeds were exposed to low water potentials at various temperatures for 7, 14, 21, or 28 days then re-hydrated for 28 days. In the second study, seeds were incubated at 20°C at four nominal water potentials (-4, -10, -40, or -150 MPa) following 8 or 24 hours of initial hydration. Seeds were dehydrated for 1, 7, 14, or 21 days, then re-hydrated. In study three, inoculated seeds were chemically fixed between days 0 and 21 and viewed with a scanning electron microscope. In the fourth study, infected seeds were frozen with liquid nitrogen following 3, 8, and 14 days of disease development, then cross sectioned longitudinally and laterally prior to chemical fixation. Results indicate that non-dormant seeds escape death by germinating rapidly under favorable conditions, that incubation at low water potentials greatly increases seed mortality, that -10 MPa is near the threshold for full pathogen activity, and at water potentials lower than -40 MPa, P. semeniperda may successfully survive severe dehydration if previous hydration resulting in infection has occurred. SEM images indicate that mycelia penetration occurs within 8-24 hours, and that mycelium may penetrate all opening in the seed (i.e., stomata, cracks). Development of P. semeniperda is shown to cause significant damage to the endosperm and embryo within 8 days. As starch is consumed, the endosperm collapses leaving a hollow middle. The embryo is more resilient, but gradually deforms and deteriorates.

Whole-plant corn silage (WPCS) is a major source of forage for lactating dairy cattle in Brazil. Improved kernel processing may be especially advantageous when feeding corn hybrids with vitreous endosperm, which are more difficult to be broken. Two experiments were conducted to evaluate the effects of theoretical length of cut (TLOC) and ensiling time on whole-plant corn silage (WPCS) particle size and kernel processing with two types of forage harvesters. The same vitreous corn hybrid DKB 177 VT PRO 2 was used in both experiments. In the first experiment, the whole-plant corn was harvested by a pull-type forage harvester (PTFH) at TLOC of 3, 6 and 9-mm. In the second experiment, the harvesting was performed by a self-propelled forage harvester (SPFH) at the following TLOC settings: 6, 12 and 18-mm with a roll gap of 3-mm. The WPCS were stored for 0, 35 and 140 d. Vitreousness, measured by dissection in unfermented kernels, averaged 65.6%. Data from both trials were analyzed as a split-plot design using the procedure MIXED of SAS (SAS Institute Inc., Cary, NC). The model included the fixed effects of TLOC, ensiling time and the interaction TLOC × ensiling time. In PTFH, the TLOC of 3 and 6-mm did not differ WPCS particle size distribution and mean particle length (MPL). However, the TLOC of 9-mm increased particles above the top 2 sieves and, as a consequence, the MPL. The rise of TLOC in SPFH led to a higher MPL and percentage of long particles (< 19-mm). The ensiling time increased MPL and long particles only for WPCS harvested by SPFH. The strategy of reducing TLOC in SPFH increased the percentage of kernels smaller than 4.75-mm. Furthermore, The TLOC of 6-mm led to the best kernel processing for SPFH. The ensiling time reduced the particle size of kernel fraction for both forage harvesters. The corn silage processing score only improved with 140 d of ensiling for SPFH samples. In the third experiment the objective was to evaluate the impact of two types of forage harvesters and two TLOC settings on the physical characteristics of WPCS. A vitreous corn silage hybrid (BM 709, Sementes Biomatrix) was cultivated and harvested when whole-corn plant DM achieved 34.8%. The whole-plant was harvested with a conventional pull-type forage harvester (PTFH) without kernel processor (KP) at 6 and 10 of theoretical lengths of cut (TLOC) or by a new PTFH with KP at same TLOC settings. Whole-plant corn silage (WPCS) samples were stored for 35 d. Vitreousness, measured by dissection in unfermented kernels, averaged 62.4%. Data were analyzed as completely randomized design in a factorial arrangement: 2 harvesters × 2 TLOC using the procedure MIXED of SAS (SAS Institute Inc., Cary, NC). The model included the fixed effects of harvester, TLOC and the interaction harvester × TLOC. The major differences occurred only in TLOC of 10-mm. KP reduced the material above the 19-mm sieve from 4.7 to 1.8% which, in turn, increased the percentage of particles below 8-mm sieve while MPL was decreased. This TLOC displayed the highest value of material retained in the 8-mm and the lowest values in the 4-mm sieve for unprocessed WPCS. Kernel processing and short TLOC led to a rise in particles retained in a 4-mm sieve. KP increased the percentage of kernels smaller than 4.75-mm from 56.4 to 80.0% and the starch content below 8-mm sieve. The short TLOC and kernel processing, together, led to a reduction of kernel geometrical mean particle size (GMPS) followed by an increase of surface area. The new PTFH with KP promoted more extensive and effective kernel breakage. The impact of this harvester in WPCS particle size distribution was pronounced in the TLOC of 10-mm which led to a drop in MPL. The reduction of TLOC for both forage harvesters may be a good strategy to fracture corn kernels for WPCS at 34.8% of DM. In the fourth experiment, the objectives of this study were: 1) to evaluate the effect of kernel processing on a Brazilian vitreous endosperm corn hybrid and 2) the increment of particle size in WPCS on intake, lactation performance, total-tract nutrient digestibility, feeding behavior and milk fatty acids profile. The following treatments were performed during the harvest: 1) pull-type forage harvester (without kernel processor, JF AT 1600) set for a 6-mm theoretical length of cut (TLOC) - PT6; 2) self-propelled forage harvester (New Holland, FR 9050) set for a 6-mm TLOC - SP6; 3) self-propelled forage harvester set for a 12-mm TLOC - SP12; and 4) self-propelled forage harvester set for a 18- mm TLOC - SP18. The WPCS of treatments were storaged for 9 months. The CSPS of the WPCS were: 32.1% (PT6), 53.9% (SP6), 49.0% (SP12) and 40.1% (SP18). Holstein cows (n = 24; 139 ± 63 DIM) were blocked and assigned to six 4 × 4 Latin squares, with 24-d period (18 d of adaptation). Diets were formulated to contain (% DM) 48.5% WPCS, 9.5% soybean meal, 6.9% soybean meal non- enzymatic browned, 15.1% dry ground corn, 15.5% citrus pulp, 1.7% minerals and vitamins mix, 1.8% calcium soap of palm fatty acids, and 1% urea. Nutrient composition of the diets (% DM) was: 16.5% CP, 28.9% NDF and 25.4% starch. Three orthogonal contrasts were used to compare treatments: C1 = PT6 vs. SP6 (effect of kernel processing), C2 = SP6 vs. SP12 (effect of particle size) and C3 = SP12 vs. SP18 (effect of particle size). Cows fed SP6 WPCS had greater 1.2 kg/d milk yield with no changes in DMI, resulting in greater feed efficiency when compared with PT6. The total-tract starch digestibility (TTSD) and plasma glucose was also improved when cows were fed SP6. Moreover, the higher milk protein (+ 36 g/d), lactose (+ 61 g/d) and solids (+ 94 g/d) secretions were a result of the improvement in milk yield for SP6. In addition, the mechanism apparently involved a better nutrient digestibility and glucose availability for the synthesis of lactose by the mammary gland. There was no evidence for differences in DMI (kg/d) among self-propelled treatments. The SP12 cows achieved the same milk yield of SP6; however they tended to reduce plasma D-lactate and serum amiloyd A (SAA) while maximizing chewing time and selecting for fine particles. In SP6, cows selected against fine particles and tended to show higher levels of SAA than PT6. The SP18 reduced TTSD and tended to reduce milk production, and plasma glucose. The SP6 raised milk linear-odd chain fatty acids content in comparison with PT6. Nonetheless, a reduction of these same fatty acids occurred in SP12 in relation to SP6. SP6 cows had higher content of some monounsaturated fatty acids (C14:1 and C16:1), however the opposite occurred for SP12. Kernel processing at TLOC settings of 6 and 12-mm enhanced nutrient digestibility, plasma glucose and milk yield, whereas the 18-mm TLOC impaired lactation performance and TTSD. The TLOC of 12-mm improved chewing time and reduced blood sub- acute acidotic markers. The findings of this set of studies suggest that ensiling time and low TLOC in SPFH (6-mm) may be strategies to increase kernel damage and thus starch digestibility in WPCS. Despite 6-mm TLOC has improved kernel processing, the TLOC of 12-mm appeared to be the best setting to harvest whole-corn plant to feed dairy cows. / Silagem de milho (SM) é a principal fonte de forragem para vacas leiteiras em sistemas intensificados de produção no Brasil. O processamento adequado dos grãos é crucial principalmente quando SM são oriundas de híbridos com endosperma vítreo, os quais são mais difíceis de serem quebrados. Nesse sentindo dois experimentos foram conduzidos para avaliar os efeitos do tamanho teórico de corte (TTC) e do tempo de estocagem sobre o tamanho de partículas e o processamento dos grãos em SM colhida com diferentes tipos de colhedoras. O mesmo híbrido de milho vítreo DKB 177 VT PRO 2 foi usado em ambos os experimentos. A média do teor de matéria seca da planta no momento da colheita foi de 34,2%. No primeiro experimento, a lavoura de milho foi colhida por uma colhedora de forragem tracionada por trator (CFTT; JF AT 1600, sem processador de grãos) nos TTC de 3, 6 e 9-mm. No segundo experimento, a colheita foi realizada por uma colhedora de forragem auto propelida (CFAP; New Holland, FR 9050) nos seguintes TTC: 6, 12 e 18-mm com distância entre os rolos de 3-mm. As SM foram armazenadas em mini-silos (4 repetições por tratamento) por 0, 35 e 140 dias. A vitreosidade foi medida por dissecação em grãos não fermentados e foi de 65,6%. Os dados de ambos os ensaios foram analisados como arranjo em parcelas subdivididas usando o procedimento MIXED do SAS (SAS Institute Inc., Cary, NC). O modelo incluiu os efeitos fixos do TTC, tempo de estocagem e a interação entre os mesmos. Na CFTT, os TTC de 3 e 6-mm não impactaram na distribuição das partículas e no tamanho médio de partículas (TMP). No entanto, o TTC de 9-mm aumentou a porcentagem de partículas acima das 2 peneiras superiores do separador de partículas da Penn State e consequentemente, o TMP. O aumento do TTC na CFAP levou a aumento da porcentagem de partículas longas (< 19-mm) e do TMP. O tempo de estocagem também aumentou porcentagem de partículas longas e TMP apenas para SM colhida por CFAP. A estratégia de redução de TTC na CFAP melhorou a porcentagem de grãos menores que 4,75-mm. Além disso, o TTC de 6-mm proporcionou o melhor processamento de grãos nesta mesma colhedora. O tempo de estocagem reduziu o tamanho de partículas da fração grãos para ambas as colhedoras. O corn silage processing score (CSPS) aumentou apenas após 140 dias de estocagem somente para as amostras colhidas por CFAP. No terceiro experimento, o objetivo foi avaliar o efeito de uma CFTT equipada com processador de grãos (PG) regulado em diferentes tamanhos de corte sobre características físicas de SM. Um híbrido de endosperma vítreo (BM 709, Sementes Biomatrix) foi cultivado e colhido quando a MS da planta de milho atingiu 34,8%. A plantação foi colhida com uma CFTP sem PG regulada para TTC de 6 e 10-mm ou por uma CFTP com PG ajustada para os mesmos TTC. As amostras de SM foram armazenadas por 35 dias. A vitreosidade foi de 62,4%. Os dados foram analisados em delineamento inteiramente casualizado em arranjo fatorial: 2 tipos de colhedoras × 2 TTC utilizando o procedimento MIXED da SAS (SAS Institute Inc., Cary, NC). O modelo incluiu os efeitos fixos de colhedora, do TTC e da interação dos mesmos. As seguintes diferenças ocorreram apenas no TTC de 10-mm. O PG reduziu o material retido acima da peneira de crivo de 19 mm de 4,7 para 1,8%, o que por sua vez aumentou a percentagem de partículas abaixo da peneira de crivo de 8-mm e diminuiu o TMP. Este TTC exibiu o valor mais alto de material retido na peneira de 8-mm e os valores mais baixos na peneira de 4 mm para SM não processada. Tanto o processamento de grãos e como a redução do TTC levaram a um aumento de partículas retidas na peneira de 4 mm. O PG aumentou a porcentagem de grãos menores que 4,75 mm de 56,4 para 80,0% e o teor de amido abaixo da peneira de 8-mm. O TTC de 6-mm e o PG levaram a uma redução do tamanho geométrico dos grãos, com subsequente aumento da área superficial. A nova CFTT com PG promoveu um processamento mais intenso nos grãos. No quarto experimento, os objetivos do estudo foram: 1) avaliar o efeito do processamento de grãos em um híbrido brasileiro de endosperma vítreo e 2) aumento no tamanho de partículas da SM no consumo, desempenho, digestibilidade dos nutrientes, comportamento alimentar e perfil de ácidos graxos no leite de vacas leiteiras. Os seguintes tratamentos foram realizados durante a colheita: 1) CFTT (sem PG, JF AT 1600) ajustada para um TTC de 6-mm - PT6; 2) CFAP (New Holland, FR 9050) ajustada para um TTC de 6-mm - SP6; 3) CFAP ajustada para um TTC de 12-mm - SP12; e 4) CFAP ajustada para um TTC de 18-mm -SP18. As SM foram estocadas por 9 meses. Os CSPS das SM dos tratamentos foram: 32,1% (PT6), 53,9% (SP6), 49,0% (SP12) e 40,1 (SP18). Vinte e quatro vacas da raça Holandesa (139 ± 63 DEL) foram blocadas e distribuídas em seis quadrados latinos 4 × 4, com período de 24 dias (18 dias de adaptação). As dietas foram formuladas para conter (% MS) 48,5% SM, 9,5% farelo de soja, 6,9% farelo de soja protegido, 15,1% milho moído seco, 15,5% polpa cítrica, 1,7% de minerais e vitaminas, 1,8% de sabão de cálcio de ácidos graxos de palma e 1% de ureia. A composição nutricional das dietas (% MS) foi: 16,5% PB, 28,9% FDN e 25,4% amido. Três contrastes ortogonais foram usados para comparar os tratamentos: C1 = PT6 vs. SP6 (efeito do processamento de grãos), C2 = SP6 vs. SP12 (efeito do tamanho de partículas) e C3 = SP12 vs. SP18 (efeito do tamanho de partículas). Vacas alimentadas com SP6 apresentaram maior produção de leite de 1,2 kg / d, sem alterações no consumo de matéria seca, resultando em maior eficiência alimentar quando comparadas com PT6. A digestibilidade do amido no trato total e a glicose plasmática também foram maiores para as vacas alimentas com SP6. Além disso, as maiores secreções de proteína (+ 36 g/d), lactose (+ 61 g/d) e sólidos do leite (+ 94 g/d) foram resultado da melhora na produção de leite para as vacas do SP6. O mecanismo envolve uma melhor digestibilidade dos nutrientes e disponibilidade de glicose para a síntese de lactose pela glândula mamária. Não houve evidência de diferenças no consumo de matéria seca (kg/d) entre os tratamentos colhidos por SPFH. As vacas do SP12 alcançaram a mesma produção de leite em relação ao SP6; no entanto, elas tenderam a reduzir a concentração plasmática de D-lactato e o amiloide sérico A (SAA), maximizando o tempo de mastigação e selecionando a favor de partículas finas. No SP6, vacas selecionaram contra partículas finas e tenderam a aumentar SAA em relação a PT6. O SP18 reduziu a digestibilidade do amido no trato total e tendeu a reduzir a produção de leite e a glicose plasmática. O SP6 elevou o teor de ácidos graxos da cadeia linear ímpar do leite em comparação ao PT6. No entanto, uma redução desses mesmos ácidos graxos ocorreu no SP12 em relação ao SP6. As vacas SP6 apresentaram maior teor de alguns ácidos graxos monoinsaturados (C14:1 e C16:1), porém o oposto ocorreu para SP12. O processamento de grãos nos TTC de 6 e 12-mm aumentou a digestibilidade de amido, glicose plasmática e produção de leite, enquanto o TTC de 18-mm prejudicou o desempenho e a digestibilidade de amido. O TTC de 12-mm melhorou o tempo de mastigação e reduziu os marcadores de acidose subclínica no sangue. Estes resultados sugerem que o tempo de estocagem de pelo menos 140 dias e TTC curto (6-mm) para SPFH podem ser estratégias para maximizar o processamento de grãos e, assim, a digestibilidade do amido em SM. Porém, para promover desempenho e saúde de vacas leiteiras, o TTC de 12-mm é o indicado para colheita de híbridos de milho endosperma vítreo para SM.

A semente de milho representa uma importante fonte de proteínas para alimentação humana e de animais monogástricos. Porém, como membros da família dos cereais não apresentam proteínas com um balanço nutricional adequado, devido principalmente ao baixo conteúdo de lisina. As proteínas de reserva da semente de milho são classificadas como fração não-zeína (albumina, globulina e glutelina) e zeína. Mutantes de endosperma de milho, como o2 apresentam quantidades maiores de lisina na semente. Porém, muitos mutantes considerados "alta lisina" não foram ainda caracterizados bioquimicamente. Uma série de mutantes, opaco (o1, o2, o5, o7, o10, o11 e o13) e floury (fl1 e fl2), foram estudadas para determinar as quantidades de proteínas de reserva, o perfil electroforético das proteínas e o conteúdo de LYS na semente, endosperma e embrião. Foi observado que os mutantes apresentaram redução no conteúdo de zeína e aumentos da fração não-zeína com variações dependendo do mutante, do background genético e do tecido analisado. A análise da semente determinou aumentos principalmente da fração albumina e globulina nos mutantes, exceto para o5 com aumentos apenas da fração glutelina. No endosperma foi observado aumento principalmente de albumina em o2, o7 e o5; e globulina em fl2, o10, o11 e o13. No embrião foram registrados os níveis maiores de albumina e globulina da semente, porém a quantidade de proteínas de reserva foi similar entre os genótipos. As quantidades de lisina presentes nas frações protéicas foram sempre maiores nos mutantes, porém para o10, o11 e o13 diferenças significativas, foram observadas principalmente para LYS na fração glutelina. O perfil SDS-PAGE revelou a presença de numerosas bandas protéicas variando entre 100kDa e 10kDa, sendo que a fração não-zeína revelou maior heterogeneidade no número de bandas. Bandas protéicas de maior intensidade foram observadas nos mutantes. Análise 2D-PAGE de proteínas de reserva do endosperma de mutantes o1, o2, fl1 e fl2, revelou padrões similares de distribuição de proteínas, aumentos de intensidade de spots protéicos e a presença de spots restrita ao perfil dos mutantes. Os resultados sugerem que os mutantes opacos e floury avaliados apresentam quantidades maiores de LYS na semente quando comparados aos genótipos selvagens que lhes deram origem. A futura análise dos spots que apresentaram alterações altamente significativas permitirá uma maior compreensão dos efeitos específicos dessas mutações sobre a regulação da biossíntese das proteínas de reserva e do acúmulo de lisina no grão. / The maize seed is an important source of proteins for humans and monogastric animals. However, such as all cereals, maize storage proteins is nutritionally poor mainly due to the low content of lysine. Maize seed storage proteins can be classified as non-zeins (albumins, globulins and glutelins) and zein. Some storage proteins mutants such as the o2 mutants exhibit higher contents of lysine. However, many of these high-lysine mutants have not yet been biochemically characterized. The opaque mutants o1, o2, o5, o7, o10, o11, o13 and floury mutants fl1 and fl2 were analyzed for storage proteins contents, eletrophoretic profile and lysine content in the seeds, endosperm, and embryo. It was observed that the mutants exhibited reduction in the zein fraction and increase in the non-zein fraction which varied according to the mutant, genetic background and tissue analyzed. The whole seed analysis revealed increases mainly in albumins and globulins, with the exception of the o5 mutants which exhibited a higher increase in the glutelin fraction. In the endosperm it was observed increase in the albumin fraction in o2, o7 and o5 and in the globulin fraction in fl2, o10 and o13. Higher concentrations of albumin and globulin were observed in the embryo, but with similar concentrations of total proteins among the mutants. The lysine content was higher always higher in the storage proteins of the mutants, however, in o10, o11 and o13 significant differences for lysine content were observed mainly in the glutelin fraction. SDSPAGE analysis revealed the presence of several band varying from 10kDa to 100kDa, with a higher heterogeneity among the non-zein fraction. Bands with higher intensity have been observed in the mutants. 2D-PAGE analysis revealed that the o1, o2, fl1 and fl2 mutants exhibited similar band patterns with increases in similar spots and mutant specific bands. The results suggest that the opaque and floury mutants exhibited higher lysine content when compared to their respective wild-type counterparts. Future analysis of the protein spots that exhibited significant variations will allow a better understanding of the specific effects of each mutation on the regulation of storage protein synthesis and lysine accumulation in the seeds.

Made available in DSpace on 2015-03-26T13:42:19Z (GMT). No. of bitstreams: 1 texto completo.pdf: 525415 bytes, checksum: 988d62e0043ef473ad492ea9ce941239 (MD5) Previous issue date: 2006-09-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / In the genetic mapping in outbred populations not always it is possible to determine the linkage phase of the alleles. Thus, heterozygous individuals are discarded from these analyses due to the lack of information, once it is not possible, through their genotype, to distinguish the origin of their parental alleles. In this way, the main objective of this work was to propose the endosperm genotyping as a strategy to identify the allelic origin of those heterozygotes individuals. Initially, fragments from the endosperm representing 10, 25 and 50% of the corn seeds weight were extracted and the seeds were submitted to the germination test. The results suggest that the elimination of up to 50% of the endosperm did not affected the seed germination. The methodology of semiquantitative PCR was optimized to differentiate doses of the alleles in the mixtures of DNA derived from leaves of two maize inbred lines (L3 and L1113- 01). It was represented different allelic proportions observed in the endosperm of their reciprocal crosses, based on the maximum amount of endosperm that could be used for DNA extraction. SSR markers were generated by semiquantitative PCR technique and the amplified fragments were evaluated in both agarose gels treated with ethidium bromide and poliacrylamide gels using fluorescently labeled primers. Gel resolution using agarose did not allow the differentiation of the mixtures of parental DNAs. However, through the regression analysis and comparison of the band intensity corresponding to the same allele in the different mixtures, the initial concentration of each one of the alleles could be inferred. The requirement of an allelic pattern limited the use of this technique to QTL analysis in populations where at least one of the genitors is known. Although the resolution of poliacrylamide gels using fluorescent markers was more efficient in the endosperm genotyping, once it was allowed to differentiate the maternal origin of reciprocal hybrids seed´s. So, the strategy of endosperm genotyping using fluorescent SSR primer amplified by semiquantitative PCR allowed the determination of allelic origin in the heterozygous offspring derived from outbred populations, including these individuals in the QTL detection, and consequently, increasing the precision of this analysis. / No mapeamento genético e detecção de QTLs em populações exogâmicas nem sempre é possível a determinação da fase de ligação dos alelos. Assim, indivíduos heterozigotos são descartados dessas análises por serem não informativos, uma vez que não é possível, por meio do seu genótipo, distinguir a origem de seus alelos em relação aos dois genitores. Dessa forma, o objetivo principal deste trabalho foi propor a genotipagem do endosperma para identificar a origem alélica dos indivíduos heterozigotos. Inicialmente, fragmentos do endosperma representando 10, 25 e 50% do peso das sementes de milho foram retirados, sendo as sementes submetidas ao teste de germinação. Observou-se que a remoção de até 50% do endosperma não afetou a taxa de germinação das sementes. A metodologia de PCR semiquantitativo foi otimizada para diferenciar doses dos alelos nas misturas de DNA foliar de duas linhagens de milho (L3 e L1113-01), representando as diferentes proporções alélicas observadas no tecido endospermático dos seus cruzamentos recíprocos, tendo como base a quantidade máxima de endosperma que podia ser utilizada na extração do DNA. Marcadores SSR foram gerados pela técnica de PCR semiquantitativo, e os fragmentos amplificados foram avaliados tanto em gel de agarose tratado com brometo de etídio quanto em gel de poliacrilamida, usando-se primers fluorescentes. A resolução do gel de agarose não possibilitou a diferenciação das misturas dos DNAs parentais. No entanto, por meio da análise de regressão e da comparação da intensidade da banda correspondente a um mesmo alelo nas diferentes misturas, pôde-se inferir a concentração inicial de cada um dos alelos. A necessidade de um padrão de alelos limitou o uso dessa técnica nas análises de QTLs em populações nas quais pelo menos um dos genitores é conhecido. Já a resolução do gel de poliacrilamida utilizando marcadores fluorescentes foi mais eficiente na genotipagem de endospermas, uma vez que possibilitou a diferenciação da origem materna das sementes dos híbridos recíprocos. Assim, a estratégia de genotipagem do endosperma utilizando primers SSR fluorescentes amplificados pela técnica de PCR semiquantitativo possibilitou a determinação da origem dos alelos dos descendentes heterozigotos derivados de populações exogâmicas, permitindo a inclusão destes na detecção de QTLs e, conseqüentemente, aumentando a precisão das análises.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Physic nut (Jatropha curcas L.) is an important crop due to its ability of storing high content of oil in the seeds, which can serve as raw material for biodiesel production. Because of the presence of toxic constituents like phorbol esters (PEs) and curcins, the seed cake produced as a result of oil extraction cannot be utilize for animal feed. Development of the genotypes better suited for the industrial applications and biodiesel production as well as with lower level of toxic constituents is being hampered by a lack of understanding about the a) proteins related to the biosynthesis and degradation of fatty acids (FAs) and triacylglycerides (TAGs), b) role of proteins deposited during seed development and c) proteins related to the synthesis and storage of toxic compounds during seed development. Agreeing with this, we have performed the anatomical analysis of the developing seeds of J. curcas, followed by the proteome analysis of the endosperm isolated from the seeds of J. curcas at five different developmental stages, which resulted into the identification of the 1517, 1256, 1033, 752 and 307 proteins, from Stage 6, 7, 8, 9 and 10, respectively, summing up to a total of 1760 proteins. Proteins with similar expression pattern were grouped into five different clusters and protein quantification based on spectral counts was determined. Besides identification of the proteins involved in the biosynthesis and degradation of the FAs and TAGs, we also identified a large number of proteins involved in the metabolism of the carbohydrates, which are important for supplying energy and carbon source for the synthesis of TAGs in heterotrophic seeds. Among the members of different classes of seed storage proteins (SSPs), we have identified four SSPs named as nutrients reservoir, which in contrast to the other SSPs showed decreasing deposition pattern during seeds development and revealed to have special role during seed development. In addition, peptidases belong to different mechanistic classes were identified, which have a range of functions, highlighting the role in reserve mobilization during germination. Isoforms of curcin were also identified in this proteome analysis which were absent in our previous proteome analysis of the other tissues from these seeds, suggesting that the deposition of these toxic proteins only occur in the endosperm. Similarly, several enzymes involved in the biosynthesis of diterpenoid precursors were identified in this proteome analysis but, like in our previous proteome analysis of the other tissues from J. curcas seeds,we were unable to identify any terpene synthase/cyclase, enzymes responsible for the synthesis of PEs, which collectively suggesting that the synthesis of PEs may not occur in seeds of this plant. In conclusion, the strategy used here enabled us to provide a first in depth proteome analysis of the endosperm from J. curcas developing seeds, which along with providing information regarding important aspects of the seed development, also set the foundation of a proteomic approach to study biotechnologically important plant species. / PinhÃo manso (Jatropha curcas L.) à uma cultura importante devido à sua habilidade em armazenar alto conteÃdo de Ãleo nas sementes, as quais podem servir como matÃria-prima para a produÃÃo de biodiesel. Devido à presenÃa de constituintes tÃxicos como Ãsteres de forbol e curcina, a torta da semente produzida como resultado da extraÃÃo do Ãleo nÃo pode ser utilizada na alimentaÃÃo animal. O desenvolvimento de genÃtipos mais adequados a aplicaÃÃes industriais e à produÃÃo de biodiesel assim como apresentando baixos nÃveis de constituintes tÃxicos està sendo prejudicado pela falta de entendimento sobre a) proteÃnas relacionadas a biossÃntese e degradaÃÃo de Ãcidos graxos e triacilglicerÃis, b) o papel de proteÃnas depositadas durante o desenvolvimento da semente e c) proteÃnas relacionadas à sÃntese e reserva de compostos tÃxicos durante o desenvolvimento da semente. Diante disso, nÃs realizamos uma anÃlise anatÃmica de sementes em desenvolvimento de J. curcas, seguido por uma anÃlise proteÃmica do endosperma isolado de sementes dessa espÃcie em cinco diferentes estÃgios de desenvolvimento, o que resultou na identificaÃÃo de 1517, 1256, 1033, 752 e 307 proteÃnas, dos estÃgios 6, 7, 8, 9 e 10, respectivamente, somando um total de 1760 proteÃnas. ProteÃnas com padrÃo de expressÃo similar foram agrupadas em cinco grupos diferentes e a quantificaÃÃo das proteÃnas baseada na contagem dos espectros foi determinada. AlÃm da identificaÃÃo das proteÃnas envolvidas na biossÃntese e degradaÃÃo de FAs e TAGs, nÃs identificamos um grande nÃmero de proteÃnas envolvidas no metabolismo de carboidratos, as quais sÃo importantes para o fornecimento de energia e fontes de carbono para a sÃntese de TAGs em sementes heterotrÃficas. Entre os membros de diferentes classes de proteÃnas de reservas de sementes (SSPs), nÃs identificamos quatro SSPs denominadas reservatÃrios de sementes, que em contraste as outras SSPs mostraram decrÃscimo no padrÃo de deposiÃÃo e revelaram ter um papel especial durante o desenvolvimento da semente. Em adiÃÃo, peptidases pertencentes a diferentes classes mecanÃsticas foram identificadas destacando o papel da mobilizaÃÃo de reservas durante a germinaÃÃo. Isoformas da curcina ausentes em nossas anÃlises proteÃmicas prÃvias de outros tecidos da semente foram identificadas sugerindo que a deposiÃÃo dessas proteÃnas tÃxicas sà ocorre no endosperma. Similarmente, vÃrias enzimas envolvidas na biosÃntese de precursores de diterpenÃides foram identificadas nessa anÃlise proteÃmica, mas como em nossas prÃvias anÃlises proteÃmicas de outros tecidos de sementes de J. curcas, nÃs nÃo fomos capazes de identificar sintases/ciclases de terpenos, enzimas responsÃveis pela sÃntese de PEs, o que coletivamente sugere que a sÃntese desses compostos pode nÃo ocorrer nas sementes dessa planta. Em conclusÃo, a estratÃgia utilizada nos fornece a primeira anÃlise proteÃmica profunda do endosperma de sementes em desenvolvimento de J. curcas, o que alÃm de fornecer informaÃÃes sobre aspectos importantes do desenvolvimento da semente, tambÃm estabelece a base para uma pesquisa proteÃmica com o objetivo de estudar espÃcies vegetais importantes biotecnologicamente.

A semente de milho representa uma importante fonte de proteínas para alimentação humana e de animais monogástricos. Porém, como membros da família dos cereais não apresentam proteínas com um balanço nutricional adequado, devido principalmente ao baixo conteúdo de lisina. As proteínas de reserva da semente de milho são classificadas como fração não-zeína (albumina, globulina e glutelina) e zeína. Mutantes de endosperma de milho, como o2 apresentam quantidades maiores de lisina na semente. Porém, muitos mutantes considerados “alta lisina” não foram ainda caracterizados bioquimicamente. Uma série de mutantes, opaco (o1, o2, o5, o7, o10, o11 e o13) e floury (fl1 e fl2), foram estudadas para determinar as quantidades de proteínas de reserva, o perfil electroforético das proteínas e o conteúdo de LYS na semente, endosperma e embrião. Foi observado que os mutantes apresentaram redução no conteúdo de zeína e aumentos da fração não-zeína com variações dependendo do mutante, do background genético e do tecido analisado. A análise da semente determinou aumentos principalmente da fração albumina e globulina nos mutantes, exceto para o5 com aumentos apenas da fração glutelina. No endosperma foi observado aumento principalmente de albumina em o2, o7 e o5; e globulina em fl2, o10, o11 e o13. No embrião foram registrados os níveis maiores de albumina e globulina da semente, porém a quantidade de proteínas de reserva foi similar entre os genótipos. As quantidades de lisina presentes nas frações protéicas foram sempre maiores nos mutantes, porém para o10, o11 e o13 diferenças significativas, foram observadas principalmente para LYS na fração glutelina. O perfil SDS-PAGE revelou a presença de numerosas bandas protéicas variando entre 100kDa e 10kDa, sendo que a fração não-zeína revelou maior heterogeneidade no número de bandas. Bandas protéicas de maior intensidade foram observadas nos mutantes. Análise 2D-PAGE de proteínas de reserva do endosperma de mutantes o1, o2, fl1 e fl2, revelou padrões similares de distribuição de proteínas, aumentos de intensidade de spots protéicos e a presença de spots restrita ao perfil dos mutantes. Os resultados sugerem que os mutantes opacos e floury avaliados apresentam quantidades maiores de LYS na semente quando comparados aos genótipos selvagens que lhes deram origem. A futura análise dos spots que apresentaram alterações altamente significativas permitirá uma maior compreensão dos efeitos específicos dessas mutações sobre a regulação da biossíntese das proteínas de reserva e do acúmulo de lisina no grão. / The maize seed is an important source of proteins for humans and monogastric animals. However, such as all cereals, maize storage proteins is nutritionally poor mainly due to the low content of lysine. Maize seed storage proteins can be classified as non-zeins (albumins, globulins and glutelins) and zein. Some storage proteins mutants such as the o2 mutants exhibit higher contents of lysine. However, many of these high-lysine mutants have not yet been biochemically characterized. The opaque mutants o1, o2, o5, o7, o10, o11, o13 and floury mutants fl1 and fl2 were analyzed for storage proteins contents, eletrophoretic profile and lysine content in the seeds, endosperm, and embryo. It was observed that the mutants exhibited reduction in the zein fraction and increase in the non-zein fraction which varied according to the mutant, genetic background and tissue analyzed. The whole seed analysis revealed increases mainly in albumins and globulins, with the exception of the o5 mutants which exhibited a higher increase in the glutelin fraction. In the endosperm it was observed increase in the albumin fraction in o2, o7 and o5 and in the globulin fraction in fl2, o10 and o13. Higher concentrations of albumin and globulin were observed in the embryo, but with similar concentrations of total proteins among the mutants. The lysine content was higher always higher in the storage proteins of the mutants, however, in o10, o11 and o13 significant differences for lysine content were observed mainly in the glutelin fraction. SDSPAGE analysis revealed the presence of several band varying from 10kDa to 100kDa, with a higher heterogeneity among the non-zein fraction. Bands with higher intensity have been observed in the mutants. 2D-PAGE analysis revealed that the o1, o2, fl1 and fl2 mutants exhibited similar band patterns with increases in similar spots and mutant specific bands. The results suggest that the opaque and floury mutants exhibited higher lysine content when compared to their respective wild-type counterparts. Future analysis of the protein spots that exhibited significant variations will allow a better understanding of the specific effects of each mutation on the regulation of storage protein synthesis and lysine accumulation in the seeds.

The mode of reproduction can greatly influence the demography and the evolutionary success of the taxon. In the case of autonomous asexual formation seeds are apomictic taxa fully independent of pollinators and compatible partners. For sexual taxa with strict autoincompatibility it is the opposite, i.e. sexual taxa need pollinators and compatible partners for birth of offspring. Therefore, in marginal population and for more extreme situation with lower pollinating activity will have apomictic taxa a higher level of reproductive assurance than sexual taxa vascular plants. This hypothesis was tested in natural populations of apomictic and sexual taxa. In the diploma thesis we therefore investigate the method mode of reproduction and reproductive assurance of 52 taxa of the genus Hieracium s. str. (family Asteraceae) in Europe. Of these, 12 were diploid sexually diploid taxa and 42 polyploid apomictic reproductive taxa. From these taxa we harvested seeds from fully developed capitulum and we determined the potential (total number of seeds in the capitulum) and the realized (the percentage of well-developed seeds at the capitulum). The ploidy of the offspring (the embryos and the seedling) and method origins of seeds we examined using flow cytometry. The results show that the plants of diploid species...

La dureté du grain de blé est un des paramètres fondamentaux de la texture de l’albumen. Ce caractère, essentiel pour la valeur d’utilisation des farines, est fortement lié à l’absence ou à la modification des puroindolines. Afin de mieux comprendre la fonction biologique de ces protéines dans le grain de blé (Triticum aestivum L.), nous avons étudié à quatre stades de développement du grain la localisation subcellulaire des puroindolines par immunocytochimie et les protéomes dans deux lignées de blé quasi-isogéniques pour la dureté. Dès la fin de la cellularisation de l’albumen, les puroindolines sont localisées sur la face interne des membranes vésiculaires et dans les corps protéiques en formation, structures dans lesquelles s’accumulent les protéines de réserve du grain. L’analyse par AFFFF (Asymmetrical Flow Field-Flow Fractionation) des deux lignées Hard et Soft, qui diffèrent essentiellement par l’absence du gène Pina dans la lignée Hard, a montré une corrélation entre la dureté et la taille des polymères de protéines de réserve. L’analyse protéomique des fractions albumines/globulines et amphiphiles des grains en développement a révélé une augmentation des protéines de la machinerie de repliement et de réponse au stress dans la lignée Hard, par rapport à la lignée Soft. Les deux approches méthodologiques utilisées semblent également mettre en évidence une cinétique de développement du grain raccourcie dans la lignée Hard. Ces observations suggèrent que les puroindolines interagissent avec les protéines de réserve du grain et suivent le même routage cellulaire. Elles pourraient être impliquées dans les mécanismes de repliement et d’assemblage des prolamines. / Wheat grain hardness, a major trait for endosperm texture and flour end-use properties, is strongly associated to absence or modification of puroindolines. To gain insight into biological function of those proteins in bread wheat kernel (Triticum aestivum L.), puroindolines’ subcellular localization was sought by immunocytochemistry and proteomes were analyzed at four stages of developing kernels in two near-isogenic lines for hardness, differing mainly in the absence of Pina in the Hardline. As early as the end of endosperm cellularisation, puroindolines were localized onto vesicular membranes and into protein bodies, where storage proteins accumulate. Using AFFFF (Asymmetrical Flow Field-Flow Fractionation), we observed a correlation between hardness and storage protein polymer size. Proteomic analyses of the albumin/globulin and amphiphilic fractions revealed an increase in folding and stress-related proteins in the Hard line, as compared to the Soft one. Both ultrastructural and proteomic studies suggested also that the Hard/Soft genotype affects the kinetics of kernel development. These lines of evidence imply that puroindolines interact with storage proteins and display the same routage. Puroindolines could therefore be involved in prolamines folding and assembly mechanisms.

Le blé est la seconde céréale la plus produite dans le monde. Il constitue une importante source de denrées alimentaires et de beaucoup d’autres usages industriels. La compréhension des mécanismes impliqués dans le développement du grain de blé est fondamentale pour développer des blés à valeur ajoutée. La physiologie du grain de blé et les mécanismes moléculaires impliqués dans son développement nécessitent d’être mieux connus et ces connaissances pourront être très utiles pour l’amélioration du blé mais aussi des autres céréales. L’approche protéomique a été aussi utilisée dans ce contexte mais aucun travail n’avait jusqu’ici été réalisé sur la totalité des phases de développement des tissus et sur des intervalles de temps très courts. La caractérisation des changements d’expressions protéiques dans les couches périphériques du grain et de l’albumen est présentée dans cette étude. Nous avons utilisé les grains de Triticum aestivum de la variété Récital, cultivés à l’INRA de Clermont-Ferrand. Les grains ont été prélevés tous les 50°C jour (°Cj) depuis la fécondation jusqu’à la maturité sur 15 stades de développement pour les couches périphériques et sur 21 stades pour l’albumen amylacé. Pour chaque échantillon, les couches périphériques des grains ont été disséquées et les protéines totales extraites. L’analyse des protéines en électrophorèse bidimensionnelle puis par spectrométrie de masse MALDI-TOF a permis d’identifier via l’interrogation des bases de données, 207 protéines différentiellement exprimées sur 15 stades de développement (0°Cj-700°Cj). Ces protéines ont ensuite été classées en 16 classes fonctionnelles. L’analyse en cluster a révélé 5 profils d’expression au cours du temps. Parallèlement, l’albumen amylacé a été isolé des grains et les protéines métaboliques de ce tissu extraites. Après électrophorèse bidimensionnelle des protéines, 487 protéines variant significativement dans l’albumen sur l’ensemble des stades de développement (0°Cj-1006°Cj) ont été identifiées par utilisation de la LC-MS. Les protéines ont été réparties sur neuf profils d’expression et 17 fonctions biochimiques. Le protéome des couches périphériques a ensuite été comparé au protéome de l’albumen dans le but de comprendre si l’évolution des processus biochimiques diffère dans chacun de ces tissus. Au final, nous avons optimisé l’intégration des données protéomiques avec celles du transcriptome (en se focalisant sur les protéines du métabolisme carboné). Seulement 32% des profils d’expression protéome/transcriptome montrent une corrélation significative au cours du développement (152°Cj-700°Cj). Les profils d’expression des enzymes ont été comparés sur les deux niveaux. Ils devraient permettre de distinguer les processus régulés au niveau du transcriptome de ceux régulés au niveau du protéome. L’ensemble de ces données pourra être compilé dans une base de données propre de la variété Récital et utilisé comme référence dans l’étude des maladies et des stress abiotiques des tissus du grain de blé en développement. / Wheat is the second most produced cereal in the world, important for food, feed and many industrial uses. Understanding of the mechanisms involved in grain development is fundamental for developing high quality wheat. In particular, detailed knowledge of the wheat grain physiology and molecular mechanisms involved in its development would help in breeding not only of wheat but also many other cereals. A proteomic approach has been used in this context but, up to now, there had been no work on developing tissues at very short temporal distances. This thesis presents, firstly, a proteomic study to characterize protein expression changes in peripheral layers and in starchy endosperm of wheat, during kernel development. We used grains of Triticum aestivum cv Récital, cultivated at INRA, Clermont-Ferrand. Grains were harvested at each 50°Cd from fertilization to maturity at fifteen stages for peripheral layers and at twenty-one stages for starchy endosperm. After grain dissection, protein extraction and 2DE- MALDI-TOF MS and data mining, we identified 207 differentially expressed proteins at fifteen stages (0°Cd-700°Cd) of peripheral layers during kernel development. These proteins were then classed in sixteen different functional classes. HCA revealed five different expression profiles during development. Similarly after obtaining starchy endosperm from dissected grains, we performed protein extraction specific to metabolic proteins. After 2DE, 487 proteins were identified from fertilization to grain maturity (0°Cd-1006°Cd), using LC-MS and data mining. Proteins were grouped in nine different expression profiles and were classed in seventeen biochemical functions. We have constructed proteome maps of these two important grain tissues during kernel development. Further, the comparison of peripheral layers and starchy endosperm proteomic data was made, with an objective to understand whether the changes in different biochemical processes differ between these tissues.Finally, we performed an integration of our proteomic data (focusing our approach on proteins involved in carbohydrate metabolism) with that of transcriptomics. Only 32% of proteome/transcriptome expression profiles showed a significant correlation during development (from 152°Cd-700°Cd). Comparison of enzyme expression profiles with those of proteome and transcriptome would help to distinguish the processes regulated at transcriptome level and those controlled at the proteome level. This comprehensive grain development data could further help in construction of a Récital databank, which may be used as reference for studies of diseased and stressed grain tissues during development.