Sucrose-phosphate phosphatase from sugarcane reveals an ancestral tandem duplication.

BACKGROUND: Sugarcane is capable to store large amounts of sucrose in the culm at maturity hence it became a major source of sucrose for the food and the renewable energy industries. Sucrose, the main disaccharide produced by photosynthesis, is mainly stored in the vacuole of the cells of non-photosynthetic tissues. Two pathways are known to release free sucrose in plant cells, one is de novo synthesis dependent on sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (S6PP) while the other is regulatory and dependent on sucrose synthase (SuSy) activity. The molecular understanding of genes that give rise to the expression of the enzyme sucrose phosphate phosphatase, responsible for the release of sucrose in the last synthetic step lag behind the regulatory SuSy gene. RESULTS: Sugarcane genome sequencing effort disclosed the existence of a tandem duplication and the present work further support that both S6PP.1 and S6PP_2D isoforms are actively transcribed in young sugarcane plants but significantly less at maturity. Two commercial hybrids (SP80-3280 and R570) and both Saccharum spontaneum (IN84-58) and S.officinarum (BADILLA) exhibit transcriptional activity at three-month-old plants of the tandem S6PP_2D in leaves, culm, meristem and root system with a cultivar-specific distribution. Moreover, this tandem duplication is shared with other grasses and is ancestral in the group. CONCLUSION: Detection of a new isoform of S6PP resulting from the translation of 14 exon-containing transcript (S6PP_2D) will contribute to the knowledge of sucrose metabolism in plants. In addition, expression varies along plant development and between sugarcane cultivars and parental species.

Root growth and antioxidant enzyme responses to aluminium stress in sugarcane / Crescimento radicular e resposta de enzimas antioxidantes ao estresse por alumínio em cana-de-açúcar

Aluminium (Al) toxicity in acid soils is a major abiotic stress that can limit plant production worldwide. Al toxicity directly inhibits root development and exacerbates oxidative stress in the plant. Sugarcane is mostly cultivated in tropical regions and is often exposed to phytotoxic concentrations of soil Al. In this study, our objectives were to evaluate nine sugarcane cultivars on their tolerance to Al in a hydroponic system, investigating the effects of 143µM {Al3+} on root growth and on activity of the antioxidant enzymes ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD). The screening method proposed was suitable for a rapid, reliable and reproducible procedure of the sugarcane cultivars. Exposure to Al for three days altered root growth and activity of enzymes of the nine sugarcane cultivars. However, the magnitude of the alterations varied significantly among cultivars. The cultivar RB928064 was classified as Al-tolerant and the cultivar RB835486 as Al-sensitive. Increases in enzyme activity after Al exposure varied from 4 to 46%, with average increases of 19% in APX, 20% in CAT, and 8% in SOD. The variations induced by Al in enzyme activity, however, did not correlate significantly with those variations induced by Al in the root growth.(AU)

A toxidez por alumínio (Al) em solos ácidos é um dos principais estresses abióticos que podem limitara produção vegetal pelo mundo todo. A toxidez por Al inibe diretamente o desenvolvimento radicular e aumenta a produção de espécies reativas de oxigênio na planta. A cana-de-açúcar é mais cultivada em regiões tropicais e frequentemente está exposta a concentrações fitotóxicas de Al no solo. Neste estudo, nossos objetivos foram avaliar a tolerância ao Al de nove cultivares de cana-de-açúcar em um sistema hidropônico e investigar os efeitos de 143µM {Al3+} sobre o crescimento radicular e a atividade das enzimas antioxidantes ascorbato peroxidase (APX), catalase (CAT) e superóxido dismutase (SOD). O método de triagem proposto foi adequado para a avaliação de tolerância ao Al de forma rápida, confiável e reprodutível. A exposição ao Al por três dias alterou o crescimento radicular e a atividade das enzimas nas nove cultivares de cana-de-açúcar. Entretanto, a magnitude das alterações variou significativamente entre as cultivares. A cultivar RB928064 foi classificada como tolerante e a cultivar RB835486 como sensível ao Al. Os incrementos de atividade enzimática após exposição ao Al variaram entre 4 e 46%,com incrementos médios de 19% para APX, 20% para CAT, e 8% para SOD. As variações induzidas pelo Al sobre a atividade enzimática, entretanto, não apresentaram correlações significativas com aquelas induzidas pelo Al sobre o crescimento radicular.(AU)

Root growth and antioxidant enzyme responses to aluminium stress in sugarcane / Crescimento radicular e resposta de enzimas antioxidantes ao estresse por alumínio em cana-de-açúcar

Aluminium (Al) toxicity in acid soils is a major abiotic stress that can limit plant production worldwide. Al toxicity directly inhibits root development and exacerbates oxidative stress in the plant. Sugarcane is mostly cultivated in tropical regions and is often exposed to phytotoxic concentrations of soil Al. In this study, our objectives were to evaluate nine sugarcane cultivars on their tolerance to Al in a hydroponic system, investigating the effects of 143µM {Al3+} on root growth and on activity of the antioxidant enzymes ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD). The screening method proposed was suitable for a rapid, reliable and reproducible procedure of the sugarcane cultivars. Exposure to Al for three days altered root growth and activity of enzymes of the nine sugarcane cultivars. However, the magnitude of the alterations varied significantly among cultivars. The cultivar RB928064 was classified as Al-tolerant and the cultivar RB835486 as Al-sensitive. Increases in enzyme activity after Al exposure varied from 4 to 46%, with average increases of 19% in APX, 20% in CAT, and 8% in SOD. The variations induced by Al in enzyme activity, however, did not correlate significantly with those variations induced by Al in the root growth.

A toxidez por alumínio (Al) em solos ácidos é um dos principais estresses abióticos que podem limitara produção vegetal pelo mundo todo. A toxidez por Al inibe diretamente o desenvolvimento radicular e aumenta a produção de espécies reativas de oxigênio na planta. A cana-de-açúcar é mais cultivada em regiões tropicais e frequentemente está exposta a concentrações fitotóxicas de Al no solo. Neste estudo, nossos objetivos foram avaliar a tolerância ao Al de nove cultivares de cana-de-açúcar em um sistema hidropônico e investigar os efeitos de 143µM {Al3+} sobre o crescimento radicular e a atividade das enzimas antioxidantes ascorbato peroxidase (APX), catalase (CAT) e superóxido dismutase (SOD). O método de triagem proposto foi adequado para a avaliação de tolerância ao Al de forma rápida, confiável e reprodutível. A exposição ao Al por três dias alterou o crescimento radicular e a atividade das enzimas nas nove cultivares de cana-de-açúcar. Entretanto, a magnitude das alterações variou significativamente entre as cultivares. A cultivar RB928064 foi classificada como tolerante e a cultivar RB835486 como sensível ao Al. Os incrementos de atividade enzimática após exposição ao Al variaram entre 4 e 46%,com incrementos médios de 19% para APX, 20% para CAT, e 8% para SOD. As variações induzidas pelo Al sobre a atividade enzimática, entretanto, não apresentaram correlações significativas com aquelas induzidas pelo Al sobre o crescimento radicular.

Decarboxylation mechanisms of C4 photosynthesis in Saccharum spp.: increased PEPCK activity under water-limiting conditions.

BACKGROUND: C4 plants have been classified into three subtypes based on the enzymes used to decarboxylate C4 acids in the bundle sheath cells (NADP-ME, NAD-ME and PEPCK pathways). Evidences indicate that, depending on environmental factors, C4 plants may exhibit a certain degree of flexibility in the use of the decarboxylation mechanisms. In this context, the objective was to extend the knowledge on the degree of flexibility between the pathways of decarboxylation in sugarcane, a NADP-ME species, at different levels of water deficit. RESULTS: An experiment was carried out with two cultivars - RB92579 (tolerant to water deficit) and SP80-3280 (susceptible to water deficit) subjected to moderate level (- 1.5 to - 1.8 MPa), severe level (below - 2.0 MPa) and recovery (48 h after rehydration) and changes in the activities of the enzymes involved in the three C4 mechanisms and in gene expression were investigated. Our results showed that sugarcane uses the PEPCK pathway as a decarboxylation mechanism in addition to the NADP-ME, which was more evident under water deficit conditions for both cultivars. CONCLUSIONS: The results obtained here, show that sugarcane increases the use of the PEPCK pathway as a decarboxylation mechanism, in addition to the NADP-ME pathway, under conditions of water deficit, particularly in the tolerant cultivar.

The control of endopolygalacturonase expression by the sugarcane RAV transcription factor during aerenchyma formation.

The development of lysigenous aerenchyma starts with cell expansion and degradation of pectin from the middle lamella, leading to cell wall modification, and culminating with cell separation. Here we report that nutritional starvation of sugarcane induced gene expression along sections of the first 5 cm of the root and between treatments. We selected two candidate genes: a RAV transcription factor, from the ethylene response factors superfamily, and an endopolygalacturonase (EPG), a glycosyl hydrolase related to homogalacturonan hydrolysis from the middle lamella. epg1 and rav1 transcriptional patterns suggest they are essential genes at the initial steps of pectin degradation during aerenchyma development in sugarcane. Due to the high complexity of the sugarcane genome, rav1 and epg1 were sequenced from 17 bacterial artificial chromosome clones containing hom(e)ologous genomic regions, and the sequences were compared with those of Sorghum bicolor. We used one hom(e)olog sequence from each gene for transactivation assays in tobacco. rav1 was shown to bind to the epg1 promoter, repressing ß-glucuronidase activity. RAV repression upon epg1 transcription is the first reported link between ethylene regulation and pectin hydrolysis during aerenchyma formation. Our findings may help to elucidate cell wall degradation in sugarcane and therefore contribute to second-generation bioethanol production.

The FBH family of bHLH transcription factors controls ACC synthase expression in sugarcane.

Ethylene is a phytohormone involved in the regulation of several aspects of plant development and in responses to biotic and abiotic stress. The effects of exogenous application of ethylene to sugarcane plants are well characterized as growth inhibition of immature internodes and stimulation of sucrose accumulation. However, the molecular network underlying the control of ethylene biosynthesis in sugarcane remains largely unknown. The chemical reaction catalyzed by 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is an important rate-limiting step that regulates ethylene production in plants. In this work, using a yeast one-hybrid approach, we identified three basic helix-loop-helix (bHLH) transcription factors, homologs of Arabidopsis FBH (FLOWERING BHLH), that bind to the promoter of ScACS2 (Sugarcane ACS2), a sugarcane type 3 ACS isozyme gene. Protein-protein interaction assays showed that sugarcane FBH1 (ScFBH1), ScFBH2, and ScFBH3 form homo- and heterodimers in the nucleus. Gene expression analysis revealed that ScFBHs and ScACS2 transcripts are more abundant in maturing internodes during afternoon and night. In addition, Arabidopsis functional analysis demonstrated that FBH controls ethylene production by regulating transcript levels of ACS7, a homolog of ScACS2. These results indicate that ScFBHs transcriptionally regulate ethylene biosynthesis in maturing internodes of sugarcane.

Development and production of sugarcane under different levels straw after four years of cultivation / Desenvolvimento e produção de cana-de-açúcar sob diferentes quantidades de palhada após quatro anos de cultivo

Environmental constraints, labour shortages and the search for greater efficiency have induced the mechanisation of sugarcane harvesting, providing a large amount of straw in the system, which provides benefits to the soil, the crop and the environment. The objective of this work was to evaluate the effect of the removal of different amounts of straw from the soil surface on the development and productivity of sugarcane in eutrophic Red Latosol. The experiment was conducted in an area belonging to the Usina in Bandeirantes, PR, using a randomised block design with four replications. The effects of six treatments (0, 25%, 50%, 75%, 100% of straw and sugarcane) were evaluated after four years of cultivation on the LAI (leaf area index), tillering, diameter, length and fresh mass of the stalks, in five periods: 60, 120, 180, 240 and 370 days after harvest (DAC). The results were submitted to analysis of variance and the means compared by Tukeys test. The straw on the soil surface provided benefits to the development and productivity of sugarcane. The management of sugarcane burning, total removal of the straw (0% of soil cover) or 75% of straw resulted in a lower IAF and stalk diameter and a 37% decrease in final yield under conditions of water deficiency. The maintenance of 50% of straw is sufficient to favour the development and to provide greater productivity of the sugarcane, as it is possible to use the 50%surplus of the field for the production of ethanol of second generation or electric energy, without damage to the productivity of the culture.(AU)

As restrições ambientais, a falta de mão-de-obra e a busca por maior eficiência induzem a mecanização da colheita de cana-de-açúcar, disponibilizando grande quantidade de palhada no sistema, que proporciona benefícios ao solo, à cultura e ao meio ambiente. O objetivo desse trabalho foi avaliar o efeito da retirada de diferentes quantidades de palhada da superfície do solo sobre o desenvolvimento e a produtividade da cana-de-açúcar em Latossolo Vermelho eutroférrico. O experimento foi instalado em área pertencente a Usina em Bandeirantes, PR, utilizando delineamento em blocos casualizados com quatro repetições. Foram avaliados os efeitos de seis tratamentos (0, 25%, 50%, 75%, 100% de palhada e cana-queimada), após quatro anos de cultivo, sobre o IAF (Índice de área foliar), perfilhamento, diâmetro, comprimento médio e massa fresca dos colmos, em cinco períodos: 60, 120, 180, 240 e 370 dias após a colheita (DAC). Os resultados foram submetidos à análise de variância e as médias comparadas pelo teste Tukey. A palhada sobre a superfície do solo proporcionou benefícios ao desenvolvimento e a produtividade da cana-de-açúcar. Os manejos queima da cana-de-açúcar, retirada total da palhada (0% de cobertura do solo) ou de 75% de palhada resultaram em menor índice de área foliar, diâmetro de colmos e diminuição de 37% na produtividade final, em condições de deficiência hídrica. A manutenção de 50% de palhadaé suficiente para favorecer o desenvolvimento e proporcionar maior produtividade da cana-de-açúcar,sendo possível utilizar os 50% excedente do campo para produção de etanol de segunda geração ouenergia elétrica, sem prejuízos à produtividade da cultura.(AU)

Production of cellulases by Penicillium oxalicum through solid state fermentation using agroindustrial substrates / Produção de celulases por Penicillium oxalicum em fermentação em estado sólido usando diferentes substratos agroindustriais

The purpose of this study was to define the factors that influence the production of cellulases by Penicillium oxalicum, a strain obtained from a leaf-cutting ant colony and identified based on the ITS gene. The experimental design included solid state fermentation using sugarcane bagasse and lignocellulosic sorghum as the lignocellulosic substrate. The variables were analyzed using a 25-1 fractional factorial design, with three replicates on the central point. All the variables analyzed influenced the production of at least one of the three cellulose types analyzed. The highest values observed were: FPase 4.2 U g-1, CMCase 9.2 U g-1 and Avicelase 8.4 U g-1 using lignocellulosic sorghum as the substrate. The best conditions for enzyme production were: incubation temperature at 40ºC, initial moisture of 60%, pH of 4.0 and a growth period of four days using lignocellulosic sorghum as the substrate.(AU)

O estudo teve como foco a determinação de fatores que influenciam a produção de celulases por uma cepa isolada de ninho de formigas cortadeiras e identificada por meio do gene ITS como Penicillium oxalicum. O processo produtivo foi Fermentação em Estado Sólido utilizando como substrato lignocelulósico bagaço de cana-de-açúcar e sorgo lignocelulósico. As variáveis foram analisadas através de um planejamento fatorial fracionário 25-1, com três repetições no ponto central. Todas as variáveis analisadas influenciaram a produção de pelo menos um dos três tipos de celulases analisados. As maiores atividades observadas foram: FPase 4,2 U g-1; CMCases 9,2 U g-1 e avicelase 8,4 U g-1, utilizando sorgo lignocelulósico como substrato. As melhores condições para produção foram: temperatura de incubação a 40oC, umidade inicial 60%, pH 4,0, tempo de cultivo de quatro dias, utilizando como substrato sorgo lignocelulósico.(AU)

S-nitrosoglutathione spraying improves stomatal conductance, Rubisco activity and antioxidant defense in both leaves and roots of sugarcane plants under water deficit.

Water deficit is a major environmental constraint on crop productivity and performance and nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. This study aims to test the hypothesis that leaf spraying of S-nitrosoglutathione (GSNO), an NO donor, improves the antioxidant defense in both roots and leaves of sugarcane plants under water deficit, with positive consequences for photosynthesis. In addition, the roles of key photosynthetic enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) in maintaining CO2 assimilation of GSNO-sprayed plants under water deficit were evaluated. Sugarcane plants were sprayed with water or GSNO 100 µM and subjected to water deficit, by adding polyethylene glycol (PEG-8000) to the nutrient solution. Sugarcane plants supplied with GSNO presented increases in the activity of antioxidant enzymes such as superoxide dismutase in leaves and catalase in roots, indicating higher antioxidant capacity under water deficit. Such adjustments induced by GSNO were sufficient to prevent oxidative damage in both organs and were associated with better leaf water status. As a consequence, GSNO spraying alleviated the negative impact of water deficit on stomatal conductance and photosynthetic rates, with plants also showing increases in Rubisco activity under water deficit.

Development and nitrate reductase activity of sugarcane inoculated with five diazotrophic strains.

Diazotrophs are able to stimulate plant growth. This study aimed at evaluating the effect of inoculation of five diazotrophic strains on growth promotion and nitrate reductase (NR, EC 1.7.1.1) activity in sugarcane. An experiment was carried out from three stages of cultivation: sprouting, tubes, and in hydroponics. On the first two stages, seven treatments were adopted: uninoculated control; mixed inoculation with five strains; and individual inoculation with Gluconacetobacter diazotrophicus (Gd), Herbaspirillum rubrisubalbicans (Hr), Herbaspirillum seropedicae (Hs), Nitrospirillum amazonense (Na), and Paraburkholderia tropica (Pt). The four treatments showing the best performance were transferred to the hydroponic system for analysis of NR activity. Hs, Pt, and the mixture of all strains led to the highest seedling biomass in tubes, followed by Hr. In hydroponics, the mixture and the strain Hr had the highest growth-promoting effect. NR activity was influenced by inoculation only under low N supply conditions, with positive effect of Hr, Pt, and the mixture.

Biochemical composition of symplastic sap from sugarcane genetically modified to overproduce proline.

Global interest in sugarcane has increased significantly in recent years because of its economic impact on sustainable energy production. The purpose of the present study was to evaluate changes in the concentrations of total sugars, amino acids, free proline, and total proteins by colorimetric analyses and nuclear magnetic resonance (NMR) to perform a metabolic profiling of a water-soluble fraction of symplastic sap in response to the constitutive expression of a mutant Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene from Vigna aconitifolia. However, there was not a significant increase in the free proline content in the sap of transgenic plants compared to the non-transformed control plants. The most noticeable difference between the two genotypes was an almost two-fold increase in the accumulation of sucrose in the stem internodes of P5CS transgenic sugarcane plants. The results presented in this work showed that transgenic sugarcane plants with increased levels of free proline accumulates high soluble sugar content and, therefore, may represent a novel genotype for improving sugarcane cultivars.

Development and production of sugarcane under different levels straw after four years of cultivation / Desenvolvimento e produção de cana-de-açúcar sob diferentes quantidades de palhada após quatro anos de cultivo

Environmental constraints, labour shortages and the search for greater efficiency have induced the mechanisation of sugarcane harvesting, providing a large amount of straw in the system, which provides benefits to the soil, the crop and the environment. The objective of this work was to evaluate the effect of the removal of different amounts of straw from the soil surface on the development and productivity of sugarcane in eutrophic Red Latosol. The experiment was conducted in an area belonging to the Usina in Bandeirantes, PR, using a randomised block design with four replications. The effects of six treatments (0, 25%, 50%, 75%, 100% of straw and sugarcane) were evaluated after four years of cultivation on the LAI (leaf area index), tillering, diameter, length and fresh mass of the stalks, in five periods: 60, 120, 180, 240 and 370 days after harvest (DAC). The results were submitted to analysis of variance and the means compared by Tukeys test. The straw on the soil surface provided benefits to the development and productivity of sugarcane. The management of sugarcane burning, total removal of the straw (0% of soil cover) or 75% of straw resulted in a lower IAF and stalk diameter and a 37% decrease in final yield under conditions of water deficiency. The maintenance of 50% of straw is sufficient to favour the development and to provide greater productivity of the sugarcane, as it is possible to use the 50%surplus of the field for the production of ethanol of second generation or electric energy, without damage to the productivity of the culture.

As restrições ambientais, a falta de mão-de-obra e a busca por maior eficiência induzem a mecanização da colheita de cana-de-açúcar, disponibilizando grande quantidade de palhada no sistema, que proporciona benefícios ao solo, à cultura e ao meio ambiente. O objetivo desse trabalho foi avaliar o efeito da retirada de diferentes quantidades de palhada da superfície do solo sobre o desenvolvimento e a produtividade da cana-de-açúcar em Latossolo Vermelho eutroférrico. O experimento foi instalado em área pertencente a Usina em Bandeirantes, PR, utilizando delineamento em blocos casualizados com quatro repetições. Foram avaliados os efeitos de seis tratamentos (0, 25%, 50%, 75%, 100% de palhada e cana-queimada), após quatro anos de cultivo, sobre o IAF (Índice de área foliar), perfilhamento, diâmetro, comprimento médio e massa fresca dos colmos, em cinco períodos: 60, 120, 180, 240 e 370 dias após a colheita (DAC). Os resultados foram submetidos à análise de variância e as médias comparadas pelo teste Tukey. A palhada sobre a superfície do solo proporcionou benefícios ao desenvolvimento e a produtividade da cana-de-açúcar. Os manejos queima da cana-de-açúcar, retirada total da palhada (0% de cobertura do solo) ou de 75% de palhada resultaram em menor índice de área foliar, diâmetro de colmos e diminuição de 37% na produtividade final, em condições de deficiência hídrica. A manutenção de 50% de palhadaé suficiente para favorecer o desenvolvimento e proporcionar maior produtividade da cana-de-açúcar,sendo possível utilizar os 50% excedente do campo para produção de etanol de segunda geração ouenergia elétrica, sem prejuízos à produtividade da cultura.

Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding.

Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory.

Cell wall proteome of sugarcane stems: comparison of a destructive and a non-destructive extraction method showed differences in glycoside hydrolases and peroxidases.

BACKGROUND: Sugarcane has been used as the main crop for ethanol production for more than 40 years in Brazil. Recently, the production of bioethanol from bagasse and straw, also called second generation (2G) ethanol, became a reality with the first commercial plants started in the USA and Brazil. However, the industrial processes still need to be improved to generate a low cost fuel. One possibility is the remodeling of cell walls, by means of genetic improvement or transgenesis, in order to make the bagasse more accessible to hydrolytic enzymes. We aimed at characterizing the cell wall proteome of young sugarcane culms, to identify proteins involved in cell wall biogenesis. Proteins were extracted from the cell walls of 2-month-old culms using two protocols, non-destructive by vacuum infiltration vs destructive. The proteins were identified by mass spectrometry and bioinformatics. RESULTS: A predicted signal peptide was found in 84 different proteins, called cell wall proteins (CWPs). As expected, the non-destructive method showed a lower percentage of proteins predicted to be intracellular than the destructive one (33% vs 44%). About 19% of CWPs were identified with both methods, whilst the infiltration protocol could lead to the identification of 75% more CWPs. In both cases, the most populated protein functional classes were those of proteins related to lipid metabolism and oxido-reductases. Curiously, a single glycoside hydrolase (GH) was identified using the non-destructive method whereas 10 GHs were found with the destructive one. Quantitative data analysis allowed the identification of the most abundant proteins. CONCLUSIONS: The results highlighted the importance of using different protocols to extract proteins from cell walls to expand the coverage of the cell wall proteome. Ten GHs were indicated as possible targets for further studies in order to obtain cell walls less recalcitrant to deconstruction. Therefore, this work contributed to two goals: enlarge the coverage of the sugarcane cell wall proteome, and provide target proteins that could be used in future research to facilitate 2G ethanol production.

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites.

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus and Cortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatants of termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensional gel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.

High polymorphism in Est-SSR loci for cellulose synthase and ß-amylase of sugarcane varieties (Saccharum spp.) used by the industrial sector for ethanol production.

High and low polymorphisms in simple sequence repeats of expressed sequence tag (EST-SSR) for specific proteins and enzymes, such as ß-amylase, cellulose synthase, xyloglucan endotransglucosylase, fructose 1,6-bisphosphate aldolase, and fructose 1,6-bisphosphatase, were used to illustrate the genetic divergence within and between varieties of sugarcane (Saccharum spp.) and to guide the technological paths to optimize ethanol production from lignocellulose biomass. The varieties RB72454, RB867515, RB92579, and SP813250 on the second stage of cutting, all grown in the state of Paraná (PR), and the varieties RB92579 and SP813250 cultured in the PR state and in Northeastern Brazil, state of Pernambuco (PE), were analyzed using five EST-SSR primers for EstC66, EstC67, EstC68, EstC69, and EstC91 loci. Genetic divergence was evident in the EstC67 and EstC69 loci for ß-amylase and cellulose synthase, respectively, among the four sugarcane varieties. An extremely high level of genetic differentiation was also detected in the EstC67 locus from the RB82579 and SP813250 varieties cultured in the PR and PE states. High polymorphism in SSR of the cellulose synthase locus may explain the high variability of substrates used in pretreatment and enzymatic hydrolysis processes, which has been an obstacle to effective industrial adaptations.

Oligomerization, membrane association, and in vivo phosphorylation of sugarcane UDP-glucose pyrophosphorylase.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.

Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays.

Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp. (sugarcane) Hsp70, named SsHsp70 here, the purification of the recombinant protein and the characterization of its structural conformation in solution by chemical cross-linking coupled to mass spectrometry. The in vivo expression of SsHsp70 in sugarcane extracts was confirmed by Western blot. Recombinant SsHsp70 was monomeric, both ADP and ATP binding increased its stability and it was efficient in cooperating with co-chaperones: ATPase activity was stimulated by Hsp40s, and it aided the refolding of an unfolded polypeptide delivered by a member of the small Hsp family. The structural conformation results favor a model in which nucleotide-free SsHsp70 is highly dynamic and may fluctuate among different conformations that may resemble those in which nucleotide is bound. BIOLOGICAL SIGNIFICANCE: Validation of a sugarcane EST as a true mRNA that encodes a cytosolic Hsp70 (SsHsp70) as confirmed by in vivo expression and characterization of the structure and function of the recombinant protein. SsHsp70 was monomeric, both ADP and ATP binding increased its stability and was efficient in interacting and cooperating with co-chaperones to enhance ATPase activity and refold unfolded proteins. The conformation of nucleotide-free SsHsp70 in solution was much more dynamic than suggested by crystal structures of other Hsp70s. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Identification, characterisation and molecular modelling of two AP endonucleases from base excision repair pathway in sugarcane provide insights on the early evolution of green plants.

Unlike bacteria and mammals, plant DNA repair pathways are not well characterised, especially in monocots. The understanding of these processes in the plant cell is of major importance, since they may be directly involved in plant acclimation and adaptation to stressful environments. Hence, two sugarcane ESTs were identified as homologues of AP endonuclease from the base-excision repair pathway: ScARP1 and ScARP3. In order to understand their probable function and evolutionary origin, structural and phylogenetic studies were performed using bioinformatics approaches. The two predicted proteins present a considerable amino acid sequence similarity, and molecular modelling procedures indicate that both are functional, since the main structural motifs remain conserved. However, inspection of the sort signal regions on the full-length cDNAs indicated that these proteins have a distinct organelle target. Furthermore, variances in their promoter cis-element motifs were also found. Although the mRNA expression pattern was similar, there were significant differences in their expression levels. Taken together, these data raise the hypothesis that the ScARP is an example of a probable gene duplication event that occurred before monocotyledon/dicotyledon segregation, followed by a sub-functionalisation event in the Poaceae, leading to new intracellular targeting and different expression levels.

Water stress reveals differential antioxidant responses of tolerant and non-tolerant sugarcane genotypes.

The biochemical responses of the enzymatic antioxidant system of a drought-tolerant cultivar (IACSP 94-2094) and a commercial cultivar in Brazil (IACSP 95-5000) grown under two levels of soil water restriction (70% and 30% Soil Available Water Content) were investigated. IACSP 94-2094 exhibited one additional active superoxide dismutase (Cu/Zn-SOD VI) isoenzyme in comparison to IACSP 95-5000, possibly contributing to the heightened response of IACSP 94-2094 to the induced stress. The total glutathione reductase (GR) activity increased substantially in IACSP 94-2094 under conditions of severe water stress; however, the appearance of a new GR isoenzyme and the disappearance of another isoenzyme were found not to be related to the stress response because the cultivars from both treatment groups (control and water restrictions) exhibited identical changes. Catalase (CAT) activity seems to have a more direct role in H2O2 detoxification under water stress condition and the shift in isoenzymes in the tolerant cultivar might have contributed to this response, which may be dependent upon the location where the excessive H2O2 is being produced under stress. The improved performance of IACSP 94-2094 under drought stress was associated with a more efficient antioxidant system response, particularly under conditions of mild stress.