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

Germination, carbohydrate composition and vigor of cryopreserved Caesalpinia echinata seeds

The present study investigated the germination and vigor of Caesalpinia echinata (Brazilwood) seeds stored at negative temperatures. Recently harvested seeds were cryopreserved at -18º or -196ºC and periodically evaluated for germination, seed vigor and carbohydrate composition. The temperatures did not influence the germination percentages or vigor. The germination percentage decreased from 88% in recently harvested seeds to 60% after 730 days of storage. The different temperature and storage times tested did not affect the vigor seed germination as indicated by the measures of plant growth and survival. The different temperatures used did not cause changes in the carbohydrate composition. The tegument cell walls were rich in lignin, arabinose and xylose. The cytoplasm of the cotyledons and embryos had high levels of glucose, fructose, and sucrose. The cryopreservation technique here presented was effective in the conservation of Brazilwood seeds for the medium term

Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production - part 1

Background: In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass fromgrass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM). Results: Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones. Conclusion: Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.FAPESP (10/11135-6, 09/18354-8, 10/08370-3, 08/56255-9, 10/52362-5)CNPq (159341/2011-6, 482166/2010-0, 490022/2009-0)European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013)INCT do BioetanolNúcleo de Apoio à Pesquisa em Bioenergia e Sustentabilidade (NAPBS)Centro de Instrumentação para Estudos Avançados de Materiais Nanoestruturados e Biossistemas (CIEA_MNB

Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production – part 1

Abstract\ud \ud \ud \ud Background\ud \ud In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass from grass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM).\ud \ud \ud \ud Results\ud \ud Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones.\ud \ud \ud \ud Conclusion\ud \ud Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.The authors are grateful to FAPESP and CNPq for the financial support for this work via grants # 2010/11135-6; 2009/18354-8; 2010/08370-3; 2008/ 56255-9 and 2010/52362-5 (FAPESP); grants # 159341/2011-6; 482166/ 2010-0 and 490022/2009-0 (CNPq), Projeto INCT do Bioetanol (CNPq/ FAPESP), to USP for the financial via NAP Centro de Instrumentação para Estudos Avançados de Materiais Nanoestruturados e Biossistemas and NAP de Bioenergia e Sustentabilidade, and European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013) under the grant agreement n° 251132. We are also grateful to Valeria Gazda for the help with furfural and 5-HMF chromatographic analysis and Caragh Whitehead for the paper review. The electron microscopy work has been performed on the microscopes JSM-5900LV and Quanta 650-FEG at LME/LNNano/ CNPEM, Campinas

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

Evaluation of the potential use of Eucalyptus spp. bark for bioethanol production

A utilização de fontes renováveis para a produção de biocombustíveis tem sido incentivada no mundo todo. Assim, na proposta de um novo cenário energético mundial, aliado as condições ambientais, surge a necessidade de se procurar outras fontes alternativas de matéria primas renováveis. Neste contexto, o Brasil possui condições especiais, se considerarmos os resíduos lignocelulósicos do setor florestal. Atualmente, o Brasil é o maior produtor mundial de celulose à partir de fibra curta de Eucalyptus spp, com um setor bem desenvolvido e em plena expansão. Toda esta atividade industrial produz anualmente cerca de 2,8 a 5,7 milhões de toneladas resíduos sólidos na forma de casca (principalmente de eucaliptos). Em muitos casos o destino aplicado para essas biomassas é pouco eficiente e representa uma perda significativa do potencial energético, pois estes resíduos lignocelulósicos são passíveis de biotransformação a compostos com elevado valor agregado, tais como os biocombustíveis (etanol). Portanto, o objetivo deste trabalho foi avaliar o potencial das cascas de eucalipto frente a produção de etanol combustível. Desta maneira, as cascas de 5 clones comerciais (E.urophylla x E. grandis e E. grandis) foram caracterizadas quanto a composição química. As cascas de eucalipto foram submetidas a uma série de pré-tratamentos ácidos e alcalinos, avaliados em planejamento fatorial com o objetivo de recuperar os açúcares potencialmente fermentecíveis. As cascas de eucalipto apresentaram aproximadamente 20% de carboidratos solúveis totais CST (glicose, frutose e sacarose). Os CST foram extraídos com água quente à temperatura de 80oC e em seguida fermentados com leveduras convencionais (Saccharomyces cerevisiae). A produção de etanol por tonelada de casca seca foi de 106 litros (etanol de primeira geração). Após a extração dos CST, as biomassas residuais das cascas de eucalipto foram submetidas a uma série de pré-tratamentos. O pré-tratamento alcalino (NaOH) apresentou uma eficiência enzimática de conversão da glicose de aproximadamente 30% após 24 horas de incubação. Com os resultados obtidos da hidrólise enzimática, estima-se que possam ser produzidos mais 94 litros de etanol por tonelada de casca livre de extrativos (etanol celulósico).The use of no fossil source for biofuels production has been stimuled in the all world. Proposing a new global scenario related to the energy matrix, together with the environmental conditions, there is the need to search alternative renewable raw materials. In this context, Brazil presents special conditions, considering the lignocellulosic residues from the forestry industry. Nowadays, Brazil is the largest cellulose short fiber producer from Eucalyptus spp, having a strong sector in expansion. This industrial activity produces in average 2,8 to 5,7 million tons of solid waste in the form of bark (mostly from eucalyptus). Usually, the destiny of this biomass is inefficient and represents a significant loss of the energetic potential, since these lignocellulosic residues can suffer biotransformation and produce high value components, like the biofuels (ethanol). Therewith, the aim of this work was to evaluate the potential of the eucalyptus bark related to ethanol fuel production. In this way, barks from 5 commercial clones (E. urophylla x E. grandis e E. grandis) were characterized due to the chemical composition. Eucalyptus barks were subjected to a series of acid and alkaline pretreatments, evaluated in factorial design aiming to recover potentially fermentable sugars. Eucalyptus bark presented on average 20% of total soluble carbohydrates TSC (glucose, fructose and sucrose). TSC were extracted with hot water (80 °C) and fermented by Saccharomyces cerevisiae. Ethanol production per ton of dry bark was 106 liters (first generation ethanol). After TSC extraction, the residual biomass from eucalyptus bark were subjected to several pretreatments. The alkaline pretreatment (NaOH) presented a high enzymatic efficiency of glucose conversion of approximately 30% after 24 hours of incubation. With the results obtained in enzymatic analysis, is estimated that it can be produced more than 94 liters of ethanol per ton of bark (cellulosic ethanol)

Evaluation of the chemical composition of the cell wall of transgenic tobacco plants (Nicotiana tabacum) that superexpress the gene ugdh, that codes for the enzyme UDPglucose dehydrogenase (EC 1.1.1.22)

Os elementos celulares que constituem o tecido xilemático de várias espécies vegetais são amplamente utilizados em diversos setores industriais, com inúmeras aplicações, como por exemplo, a geração de energia e produção de celulose e papel. A parede celular das células vegetais é formada basicamente por celulose, hemiceluloses e lignina. A formação dos polímeros de celulose e hemicelulose dependem exclusivamente do suprimento de precursores chamados de nucleotídeos-açúcares, tais como UDP-glicose, UDP-glucuronato, UDP-xilose, UDP-arabinose, UDP-manose e UDP-galactose. A biossíntese da parede celular é altamente regulada do ponto de vista metabólico, e envolve a participação de várias enzimas que catalisam uma série de reações. Estratégias para alterar o fluxo metabólico destes precursores, podem originar modificações na deposição dos polissacarídeos na parede celular. Em particular, para o setor de celulose e papel tal estratégia pode resultar em fibras com determinadas características, melhorando a qualidade da polpa celulósica ou do papel produzido. O UDP-glucuronato é um dos principais precursores de polissacarídeos hemicelulósicos da parede celular, sendo formado a partir de UDP-glucose pela ação da enzima UDP-glicose desidrogenase (EC. 1.1.1.22). Esta enzima é chave na regulação da biossíntese das pentoses e hexoses da parede celular de plantas superiores. Com o objetivo de modular a síntese dos polissacarídeos hemicelulósicos na parede celular, o presente trabalho analisou o impacto da superexpressão do gene ugdh em plantas transgênicas de tabaco. Foram realizadas análises da composição química da parede celular primária e secundária de folhas e caules, bem como avaliações morfológicas das fibras do tecido xilemático, e também cortes histológicos da região basal do caule. Da parede secundária do tecido xilemático determinou-se o conteúdo de lignina klason e solúvel, bem como a concentração dos carboidratos via HPAE-PAD. No tecido xilemático, todas as plantas transgênicas apresentaram aumento do conteúdo de xilose, embora não significativo. Juntamente, ocorreu um aumento de arabinose significativo em três linhagens transgênicas. Paralelamente, todas as plantas transgênicas tiveram redução do conteúdo de lignina klason, embora significativo em apenas uma linhagem. A relação hexoses/pentoses reduziu em todas as linhagens transgênicas, sendo significativa em três linhagens. As análises histológicas do caule mostraram que os transformantes apresentaram um aumento do tecido xilemático em relação às plantas controles. As análises morfológicas das fibras mostraram que todas as plantas transgênicas apresentaram reduções significativas no comprimento, em relação às plantas controles. No tecido foliar, o conteúdo de polissacarídeos da parede celular primária apresentou uma redução significativa em todas as plantas transgênicas.The cellular elements that constitute the xylematic tissue of various plant species are widely used in diverse industrial sectors, with numerous applications, for example, the generation of energy and production of cellulose and paper. Plant cell walls are basically formed by cellulose, hemicellulose and lignin. The formation of cellulose and hemicellulose polymers depend exclusively on the supply of the precursors called nucleotide sugars, such as UDPglucose, UDP-glucuronate, UPD-xylose, UDP-arabinose, UDP-mannose and UDP-galactose. The biosynthesis of the cell wall is highly regulated from the metabolic point of view and involves the participation of various enzymes that catalyse a series of reactions. Strategies to alter the metabolic flux of these precursors could give rise to modifications in the deposition of polysaccharides in the cell wall. In particular, for the cellulose and paper sector these strategies could result in fibers with determined characteristics, improving the quality of the cellulose pulp or the paper produced. UDP-glucuronate is one of the principal precursors of the hemicellulose polysaccharides of the cell wall, which is formed from UDP-glucose by the action of UDPglucose dehydrogenase (EC1.1.1.22). This is a key enzyme in the regulation of the biosynthesis of the pentoses and hexoses in the cell walls of higher plants. With the objective of modulating the synthesis of the hemicellulose polysaccharides in the cell wall, the present study analysed the impact of the superexpression of the ugdh gene in transgenic tobacco plants. Chemical analyses were performed to determine the chemical composition of the primary and secondary cell walls of leaves and stems, as well as morphological evaluations of the fibers of the xylematic tissue and histological cuts through the base of the stem. The Klason and soluble lignin content as well as the carbohydrate concentrations (using HPAE-PAD) were determined in the secondary cell walls of the xylematic tissue. All of the transgenic plants showed an increase in xylose content, albeit not significant. A significant increase in arabinose content was observed in three transgenic lines. In parallel all the transgenic plants presented a reduction in Klason lignin, but only significant in one line. The ratio hexose/pentose was reduced in all transgenic lines, being significant in three. Histological analyses on the stem showed that the transformants presented an increase in the xylematic tissue when compared to the controls. The morphological analyses of the fibers showed that all the transgenic plants presented significant reductions in length when compared to the controls. In the leaf tissue, the polysaccharide content of the primary cell wall showed a significant reduction in all of the transgenic plants