Thesis (MSc (Biochemistry))--University of Stellenbosch, 2006. / This thesis is about mathematical modelling of sucrose accumulation in the storage perenchyma of Saccharum officinarum (sugarcane) In 2001, Rohwer & Botha (76) published a kinetic model that described the defining feature of this process ...

Thesis (MSc (Botany and Zoology))--University of Stellenbosch, 2005. / The main substrate use during shoot establishment from the lateral bud of sugarcane setts and enzymes involved in sucrose metabolism were investigated in the shoots and the internodes acting as source of carbohydrates. Radiolabelling studies were conducted to investigate the metabolism of sucrose and glucose during shoot establishment. The internode’s total dry mass over the 21-day of shoot establishment period was reduced by 25% and 30% in plants incubated in dark/light and total darkness, respectively.

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The cellular expression of the enzymes implicated in regulating sucrose metabolism and accumulation in sugarcane is poorly understood. The present study was therefore aimed at the development of an in situ hybridisation (ISH) technique to study differential gene expression among the various cell types of the sugarcane culm. This technique in conjunction with northern and western blotting was then used to determine the sites of cellular and tissue specific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphate dependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrose metabolism. This study revealed that the determination of the influencing parameters associated with the development of an ISH protocol was essential for the successful detection of the endogenous RNA sequences in sugarcane internodal tissues. The parameters that were investigated included the type of embedding medium, duration of fixation period, pre-treatment procedures and hybridisation temperature. It further revealed that fresh internodal tissue sections, fixed for a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated the analysis of cytological gene expression at all stages of sugarcane development. The second part of this study revealed very localised transcript expression for UDP-Glc DH, PFP and UGPase in the different internodal tissue and cell types. The UDP-Glc DH and UGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressed the UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were all expressed in the parenchyma cells that were associated with the vascular bundles and the stem storage compartment, suggesting that the parenchyma cells distributed throughout the stem in the different tissue types complement each other in function for the purposes of phloem loading, unloading and assimilate transport processes. Complimentary northern and western hybridisations demonstrated that internode 7 represents a shift in the sink from utilisation to storage. This is evident by the observed decline in both the relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stage of development. The relative mRNA and protein abundances for the three enzymes showed a similar trend. Higher levels of the gene transcripts and translated products were observed in the younger sucrose importing tissues, than in the older sucrose accumulating internodes. At a cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPase expression differed marginally. Whilst UDP-Glc DH was expressed in the phloem, xylem and parenchyma cells of the vascular complex and in storage parenchyma cells, PFP was expressed exclusively in parenchyma cells that were associated with the vascular bundles and those serving a storage function in the stem pith and UGPase was found to be localised in the phloem and parenchyma of the vascular bundles and the storage parenchyma cells. Such findings have demonstrated an increase in resolution with which gene expression can be examined at a cellular level. Hence, the results from this study have demonstrated that the knowledge of metabolic compartmentation between different tissue and cell types is a requisite to understanding the function(s) of individual enzymes within complex structures such as the sugarcane culm. / AFRIKAANSE OPSOMMING: Die sellulêre lokalisering van die ensieme wat geïmpliseer word in die regulering van sukrose metabolisme is onbekend. Met dit in gedagte, was hierdie studie gefokus op die ontwikkeling van 'n in situ hibridisasie (ISH) tegniek om differensiële geenuitdrukking in die verskillende seltipes van die suikerrietstingel te ondersoek. Hierdie tegniek, tesame met RNA-en proteïen gel blots, is volgens aangewend om die areas van sellulêre-en weefselspesifieke uitdrukking van die sitosoliese ensieme UDP-glukose dehydrogenase, pirofosfaat-afhanklike fosfofruktokinase en UDP-glukose pirofosforilase, wat almal betrokke is by sukrosemetabolisme, te bepaal. Dit het duidelik geword gedurende die studie dat die bepaling van die optimale parameters van die ISH protokol vir suikerriet van deurslaggewende belang sou wees vir die opsporing van endogene RNA volgordes. Die parameters wat ondersoek is het ingesluit die tipe inbeddingsmedium, die tydsduur van fiksering, vooratbehandelings- en hibridisasiemetodes. Dit het duidelik geword dat vars internodale weefselsnitte wat vir 24 h gefikseer is en daarna voorafbehandeling en hibridisasie ondergaan het, die bepaling van geenuitdrukking tydens alle fases van suikkerrietontwikkeling moontlik gemaak het. Die tweede fase van hierdie studie het aangetoon dat al drie ensieme spesifiek gelokaliseerde uitdrukkingspatrone gehad het in verskillende internodale weefsels en seltipes. Al drie gene is konstitutief uitgedruk in internodes. Die UDP-glukose dehydrogenase en UDP-glukose pirofosforilase transkripte is gelokaliseer na die floeëm elemente, terwyl xileem slegs die UDP-glukose dehydrogenase transkripte bevat het. Al die gene is in die parenchiemselle uitgedruk wat geassosieer is met die vaatbondels en die stingel stoorkompartement, wat moontlik beteken dat die parenchiem selle wat deur die stingel versprei is 'n sentrale netwerk vorm wat direk of indirek koolstofassimileringsprosesse beïnvloed. RNA-en proteïen gel blots op dieselfde internodes het gewys dat internode sewe 'n verskuiwing, van koolstofverbruik na berging, verteenwoordig. Dit word gerllustreer deur die afname in beide transkrip en proteïen vlakke van die drie ensiem in hierdie stadium van ontwikkeling. Alhoewel beide mRNA en proteïen vlakke vir al die ensieme 'n soortgelyke tendens getoon het, het die sellulêre uitdrukking van die ensieme volgens ISH verskil, wat die krag van die tegniek illustreer. Die resultate van hierdie studie het gedemonstreer dat begrip van die kompartementalisasie van metabolisme tussen verskillende weefsel-en seltipes 'n voorvereiste is om die funksie/s van individuele ensieme in komplekse strukture soos die suikerrietstingel te bepaal.

No description available.

The fungus Fusarium sacchari (Butler) Gams causes stem rot in sugarcane especially in association with the stem borer Eldana saccharina Walker. Sugarcane plants tolerant to F. sacchari PNG40 were obtained by chemical mutagenesis and in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF), followed by selection in the greenhouse. Somaclonal variants tolerant to F. sacchari PNG40 CF were established by treatment of calli with ethyl methanesulphonate (EMS) and various selection treatments. Investigations were conducted to test the effect of varying CF concentrations and the culture developmental stages (embryo maturation, embryo germination and plantlets) that were most effective in screening calli and plants. Incorporation of CF (0-100 ppm) in the media, at either embryo maturation or germination stages, resulted in significant callus necrosis, and consequent decreased plantlet yield. The highest callus necrosis of 95.55 ± 0.9 % and the lowest plant yield of 1.4 ± 0.45 plants/0.2 g were obtained after inclusion of 100 ppm CF in the germination medium compared with 61.5 ± 3.8 % and 43.8 ± 5.6 plants/0.2 g in the maturation medium, respectively. Exposure of whole plants with trimmed roots to 0-1500 ppm CF resulted in inhibition of root re-growth, with the 1500 ppm CF treatment having the greatest negative effect. Subsequent treatments involved immersing in vitro plantlets in varying concentrations of F. sacchari conidial suspensions. This resulted in 33.3 % and 100 % mortality with 103 and 105 conidia/ml treatments, respectively. Control and EMS-treated calli and potentially tolerant regenerated plants were selected using the established CF and inoculation treatments. Plants from EMS treatments displayed more varying root length. More plants with increased root growth, in the presence of CF, were produced from these treatments than from non-EMS treatments, indicating the ability of EMS to induce somaclonal variation. These putative tolerant plants were inoculated with PNG40 and those selected using CF in vitro were symptomless whilst the positive controls (plants unexposed to CF) were symptomatic. Re-isolation of Fusarium from the inoculated plants and identifying isolates as PNG40 using ISSR analysis confirmed tolerance of the asymptomatic plants and the fungus as the causal agent of the observed symptoms. This confirmed that tolerance to CF correlates to tolerance to F. sacchari PNG40. Future work includes testing stability of tolerance in the field and after sexual reproduction, and use of this protocol to produce plants that permit endophytic PNG40 colonisation towards biological control of E. saccharina. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Two cationic antimicrobial peptides, ESF I-GR7, and pleurocidin, were assessed for their stability in plant intercellular fluid, the targeted locale for their expression in transgenic plants. Incubation of ESFI-GR7 and pleurocidin with intercellular fluid (ICF) extracted from sugarcane, tomato and tobacco leaves reduced their biotoxicity towards various pathogens, namely Camobacterium mobile DMSO and Xanthomollas campestris. It was concluded that it may be necessary to modify the aminoacid structures of the peptides in order to ensure that endogenous proteases would not degrade the peptides once expressed in a transgenic environment. The presence of pleurocidin was detected in transgenic sugarcane transformed (in a previous study) with pleurocidin gene cloned into the pUBI 510 plasmid. ICF was extracted from four month old transgenic Saccharum hybrid species (sugarcane). Western blotting verified the presence of the transgenic protein in crude protein extracts. Immunogold labelling and transmission electron microscopy were performed to investigate the localisation of transgenic pleurocidin. The peptide was localized predominantly in the intercellular spaces and cell wall sugarcane leaves. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

As gramíneas são o mais importante grupo de plantas para em todo o mundo. Inúmeras são usadas para a alimentação dos animais, além de serem utilizadas para a produção de biocombustíveis, contribuindo para a redução do consumo de combustíveis fósseis e consequentemente da poluição ambiental. A biomassa vegetal é constituída basicamente por celulose, hemicelulose e lignina. Estudos na área de bioquímica e genética tem demonstrado a lignina como um dos principais compostos responsáveis pela recalcitrância da biomassa, sendo a resistência à digestão enzimática uma importante limitação do processo de produção de bioetanol. A lignina é um polímero vegetal resultantes da polimerização desidrogenativa de três monômeros de fenilpropanóides primários, os álcoois p-coumarilico (H), coniferilico (G) e sinapilico (S). A via metabólica dos fenilpropanóides envolve a participação de 11 enzimas, nas gramíneas, tais como PAL, C4H, 4CL, C3H, F5H, CCoAOMT, CSE, COMT, HCT, CCR e CAD. Até o momento, 28 genes de cana-de-açúcar foram identificados no SUCEST e explorados para identificar os possíveis bona fide. No entanto, com a disponibilização de bancos transcriptômicos (RNASeq) e ferramentas avançadas de bioinformática, o nosso grupo de pesquisa expandiu para 37 genes após o processo de identificação, anotação e classificação pelas análises filogenéticas. Diante disso, a presente proposta teve por objetivo realizar a análise de expressão destes transcritos anotados em uma variedade de cana-de-açúcar com a composição química da parede celular caracterizada. Para identificar quais genes estão envolvidos nesta via, o presente trabalho propôs correlacionar o perfil de expressão com os quatro estágios de desenvolvimento do entrenó, separando córtex e medula, os quais estão associados aos estágios de lignificação em maior e menor grau, respectivamente. Dentre as análises realizadas, 34 transcritos tiveram os primers padronizados , 26 foram expressos no colmo nas condições testadas, sendo 7 genes adicionais aos previamente descritos na literatura. Baseado nas análises de expressão gênica, PAL1.1/1.2, COMT1, CAD2/5/8, 4CL2, F5H1, CCoAMOT1.2/2.1 e CCR1 foram apontados como possíveis candidatos principais na via de biossíntese da lignina. / Sugarcane (Saccharum spp.) is a grass of great importance for the sustainable technological and agroindustrial development of Brazil. The cell wall of the grasses is basically composed of cellulose, hemicellulose, lignin and hydroxycinnamic acids. The study of the chemical composition of the cell wall of different sugarcane hybrids revealed that low lignin content, in particular for H89 (~ 16.8%), contribute to the low recalcitrance of the biomass. These studies obtained greater information when the regions of rind and pith of H89 were dissected, which present, respectively, 21.7% and 13.9% of total lignin. Lignin is a plant polymer resulting from the dehydrogenative polymerization of three primary phenylpropanoic monomers, the p-coumaril (H), coniferyl (G) and synapylic (S) alcohols. The metabolic pathway of the phenylpropanoids involves the participation of several enzymes, totalizing in the grasses 11 sets, such as PAL, C4H, 4CL, C3H, F5H, CCoAOMT, CSE, COMT, HCT, CCR and CAD. So far, 28 unigenes of sugarcane have been identified in SUCEST and explored to identify the possible bona fide. Recently, based on bioinformatics, phylogenetic and transcriptomic (RNA-Seq) analyzes of sugarcane and related grasses, our research group identified 37 transcripts possibly involved in monolignol biosynthesis. In the presence of detailed descriptions of the composition of lignin in H89 and of the large number of genes identified, the presente proposal aimed to perform the analysis of the expression of these transcripts annotated in H89. In order to amplify and reinforce which genes are involved in the monolignol pathway, the present work proposed to correlate the expression profile with four stages of the development of the enternode, exploring the regions of rind and pith. Among the analyzes performed, 34 genes were identified, 26 genes were expressed, being 7 additional genes to those previously described in the literature. In this context, PAL1.1 / 1.2, COMT1, CAD2 / 5/8, 4CL2, F5H1, CCoAMOT1.2 / 2.1 and CCR1 were identified as potential candidates in the lignin biosynthesis pathway.

Orientador: Cecília Laluce / Coorientador: Karen F. de Oliveira. / Banca: Sandra Regina Pombeiro Sponchiado / Banca: Nilce maria Martinez Rossi / Resumo: A biomassa lignocelulósica contém quantidades significativas de fontes de carbono, sendo assim uma fonte de energia renovável. O presente trabalho teve como objetivo estabelecer meios líquidos e sólidos para o estudo dos efeitos dos inibidores presentes nos hidrolisados do bagaço de cana-de-açúcar e, por fim, estabelecer um coquetel de inibidores que permitisse o crescimento e a fermentação da levedura Saccharomyces cerevisiae no meio líquido estabelecido. Por esta razão, o conjunto de resultados deste trabalho compreende três partes. A primeira parte foi dedicada ao estabelecimento de meios sólidos e líquidos para comparação de leveduras na presença do inibidor mais abundante do hidrolisado do bagaço de cana, o ácido acético. No meio sólido YPD, as diferentes linhagens de leveduras ensaiadas sofreram menos os efeitos inibitórios deste ácido do que o meio sintético solidificado. Em meio líquido, foi necessário adicionar 2% de extrato de levedura para ativar o crescimento da levedura 63M, mas este efeito foi suprimido em presença de ácido acético. As variações em pH inicial (3,5 - 5,5) afetaram mais a produção de etanol do que a biomassa, ao passo que a viabilidade não variou. A segunda parte deste trabalho foi dedicada ao estabelecimento de um meio sintético que permitisse o crescimento e fermentação da linhagem 63M em presença de ácido acético. O aumento de 33% em biomassa na presença de 83 mmol/L de ácido acético foi devido à elevação da glicose (10-18%) e do inóculo (5-10 mg/mL). As concentrações crescentes e variadas de ácido acético em meio sintético foi utilizado para estudar seus efeitos sobre o crescimento (inibição acima de 50 mmol/L), a viabilidade (acima de 250 mmol/L) e a produção de etanol (acima de 83 mmol/L). As concentrações de outros inibidores... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Lignocellulosic biomass contains significant quantities of carbon sources, being a renewable energy source. The aim of the present work was to determine liquid and solid media for the study of the effects of inhibitors present in hydrolysates of sugarcane bagasse, and to establish a cocktail of inhibitors that allow the growth and the fermentation of the yeast Saccharomyces cerevisiae in the liquid medium established. Thus, the results of this work consist of three parts. The first part was dedicated to establishing solid and liquid media in order to compare different yeast strains in the presence of the most abundant inhibitor found in hydrolysates of sugarcane bagasse, the acetic acid. On YPD solid medium, this acid had less inhibitory effect on the different yeast strains tested than on the synthetic solid medium. In liquid medium, it was necessary to add 2% yeast extract to activate the growth of the strain 63M, but this effect was abolished in the presence of acetic acid. Variations in the initial pH (3.5 - 5.5) affected more the ethanol production than the biomass, while the viability was not influenced. The second part of this work was dedicated to establishing a synthetic medium that allows the growth and fermentation of the strain 63M in the presence of acetic acid. The 33% increase in biomass in the presence of 83 mmol/L acetic acid was due to the high glucose concentration (10-18%) and inoculum (5-10 mg/mL) used. The increasing concentrations of acetic acid in the synthetic medium were used to study its effects on growth (inhibition above 50 mmol/L), viability (over 250 mmol/L) and ethanol production (above 83 mmol/L). The concentrations of the other inhibitors had different limits of tolerance by the yeast... (Complete abstract click electronic access below) / Mestre

Este estudo visou quantificar os teores de fosfato total, inorgânico e orgânico no caldo proveniente de diferentes alturas (região basal, mediana e apical) do colmo de três variedades de cana-de-açúcar, além de buscar levantar a ocorrência de myo-inositol hexafosfato (ácido fítico) em colmos de cana-de-açúcar. A composição química e tecnológica de colmos de cana-de-açúcar está relacionada com as características genéticas da variedade. Os colmos da cana-de-açúcar são compostos de fibra e caldo absoluto. O caldo contém cerca de 25% de sólidos solúveis, dos quais a sacarose se destaca. Na área agrícola o elemento fósforo é importante principalmente no metabolismo fotossintético da planta. No caldo de cana-de-açúcar se encontra nas formas mineral e orgânica, exercendo uma função chave no processo de clarificação do caldo para produção de açúcar, sendo também importante nos processos de fermentação alcoólica. Em grãos de cereais e leguminosas, estudos com o elemento fósforo, ou mais especificamente o myo-inositol hexafosfato (ácido fítico), têm mostrado algumas características antinutricionais, antioxidantes e anticarcinogênicas, que o ácido fítico possui quando complexado com certos cátions. Foram utilizadas as variedades SP 82-3530, SP 83-5073 e RB 83-5486 de cana-de-açúcar para as análises de fosfatos, sendo os colmos das mesmas divididos em região basal (3/8), mediana (3/8) e apical (2/8). Para as análises de ácido fítico foi utilizada apenas a variedade SP 82-3530, sendo os colmos desta analisados integralmente, com e sem casca, e divididos em nós e internódios com e sem casca. O caldo dos colmos de cana foi extraído em prensa hidráulica. As variedades apresentaram teores que variaram de 370 a 936 mg P2O5 total por litro de caldo. As regiões do colmo apresentaram valores decrescentes para os três tipos de fosfato da base para o ápice do colmo. O fosfato orgânico representou aproximadamente 5% do total. As porcentagens dos teores de fosfato orgânico em relação ao total, apresentaram um gradiente crescente da região basal para a apical. A análise de ácido fítico, por este ter apresentado teores insignificantes no caldo e na cana, ficou prejudicada pela metodologia aplicada, a qual se mostrou inadequada. / The objective of the present study was to estimate total, inorganic and organic phosphate amounts in the juice of stalk segments (basal, middle and top) of three sugarcane varieties, as well as assessing the presence of myo-inositol 6-phosphate (phytic acid) in sugarcane stalks. The chemical and technological compositions of sugarcane stalks are linked to the genetic characteristics of each variety. Sugarcane stalks are composed of fiber and absolute juice. The juice contains about 25% of soluble solids, mainly sucrose. Phosphorus is an important element in agriculture, due to its participation in the photosynthetic metabolism of plants. Phosphorus is found in its mineral and organic forms in the sugarcane juice and plays a key role in the clarification and fermentation processes for the production of sugar and alcohol. Studies on phosphorus, or rather, on myo-inositol 6-phosphate (phytic acid), in cereal and leguminous grains have evidenced some anti-nutritious, antioxidant and anticarcinogenic characteristics of the phytic acid when complexed with certain cations. Basal (3/8), middle (3/8) and top (2/8) stalk segments from SP 82-3530, SP 83-5073 and RB 83-5486 sugarcane varieties were analyzed for their phosphate amounts. Only stalk segments from the variety SP 82-3530, separated in nodes and internodes, peeled or not, were analyzed for phytic acid amounts. The juice of sugarcane stalks was extracted on hydraulic press. Total P2O5 varied from 370 to 936 mg per liter of juice. The three types of phosphate showed decreasing amounts from the bottom to the top of the stalk. Organic phosphate constituted approximately 5% of total phosphate. Percentages of organic phosphate in relation to total phosphate amounts increased gradually from the basal to the top stalk segments. As phytic acid was found in insignificant amounts, the assessment of its presence was limited by the methodology used, which showed to be inappropriate.

Na região de Piracicaba, grande parte dos pequenos produtores cultivam cana-de-açúcar em áreas de alta declividade, mas a mecanização da colheita tem inviabilizado seu cultivo nesta condição. Sistemas agroflorestais (SAFs) podem ompatibilizar a produção da cultura neste cenário, com a conservação de recursos naturais, porém, há falta de pesquisa sobre seu cultivo nestes sistemas. Portanto, o uso de modelos de simulação e avaliações de campo da interface árvore-cultura podem desempenhar importante papel para investigar seus potenciais e restrições. Assim, identificamos e selecionamos terras e árvores para cultivo dos SAFs e avaliamos o crescimento e produção de plantas dos SAFs cana-de-açúcar x seringueira e cana-de-açúcar x eucalipto. Realizamos dois experimentos de campo e utilizamos o modelo WaNuLCAS e um modelo que simula a disponibilidade de radiação para culturas intercalares, acionando-o com a produção de matéria seca da cultura. SAFs em aléias em contorno são alternativa viável para cultivo de cana-de-açúcar no município de Piracicaba, havendo diversas espécies arbóreas exóticas e nativas que podem compor estes SAFs, destacando-se seringueira e eucalipto. Luz e água são limitantes ao crescimento da cultura e há aumento da competição de acordo com o crescimento das árvores. A importância relativa entre competição abaixo e acima do solo depende da distância das árvores. Contudo, o SAF cana-de-açúcar x eucalipto tem produção semelhante ao monocultivo de cada espécie. Os modelos permitiram avaliar os SAFs e orientar experimentos, mas suas indicações devem ser verificadas em experimentos de campo. A recomendação dos SAFs deve ser feita após análise econômica dos mesmos. / In Piracicaba, many smallholders grow sugarcane in areas of high solpe, but the mechanization of the harvest process has made inviable its cultivation under this situation. Agroforestry systems (AFs) are an alternative that may join the cultivation of the crop under this scenario with conservation of natural resources. However, there is lack of research of its cultivation in AFs. Therefore, the use of simulation models and evaluation of on-farm tree-crop interfaces may play an important role to investigate its potentials and restrictions. Thus, we identified and selected lands and trees to the cultivation of the AFs and assessed plant growth and yield of sugarcane x rubber and sugarcane x eucalyptus AFs. We did two field experiments and used the WaNuLCAS model and another one that simulates the available irradiance for intercrops, related with their dry matter production. Contourhedgerows are a feasible alternative for sugarcane cultivation in Piracicaba, where many tree species may be part of it, especially rubber and eucalypts. Light and water are limiting factors for crop growth and competition increases with tree growth. The relative importance of each variable depends on the distance from the trees. However, the sugarcane x eucalyptus AF has similar production of the monocropping of each specie. The models allowed to assess the AFs and to drive field trials, but their results should be verified in experiments. The recomendation of the AFs should be done after their economic analysis.