Densidade populacional de perfilhos em Brachiaria spp submetidos a fontes e doses de adubação nitrogenada: Tiller population density and tillering dynamics in Brachiaria spp. subjected to sources and levels of nitrogen fertilization

O objetivo deste trabalho foi avaliar os efeitos de fontes e doses de nitrogênio aplicadas em quatro genótipos de braquiária: Brachiaria brizantha cv. Marandu, Brachiaria brizantha cv. Piatã e dois híbridos múltiplos, intra e interespecíficos apomíticos de Brachiaria spp sobre a densidade populacional de perfilhos. O delineamento experimental foi de blocos completos casualizados, em arranjo fatorial 4 x 2 x 4, quatro genótipos de braquiária, duas fontes (ureia e nitrato de amônio) e quatro doses de nitrogênio (0, 75, 150 ou 225 mg/dm3), com cinco repetições, totalizando 160 vasos de cerâmica com capacidade para 3,34 dm3 de terra. A densidade populacional de perfilhos foi influenciada positivamente pelo uso da adubação nitrogenada, que pode alterar padrões de crescimento e estrutura do dossel. Os híbridos H69 e H12 apresentaram características estruturais em relação ao perfilhamento que os validam positivamente como novas opções para as pastagens. O nitrato de amônio foi mais eficiente que a ureia, por proporcionar maior perfilhamento basal para os híbridos H69 e H12 no primeiro corte e na rebrota de Piatã e Marandu

Modulating MIOX2 expression in Nicotiana tabacum and impacts on genes involved in cell wall biosynthesis

Cell walls are essential structures for plant development and growth. Apart from its biological functions, the polysaccharides that make cell walls (cellulose, hemicellulose and pectins) are the principal natural fibrous materials, considered the most important renewable resource on earth, used as raw material for many industrial processes among them, for pulp and paper production, charcoal, and biofuels. For all these reasons, the study of molecular composition and biosynthesis of plant cell walls has been a matter of great interest for researchers over the past few years. In this context, a full-length cDNA fragment of Miox2 gene was cloned from Arabidopsis seedlings, using RT-PCR, with an open reading frame of 954 pb and a corresponding protein subunit molecular mass of 37 kDa. The deduced amino acid sequence of the cDNA showed a high degree of homology with myo-Inosytol oxygenases from other organisms. This cDNA was used for genetic transformation of model plants (tobacco), which expressed either antisense or sense RNA. Transgenic homozygous tobacco model plants with either repression or constitutively expressed Miox2 were obtained with the number of copies varying from 1 to 7. Neither, the repression of the endogenous tobacco Miox genes or the constitutive expression of Miox2 gene, caused major impact on plant development, leaf morphology or flowering time. There was however, statistically significant (P<0.05) changes in the arabinan and D-galacturonate contents. These results clearly indicate that the modulation of the myo-Inositol pathway caused no major impact on cell wall polysaccharide biosynthesis.As paredes celulares vegetais são estruturas essenciais para o crescimento e desenvolvimento das plantas. Além de suas diversas funções biológicas, os componentes polissacarídicos constituintes das paredes celulares (celulose, hemiceluloses e pectinas) são de vital importância como fonte natural de fibras, sendo consideradas as fontes principais de recursos renováveis do planeta, utilizados como matéria prima para diversos processos industriais, dentre eles, a produção de papel e celulose, carvão vegetal e biocombustíveis. Todos esses fatores têm despertado grande interesse no estudo da composição e biossíntese das paredes celulares. Neste contexto, um fragmento de cDNA do gene Miox2 foi clonado de plântulas de Arabidopsis, via RT-PCR, com uma região aberta de leitura de 954 pb e sua proteína com massa molecular de 37kDa. A sequência deduzida de aminoácidos do cDNA apresentou alto grau de identidade com mio-Inositol oxigenases de outros organismos. Este cDNA foi usado para transformação genética de plantas modelo (tabaco) que produziram RNA antisense ou sense. Plantas de tabaco homozigotas para o transgene com repressão ou expressão constitutiva do gene Miox2 foram obtidas com um número de cópias do transgene, variando de 1 a 7. A repressão do gene Miox de tabaco endógeno assim como a expressão constitutiva do gene Miox2 de Arabidopsis não causaram alterações no desenvolvimento, morfologia foliar ou tempo de florescimento das plantas. Entretanto, alterações estatisticamente significativas (P<0.05) ocorreram no conteúdo de arabinana e de D-galacturonato. Estes resultados indicam que a modulação do metabolismo do mio-Inositol não causou grandes impactos na biossíntese dos polissacarídeos da parede celular.Fil: Defávari Nascimento, D.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Conti, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Labate, Mônica T. V.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Gutmanis, Gunta. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Bertolo, Ana L. F.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: de Andrade, Alexander. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Bragatto, Juliano. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Pagotto, Luís Otávio. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Damin, Plínio. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Moon, David H.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Labate, Carlos A.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; Brasi

Genetic diversity of asses’ five populations through the use of microsatellites markers

Genetic characterization of five asinine populations, corresponding to two Brazilian breeds (Nordestina - two populations - and Marchador Brasileiro), a local breed of Portugal (Burro de Miranda) and a diverse group of individuals from different Iberian populations was performed based on 193 animals. Fifteen microsatellites markers were analyzed in order to estimate the inter/intra population variability as well as to know the genetic relationship between Brazilian and Iberian asses. The results showed that the majority of loci were not at Hardy-Weinberg equilibrium in the investigated populations due to a significant excess of homozygotes (P&lt;0.05). Brazilian populations are not linked to European populations. The two Nordestina breed populations showed a well-defined structure and can be considered as two distinct populations. The Conservation Nucleus of Marchador Brasileiro breed, however, showed two different clusters, probably due to its reduced size with individuals from different origins (FIS = 0.2714). These donkeys are a high priority for the conservation of genetic resources. The information generated by microsatellites can be used for mating strategies in order to recover their diversity, especially regarding to the excess of homozygotes. There will be a new sampling in this Nucleus, in order to know more precisely the level of inbreeding present to supervise its diversity

Cloning and endogenous expression of a Eucalyptus grandis UDP-glucose dehydrogenase cDNA

UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis

Modulation of ugp gene and analysis of the changes in carbohydrates composition of the Nicotiana tabacum and Eucalyptus grandis primary and secondary cell wall

A associação de tecnologias como transgenia, genômica e proteômica aos programas de melhoramento convencional de árvores, pode acelerar o processo de obtenção de exemplares com características específicas da madeira para a indústria de papel e celulose. A parede celular vegetal é extremamente importante para este setor industrial, pois fornece polissacarídeos como celulose e hemiceluloses. A UDP-glucose pirofosforilase (UGPase, EC 2.7.7.9) é uma enzima chave na produção de UDP-glucose, precursor relevante na interconversão de nucleotídeoaçúcares que constituem os monômeros desses polissacarídeos. O objetivo deste trabalho foi clonar os cDNAs que codificam a UGPase, extraídos do tubérculo de batata (Solanum tuberosum L.) e da raiz de eucalipto (Eucalyptus grandis), e inseri-los por meio de transformação genética via Agrobacterium tumefaciens, respectivamente, no genoma de plantas de fumo (Nicotiana tabacum) e de eucalipto, para alterar o fluxo de carbono de modo a aumentar o conteúdo de pentosanas (xilose e arabinose). O maior teor de pentosanas é benéfico economicamente para a indústria de papel e celulose, pois diminui a energia consumida no refino da polpa além de benificiar a qualidade do papel que se torna mais resitente ao rasgo. O impacto da superexpressão do gene ugp nas plantas de tabaco e de eucalipto obtidas da transformação genética, foi avaliado por meio de análises bioquímicas e morfológicas. A análise de Southern blot indicou uma a quatro cópias do transgene no DNA genômico de dez linhagens T2 de tabaco, e uma cópia em uma linhagem T0 de eucalipto. A análise de qPCR mostrou que todas as linhagens T2 de tabaco apresentaram expressão do gene ugp exógeno, sendo que em quatro o nível de expressão foi maior que nas demais. As linhagens com menor expressão do gene ugp exógeno, com exceção de uma delas, também expressaram menos o gene ugp endógeno, sugerindo que nessas linhagens algum mecanismo de regulação atuou alterando a expressão desse gene, tanto endógeno, quanto exógeno. As linhagens transgênicas de tabaco foram morfologicamente semelhantes às plantas controle quando comparadas quanto à altura da planta, comprimento de internós, área foliar total, diâmetro do caule e massa seca da raiz. As alterações nos tabacos transgênicos, em relação aos controles, verificadas através das análises químicas, foram estatisticamente mais significativas na medula caulinar, onde houve aumento no conteúdo dos monossacarídeos e dos ácidos urônicos. As poucas diferenças encontradas no tecido xilemático sugerem que a planta, de alguma forma, tende a alterar menos a composição da parede celular secundária, do que da parede primária. Também sugerem que o metabolismo dos carboidratos pode afetar o conteúdo de lignina, embora seja sintetizada por outra rota metabólica. A parede celular primária, analizada utilizando a medula das plantas de tabaco e folhas de eucalipto, mostrou um aumento no conteúdo de pentosanas, o que não foi observado na análise de plântulas de tabaco.Assossiating technologies like transgenesis, genomics and proteomics with conventional forestry breeding can accelerate the process of obtaining new trees with specific wood properties for the paper and pulp industry. Plant cell wall is extremely important for this industry, providing polysaccharides like cellulose and hemicelluloses. UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9) is a key enzyme producing UDP-glucose, an important forerunner for nucleotide sugars that constitute the monomers of these polysaccharides. The goal in this work was to clone the cDNAs that encode UGPase, extracted from potato (Solanum tuberosum L.) tuber, and eucalypt (Eucalyptus grandis) root, and transfer through genetic transformation via Agrobacterium tumefaciens, respectively, in tobacco (Nicotiana tabacum) plants and eucalypts, in order to change the carbon flux, increasing the pentosans (arabinose and xylose) content. Higher pentosans content is economicaly important for the paper and pulp industry, as decreases energy consumption at pulp refining and also can benefit paper quality that becomes more resistant to rip. The impact of the ugp gene overexpression over tobacco and eucalypt plants obtained through genetic transformation were morphological and biochemicaly evaluated. Southern blot analysis showed one to four copies of the transgene in the genomic DNA of ten tobacco T2 lines, and one copy in one eucalypt T0 line. The qPCR analysis showed that all tobacco T2 lines expressed exogenous ugp gene, being the expression level in four of them biger than the others. Lines that expressed less exogenous ugp gene, with exception of one of them, equally had less endogenous ugp gene expression level, suggesting that in these lines, some kind of regulation acted changing the gene expression. The transgenic tobacco lines were morphologicaly similar to the control plants when compared for total height, internode lenght, total leaf area, stem diameter and root dry mass. Alterations in transgenics tobacco plants, in relation to controls, checked by chemical analysis, were statisticaly more significants in the pith, where the monosaccharides and uronic acids contents increased. The few diferences founded in the xilematic tissue sugest that the plant, somehow, tends to change less the secondary cell wall composition than the primary wall. They also suggest that the carbohydrate metabolism can affect lignin content, thought it\'s sintetizaded in another metabolic pathway. The primary cell wall, evaluated using tobbaco plants pith and eucalypt leaves, showed an increase in pentosanas content, that wasn\'t observed analising tobacco seedlings

Cloning of cdna encoding potato (Solanum tuberosum L.) udp-glucose pyrophosphorylase.

A biotecnologia está revolucionando a atividade humana nas mais diversas áreas. No setor florestal, as novas tecnologias como transgenia, genômica, proteômica e bioinformática, estão sendo rapidamente incorporadas. A associação dessas tecnologias aos programas de melhoramento convencional de árvores pode acelerar o processo de obtenção de árvores com características específicas da madeira para a indústria de papel e celulose. UDP-glicose pirofosforilase (UGPase, EC 2.7.7.9) é uma enzima chave na produção de UDP-glicose, intermediário na interconversão de carboidratos envolvidos em várias funções metabólicas, como síntese de sacarose e de celulose. No presente trabalho, foi clonado o cDNA que codifica a UGPase. Este cDNA foi sintetizado por meio de RT-PCR utilizando-se mRNA extraído de tubérculo de batata (Solanum tuberosum L.). Os primers específicos para a ORF do gene da UGPase foram definidos com base em seqüências conservadas entre genes ortólogos, já disponíveis em bancos de dados. O cDNA foi clonado no vetor pENTR-D TOPO e inserido, por meio de transformação química, em células competentes de Escherichia coli. O DNA plasmidial foi purificado e após seu sequenciamento, a presença de um inserto de 1402 pb foi confirmada. O alinhamento da seqüência deduzida de aminoácidos com outras UGPases revelou alta homologia a UGPases de batata e de outras espécies vegetais. Na seqüência do cDNA clonado foram identificados sítios de N-glicosilação, resíduos lisina e motivos conservados que possivelmente estão relacionados tanto com a capacidade de ligação ao substrato, como com a atividade catalítica da enzima. Também foi realizada uma análise de Southern blot para confirmar a presença deste gene no DNA genômico de batata e de eucalipto (Eucalyptus grandis). Posteriormente, o cDNA clonado será utilizado na avaliação do impacto de sua superexpressão na biossíntese de celulose e hemiceluloses e no desenvolvimento de plantas de fumo (Nicotiana tabacum) e de eucalipto.Biotechnology is revolutionazing human activity in several fields. In forestry, new technologies like transgenies, genomics, proteomics and bioinformatics have been quickly consolidated. Assossiating these technologies with conventional forestry breeding will accelerate the process of obtaining new trees with specific wood properties for the paper and pulp industry. UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9) is a key enzyme producing UDP-glucose, an important intermediate in carbohydrate interconversion involved in various metabolic processes like sucrose and cellulose synthesis. In this work, it was cloned the cDNA that encodes UGPase. This cDNA was synthetized through RT-PCR using RNA extracted from potato (Solanum tuberosum L.) tuber. The UGPase primers were designed based on the conserved sequences of hortologous genes, already available in public data banks. The cDNA was cloned into pENTR-D TOPO vector and introduced into Escherichia coli competent cells, by chemically transforming. The plasmid DNA was purified and the insert PCR amplified. The presence of 1402 bp insert was confirmed after sequencing. The alignment of the derived amino acids sequence with other UGPases revealed high homology to UGPases from potato and other plant species. Along the cDNA sequence N-glicolisation sites, Lysyl residues and conserved domain were identified, which probably are involved in substrate binding capacity, as well as catalytic ativity of the enzyme. Southern blot analysis was carried to confirm the presence of this gene in potato and eucalypt (Eucalyptus grandis) genomic DNA. The cloned cDNA will be used to evaluate the impact of overexpression on celullose and hemicelluloses biosynthesis and in the tobacco (Nicotiana tabacum) and eucalypt plant development

Diagnosis tools useful in the management of nitrogen nutrition in Urochloa

The N management on pasture requires methods for analyzing and predicting the need for fertilization, aiming at greater efficiency of fertilizer application, adjusting the forage requirements for biomass productivity with quality and sustainability. Nitrogen Nutrition Index (NNI) defined as the ratio between the actual N concentration and the N concentration critical, corresponding to the actual standing biomass. NNI is an indicator well connected with the physiological regulation of N concentration, but it cannot be used directly in farm conditions. Leaf N concentration has been shown to be with greenness (chlorophyll meter readings), as measured by SPAD 502, giving instantaneous values that could be used for estimating directly the N concentration and indirectly the NNI. The main of this work was to test the usefulness N concentration and SPAD to diagnose the N status of four genotypes Urochloa. The experimental design was a randomized block in a factorial 4 x 4, genotypes of Urochloa (U. brizantha cv. Piata, U. brizantha cv. Marandu, and two hybrids H69 and H12) and nitrogen levels (0, 75, 150 and 225 mg dm-3) whose source urea, with five replications, in pots (3.34 dm3) with Psament soil. Evaluations were performed on plants aged 52 days after sowing. Data were analyzed by the mixed procedure of SAS V. 9.2; average qualitative treatments were compared by Tukey test at 5% probability. The degrees of freedom related to N rates (quantitative treatment) were decomposed into orthogonal polynomials; to obtain the best equation fits the data. It is known that chlorophyll meter readings express indirectly the amount of chlorophyll in the plant tissue, and as nitrogen is a component of the chlorophyll molecule, the concentration of this nutrient in the tissue is positively correlated with the SPAD values, several studies have confirmed this fact . The SPAD values were found to be in the range 17.4 to 36.0 for H69, 24.2 to 36.6 for H12, 28.2 to 44.89 to Piata and 15.7 to 40.0 for Marandu. Without N application, Piatã registers higher values of SPAD, presenting visually better distribution of the chlorophyll more intense and uniform, followed by the hybrid H12, H69 and Marandu and finally, by application of 225 mg dm-3 N, most high SPAD values were very similar for the genotypes studied, ranging from 36.0 to 44.89. The NNI was closely associated with SPAD values: NNI = 0.05(SPAD – 23.31), R= 0.96;Piatã2 NNI = 0.024(SPAD + 2.13), R= 0.95; Marandu2NNIH69= 0.042(SPAD - 11.62), R= 0.86; NNI= 0.040(SPAD – 10.73), R= 0.87. Values of NNI close to 1 indicate that the date of the determination of N concentration or N critical of plant were non-limiting N supply. Values more than 1 indicate a luxury consumption of N. Values lower than 1 indicates an N deficiency. Then the values SPAD associated to NNI=1 were 43.3, 39.5, 35.4 and 35.7 for Piatã, Marandu, H69 and H12, respectively. SPAD values arise as an adequate method to characterizing an instantaneous, approximated, estimation of N status, thus possibiliting a prompt correction of N deficiency

Ferramentas diagnósticas úteis no manejo da nutrição nitrogenada em Urochloa

O artigo não apresenta resumo em português

Ferramentas diagnósticas úteis no manejo da nutrição nitrogenada em Urochloa

O artigo não apresenta resumo em português

Evaluation of interspecific hybrids and cultivars of Brachiaria spp. submitted to sourcers and levels of nitrogen fertilization

The management of nitrogen supply (N) is a very important issue concerned to plant growth and the environment healthy. For farmers, to know how much N the plant requires enables the application of appropriate amounts of nitrogen fertilizer, maximizing the use of this material. The availability of N has been identified as one of the main limiting factors, but also a management tool in the production of grasses. The nitrogen in the soil either, as a constituent of organic matter or in the mineral form (ammonium and nitrate), has a limited supply and can be depleted rapidly in a few crops. The aim of this study was to evaluate the effects of sources and nitrogen levels in four genotypes of Brachiaria (Brachiaria brizantha cv. Marandu, Brachiaria brizantha cv. Piata and two interspecific hybrids of Brachiaria spp) on the total and the leaf dry matter production, leaf percentage and leaf: stem ratio. The soil was an Psament-Entisol, coming from a pasture of Brachiaria decumbens with low organic matter content. The experiment was conducted in greenhouse, in Nova Odessa, Sao Paulo, from October 2009 to April 2010. The experimental design was in randomized complete blocks and the treatments were arranged in a factorial 4 x 2 x 4, four genotypes of Brachiaria , two N sources (urea and ammonium nitrate) and four nitrogen rates (0, 75, 150 or 225 mg dm-3) with five replicates, totaling 160 pots with a capacity of 3.34 dm3 of soil. Two cuts were performed in the plants. After the second cut a soil sample was collected from each experimental unit. Two evaluations were performed on plants. The first one 52 days after sowing, and the second 56 days after the first cut. Data were analyzed by the mixed procedure of SAS V. 9.2; average qualitative factors were compared by Tukey test at 5% probability. The degrees of freedom related to N rates (quantitative factor) were decomposed into orthogonal polynomials; to obtain the best equation fits the data. The variables evaluated were positively influenced by N fertilization, which can alter growth patterns and canopy structure. The hybrids H69 and H12 showed structural features in relation to all variables evaluated that positively validate as options for pasture. The ammonium nitrate was more effective than urea for dry matter production in hybrids H69 and H12 in the first cut, and for regrowth of cultivars Piata and Marandu. The results showed that nitrogen is a good tool which in turn contributes positively to increases the growth-related variables, such as dry matter (total, leaves and pseudostems). The hybrids H69 and H12 showed morphological, structural features and production of dry matter that validate them as positive options for pasture use