Akademisk avhandling--Lund. / Extra t.p., with thesis statement, inserted. Bibliography: p. 11-14.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Sugarcane is a tropical perennial grass species belonging to the Poaceae (true grasses) family. Mature sugarcane is comprised mostly of sugarcane stalks, which accumulate high amounts of sucrose, a fact that has led to its wide cultivation of sugarcane for sucrose production. Sugar yields from sugarcane have been improved in the past by either creating transgenic sugarcane or through using traditional breeding methods. Increasing sugar yields in sugarcane is still of interest and new cisgenic strategies are being considered to alleviate consumer concerns over transgenic plants. This thesis consists of two parts. The first was aimed at understanding the relation between trehalose-6-phosphate (T6P) synthesis and sucrose accumulation in sugarcane. In this study the E. coli genes involved in trehalose synthesis, otsA and otsB, were overexpressed in sugarcane in order to observe their effects on soluble sugar accumulation. Nine otsA and two otsB overexpressing lines were created, confirmed by gDNA insertion PCRs, sq-RT-PCR and immuno detection of encoded enzymes. Preliminary measurements of soluble sugars showed that four out of the nine otsA lines had significantly decreased and one line significantly increased sucrose concentrations. Correlating sq-RT-PCR results with soluble sugar measurements suggest that trehalose-6-phosphate synthase (TPS) expression affects sucrose levels in sugarcane, but further research of TPS activity is required before a conclusion can be reached. Further analysis of mature cane material in regard to relevant enzyme levels, carbohydrate levels and gene expression should contribute to more conclusive results. Three novel sugarcane TPS encoding sequences were isolated and proven to be functional through complementation of the growth defect in tps1Δ yeast grown on glucose as a carbon source. Sugarcane TPS isoforms named SoTPSa, SoTPSb and SoTPSc, were isolated by successful application of 5‟ RACE alongside standard PCR using primers based on other monocotyledonous TPS sequences. The encoded SoTPSa contains a 25 amino acid insertion within the partial TPP domain. The encoded SoTPSc contains a 126 amino acid long N terminal truncation, which removes one of the thirteen amino acids found within the active site of the TPS domain. Future characterization of the encoded enzymes will determine the effects of these modifications on TPS activity. The second part of this thesis describes initial efforts made in attempting to develop a cisgenic in vitro selectable marker system for sugarcane, S. officinarum callus, which uses a diphenylether type (DPE) herbicide as a selection agent and a sugarcane protoporphyrinogen oxidase (PPO) gene as a selection marker. Firstly the plastid targeted PPO from tobacco (NtPPO-1) was isolated and mutagenized, to mimic the double mutated Arabidopsis PPO, used by Li et al., (2003) in maize. However, sugarcane calli transformed with the double mutated NtPPO-1 and grown on media containing fomesafen herbicide, were incapable of regenerating. Future efforts will utilize a N-terminal sequence that is targeted to the plastid organelle, so as to ensure translocation of the enzyme to that subcellular location. Also, random mutations were induced in the NtPPO-1 gene to screen for mutations that confer DPE herbicide resistance, however this work is currently on hold until a heme deficient E. coli can be obtained. Secondly, attempts were made to isolate a putative sugarcane plastid targeted PPO gene, so as to eventually use this in developing a cisgenic strategy. 5‟ RACE was successful in revealing additional nucleotide sequence adding 1006 bp to the already known partial sugarcane PPO sequence. However the fragment isolated was still a partial sequence. / AFRIKAANSE OPSOMMING: Suikerriet is 'n tropiese meerjarige gras spesie wat deel is van die Poaceae (ware grasse) familie. Volwasse suikerriet bestaan hoofsaaklik uit suikerrietstamme, wat hoë hoeveelhede sukrose akkumuleer, 'n feit wat gelei het tot die wye verbouing van suikerriet vir sukrose produksie. In die verlede is suikeropbrengste vanuit suikerriet verbeter deur die skep van transgeniese suikerriet óf die gebruik van tradisionele teelmetodes. Toenemende suiker opbrengste in suikerriet is steeds van belang en nuwe cisgeniese strategieë word oorweeg om verbruikerskommer oor transgeniese plante te akkommodeer. Hierdie tesis bestaan uit twee dele. Die eerste deel is daarop gemik om die begrip van die verhouding tussen trehalose-6-fosfaat (T6P) sintese en sukrose ophoping in suikerriet te verstaan. In hierdie studie is die E. coli gene wat betrokke is in trehalose sintese, otsA en otsB, ooruitgedruk in suikerriet ten einde die uitwerking daarvan in die opgaar van oplosbare suiker te bestudeer. Nege otsA en twee otsB verhoogte uitdrukkings lyne is geskep, bevestig deur gDNA bygevoegde PKR, sq-RT-PKR en immuno opsporing van geïnkripteerde ensieme. Voorlopige metings van oplosbare suikers toon dat vier van die nege otsA lyne ʼn beduidende afname in sukrose vlakke en een lyn „n beduidende toegeneem in sukrose vlakke getoon het. Korrelerende sq-RT-PKR resultate met oplosbare suikermetings dui daarop dat trehalose-6- fosfaat sintese (TPS) geenuitdrukking sukrose vlakke sal affekteer, maar verdere navorsing van TPS aktiwiteit is nodig voordat 'n gevolgtrekking gemaak kan word. Verdere ontleding van volwasse riet materiaal met betrekking tot relevante ensiem vlakke, koolhidrate vlakke en geenuitdrukking, behoort by te dra tot meer volledige resultate. In hierdie studie is drie nuwe suikerriet TPS gene geïsoleer en dit is bewys as funksioneel deur die komplimentering van die groeidefek van tps1Δ gis, gegroei op glukose as 'n koolstof bron. Suikerriet TPS isoforme, genoem SoTPSa, SoTPSb en SoTPSc, is geïsoleer deur die suksesvolle toepassing van 5 'RACE, in kombinasie met standaard PKR, deur van spesiaal ontwerpte primers, gebaseer op ander eensaadlobbige TPS gene, gebruik te maak. Die gekodeerde SoTPSa bevat 'n 25 aminosuur invoeging binne-in die gedeeltelike TPP domein. Die gekodeerde SoTPSc bevat 'n 126 aminosuur lange N terminaal afkapping, wat een van die dertien aminosure binne die aktiewe terrein van die TPS domein verwyder. Toekomstige karakterisering van hierdie geïnkripteerde ensiemes sal die effek van hierdie veranderinge op TPS aktiwiteit bepaal. Die tweede deel van hierdie tesis beskryf die aanvanklike probeerslae wat gemaak is in 'n poging om „n cisgeniese in vitro selekteerbare merker vir suikerriet, S. officinarum kallus te ontwikkel. Hierin word gebruik gemaak van 'n difenylether tipe (DPE) onkruiddoder as 'n seleksie agent, en 'n suikerriet protoporphyrinogen oksidase (PPO) geen as 'n seleksie merker. In 'n poging om dit te bewerkstellig is daar eerstens plastied geteikende PPO van tabak (NtPPO-1) geïsoleer en geteikende mutagenese suksesvol daarop uitgevoer. Mutasies wat geinduseer is, is gegrond op die dubbele gemuteerde Arabidopsis PPO, wat gebruik was in mielies deur Li et al., (2003). Alhoewel die suikerriet kallus getransformeer is met die dubbele gemuteerde NtPPO-1 konstruk en geselekteer is op media wat fomesafen onkruiddoder bevat, was die kallus nie in staat om te regenereer nie. In toekomstige pogings sal probeer word om 'n N-terminale volgorde, geteiken op „n plastied organel, te benut sodat translokasie van die ensiem aan die plastied organel verseker kan word. So ook is toevallige mutasies veroorsaak in die NtPPO-1 gene om te soek vir nuwe mutasies wat DPE onkruiddoderweerstand verleen, maar hierdie werk is tans gestop totdat 'n heem gebrekkige E. coli mutant verkry kan word. Tweedens, is pogings aangewend om 'n vermeende suikerriet plastied geteikende PPO gene te isoleer, om uiteindelik te gebruik in die ontwikkeling van 'n cisgeniese strategie in suikeriet. 5 'RACE was suksesvol in die onthulling van bykomende nukleotiede volgorde deur 1006 bp by te voeg by die reeds bekende gedeeltelike suikerriet PPO fragment. Nie teenstaande is die fragment wat nuut geïsoleer is, steeds slegs 'n gedeeltelike volgorde volgens vergelykings met ander bekende plant PPO gene.

Made available in DSpace on 2015-03-26T13:36:46Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3795682 bytes, checksum: 12bec11cea96f50faf5ddfa5a06024dc (MD5) Previous issue date: 2007-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The soybean SBP2 (sucrose binding protein) promoter is capable to drive vascular tissue-specific expression of reporter genes in tobacco transgenic lines. This vascular-specific activity of the SBP2 promoter is confined to a distal region (-2000 to -700 sequences) designated CRD-A (cis-regulatory domain-A). Here, we first confirmed the tissue-specific activity of CRD-A through gain-of-function experiments, in which the CRD-A sequences were directly fused to 5´end of GUS cDNA, -136pSBP2-GUS and -92pSBP2-GUS constructs. In tobacco, CRD-A was able to reduce GUS activity in all organs analyzed, recapitulating in some cases the tissue-specific pattern of the full promoter. In addition, CRD-A promoted GUS transcription in the absence of the proximal TATA-containing region, which suggests that CRD-A may contain cis-regulatory elements to sustain basal transcription. In fact, this region (-2000 a -700) harbors several TATA box-like sequences, positions -790, -783 and -761, that potentially may function as alternative TATA boxes. To delimit the cis-regulatory elements responsible for the tissue-specific activity of SBP2 promoter, the -2000 to -700 sequence was divided into five fragments, which were fused to -92pSBP2-GUS construct and used to obtain transgenic lines. Histochemical analysis revealed that all the CRDA sub-fragments reduced the SBP2 promoter activity, as their fusion to the 5 end of -92pSBP2 altered its constitutive expression pattern. These results identified the presence of several potentially cis-regulatory domains. The region encompassing the sequences -1765 to -945 may contain strong shoot apex expression-repressing elements, capable to totally abolish expression, whereas the region -944 to -705 may harbor weaker repressing elements that restricted GUS expression to the vascular tissue. We also found several root expression silencers, operating in the root meristem (-1765 to -705) and in the root elongation zone (-1765 to -1485 and -1211 to -945). Furthermore, the region delimited by positions -1765 to -1485 also exhibited a strong root expressionrepressing element whose effect may be attenuated by cis-regulatory elements present in the -1485 to -705 region. Finally, a cis-element that confines GUS expression to the inner phloem of stem was identified in the region delimited by positions -1485 to -1212. The function of the identified cis-elements was evaluated through electrophoretic mobility shift assay (EMSA) that revealed sequence-specific interactions between putative transfactors from soybean and tobacco nuclear extracts and the -1765/-1485 (fragII) fragment from GmSBP2. To determine whether the SBP2 protein accumulation correlated with the tissuespecific promoter activity, a SBP2-GFP fusion was expressed in tobacco transgenic lines under the control of SBP2 promoter. Fluorescence analysis revealed that the SBP2 protein was, indeed, located in the vascular tissue, which was consistent with SBP2 promoter activity and the involvement of SBP2 in physiological process dependent of sucrose translocation. / O promotor do gene SBP2 (sucrose binding protein) de soja é capaz de dirigir a expressão tecido vascular-específica de genes repórteres em plantas transgênicas de tabaco. Esta regulação se deve à presença de domínios cisregulatórios distais (CRD-A, posição -2000 a -700) presentes no promotor. Neste trabalho, a atividade tecido-específica de CRD-A foi confirmada por meio de experimentos de ganho-de-função, nos quais o fragmento CRD-A foi diretamente fusionado ao gene repórter GUS e às construções -136pSBP2-GUS e -92pSBP2-GUS e sua atividade avaliada no sistema heterólogo de tabaco. CRDA foi capaz de reduzir a atividade de GUS em todos os órgãos analisados, restaurando, em alguns casos, o padrão tecido-específico do promotor completo. Além disso, observou-se que CRD-A é capaz de promover a transcrição de GUS, independente de promotor mínimo, indicando a presença de cis-elementos capazes de promoverem a transcrição basal. De fato, nessa região (-2000 a -700) foram identificados vários elementos TATA box, localizados nas posições -790, -783 e -761, que podem potencialmente funcionar como TATA boxes alternativos. No intuito de delimitar os cis-elementos responsáveis pelo padrão tecido-específico do promotor SBP2, a seqüência -2000 a -700 foi dividida em cinco fragmentos, os quais foram inseridos na construção -92pSBP2-GUS, e utilizados para obtenção de plantas transgênicas. Análises histoquímicas revelaram que todos os fragmentos foram capazes de reduzir a atividade do promotor SBP2, uma vez que sua inserção na extremidade 5 de -92pSBP2 alterou o padrão de expressão constitutiva do mesmo. Com base nestes resultados, diversas regiões potencialmente regulatórias foram identificadas. A região compreendida entre -1765 e -945 deve conter fortes elementos repressores para o ápice caulinar, capazes de abolir totalmente a atividade do promotor, enquanto que a região entre -944 e -705 demonstrou conter elementos repressores mais fracos, que restringiram a expressão ao tecido vascular. Foram encontrados vários elementos silenciadores para a raiz, tanto para o meristema radicular (região entre -1765 e -705), quanto para a zona de alongamento (de -1765 a -1485 e de -1211 a -945). Além disso, a região de -1765 a -1485 também apresenta um forte repressor para raiz, cujo efeito deve ser atenuado por ciselementos presentes entre -1485 e -705. Por fim, foi identificado um elemento responsável por restringir a expressão apenas ao floema interno no caule, na região entre -1485 e -1212. A funcionalidade dos cis-elementos identificados foi avaliada através do ensaio de mudança na mobilidade eletroforética (EMSA), tendo sido observada a interação seqüência-específica entre possíveis transfatores presentes em extratos nucleares de soja e de tabaco e o fragmento -1765/-1485 (fragII) de GmSBP2. A fim de verificar se o acúmulo da proteína SBP2 correlaciona-se com a atividade do promotor em tecidos específicos, foi obtida a proteína quimérica SBP2-GFP, sob o controle do promotor SBP2, em tabacos transgênicos. A análise de fluorescência revelou que a proteína SBP2 está, de fato, localizada na região de tecido vascular, consistente com o padrão de atividade do gene repórter e com seu envolvimento nos processos fisiológicos dependentes de translocação de sacarose.

Orientador: Paulo Mazzafera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T10:40:28Z (GMT). No. of bitstreams: 1 Geromel_Clara_D.pdf: 2807679 bytes, checksum: 87559525feb767ee465302f9b3380513 (MD5) Previous issue date: 2006 / Resumo: Sabendo-se que a produtividade da cultura de café está diretamente ligada a três fatores básicos de produção, climáticos, genéticos e fisiológicos, nesse estudo foram abordados alguns aspectos do metabolismo de carboidratos envolvidos no processo de enchimento dos frutos de café ao longo do seu desenvolvimento. A composição de carboidratos, principalmente de polissacarídeos do grão (endosperma) de café é conhecida, assim como a importância dos açúcares sobre a qualidade da bebida, porém a importação de sacarose a partir de folhas e seu desdobramento nos frutos ainda não são completamente esclarecidos. Os açúcares utilizados no metabolismo das sementes são de extrema importância, tendo como exemplo a regulação da relação fonte-dreno, além de controlar a expressão de genes que codificam algumas enzimas envolvidas no metabolismo de açúcares. O trabalho teve como principal objetivo estudar o metabolismo de sacarose nos frutos de café, ao longo do desenvolvimento. Análises histológicas e fornecimento de compostos marcados mostraram que não existem conexões vasculares entre os tecidos, pericarpo,perisperma e endosperma, mas vasos condutores que percorrem o funículo chegam até o perisperma, permitindo um descarregamento direto de fotoassimilados produzidos nas folhas e dele a transferência para o endosperma, além de receber fotoassimilados do pericarpo, pelo transporte de célula a célula. A fotossíntese no pericarpo diminui ao longo do desenvolvimento do fruto. Grãos de amido foram observados nos estados jovens do perisperma e à medida que surge o endosperma, esses grãos vão desaparecendo em regiões próximas ao tecido que está sendo formado. As enzimas e os teores de açúcares endógenos avaliados em tecidos separados ao longo da maturação do fruto apresentaram diferenças entre diferentes tratamentos da lavoura (¿pleno sol¿: condições normais de cultivo, sombreamento e carga reduzida). A sacarose sintase (SUS) apresentou valores de atividades bem maiores que das invertases ácidas (IAV). A sacarose fosfato sintase (SPS) acompanhou o acúmulo de sacarose no estádio inicial no pericarpo de frutos de café. Foi mostrada a existência de duas isoformas de SUS, codificadas por dos genes chamados CaSUS1 e CaSUS2 em C. arabica, que possivelmente desempenham diferentes funções metabólicas nos diferentes tecidos do fruto de café. Esses genes apresentam uma expressão diferencial, com CaSUS1 expressando-se nas fases jovens do desenvolvimento do perisperma e do endosperma; porém a expressão de CaSUS2 sobrepõe os picos de atividade SUS detectados e o acúmulo de sacarose nos estados finais de desenvolvimento do pericarpo e do endosperma. Isso sugere que a isoforma CaSUS1 está relacionada à degradação de sacarose, quando parece claro que a isoforma CaSUS2 está relacionada com a síntese desse açúcar. Foi mostrado que esses dois genes se expressam também in C. racemosa, pois as sondas CaSUS1 e CaSUS2 de C. arabica reconheceram transcritos (mRNA) no endosperma dessa espécie. Portanto, os genes CrSUS1 e CrSUS2 parecem codificar para isoformas de SUS que desempenham funções diferentes daquelas observadas em C. arábica; SUS1 parece estar relacionado com a síntese da sacarose. O sombreamento influenciou na duração do ciclo de desenvolvimento dos frutos, tornando-o mais longo que no pleno sol (respectivamente 260 e 231 dias após o florescimento) e alterando o acúmulo de sacarose nos frutos. Com base nesses resultados fica clara a complexidade do metabolismo de sacarose em frutos de café, visto que nem sempre as atividades enzimáticas seguem o mesmo padrão de acúmulo de açúcares e enzimas de degradação e ressíntese de sacarose atuam simultaneamente, assim como a transferência de açúcares entre os tecidos / Abstract: Since coffee culture productivity is straightly connected to three basic production factors: climate, genetics and physiology, herein some aspects of the carbohydrates metabolism involved in the process of coffee grains filling through its development were analyzed. It is known the carbohydrates composition, mainly the polysaccharides composition of the coffee grain (endosperm) as well as the importance of sugars in the beverage quality, nevertheless the sucrose import from the leaves and its sharing in the fruits are still to be completely clarified. The sugars utilized in the seeds metabolism are extremely important, such as in the regulation of the drain-source relation and in the expression control of genes that codify some enzymes involved in sugars metabolism. The main goal of this work was to study the sucrose metabolism in coffee fruits through their development. Histological analyses and marked compounds supply showed that there are no vascular connections among the tissues of the pericarp, perisperm and endosperm, but conduction vases that run through the funiculus get to the perisperm, enabling an unloading of photoassimilates produced in the leaves. From the perisperm, these assimilates are transferred to the endosperm; The pericarp photosynthesis diminishes through the fruit development. Starch grains were observed in juvenile stages of the perisperm and during the endosperm formation these grains start to disappear in regions close to the forming tissue. The enzymes and the endogenous sugar level evaluated in separated tissues through fruit maturation show some differences among distinct lavoura treatments (¿full sun¿: ordinary culture conditions, shadowing and reduced loading). The sucrose synthase (SUS) showed activity values much higher than the ones presented by the acid invertases (IAV). The sucrose phosphate synthase (SPS) followed the sucrose accumulation in the pericarp initial stage in coffee fruits. It was shown the existence of two isoforms of SUS codificated by genes called CaSUS1 e CaSUS2 in C. Arabica, that possibly play different metabolic roles in different tissues in coffee fruit. These genes show a differential expression, in which CaSUS1 is expressed in the juvenile stage of perisperm and endosperm development; however, CaSUS2 expression overlaps the detected activity peaks of SUS and the sucrose accumulation in the final stages of pericarp and endosperm development. This fact suggests that the CaSUS1 isoform is related to sucrose degradation while it seems clear that the CaSUS2 isoform is related to sucrose synthesis. Both genes were shown to be also expressed in C.racemosa since CaSUS1 and CaSUS2 C. arabica probes recognized transcripts (mRNA) in this species endosperm. Therefore, CrSUS1 and CrSUS2 genes seem to codify SUS isoforms that play different functions from the ones observed in C. arabica; SUS1 seems to be related to sucrose synthesis. The shadowing has influenced the fruits development cycle duration, turning it longer than in pleno sol (respectively, 260 and 231 days after flowering) and altering the sucrose accumulation in fruits. According to these results, it is clear how complex is the sucrose metabolism in coffee fruits, since it is not always that the same pattern of sugar accumulation is followed by the enzymatic activities and enzyme degradation and sucrose (re)synthesis act simultaneously as well as sugar transference among tissues / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

A cana-de-açúcar é uma gramínea C4 da família das Poaceae. Sua principal característica é a capacidade de estocar altas concentrações de sacarose no colmo. Devido à sua alta atividade fotossintética, ela consegue converter uma boa parte da radiação solar em biomassa. Assim, ela pode ser considerada um dos melhores modelos para os estudos da relação fonte-dreno. O Brasil é um dos maiores produtores e exportadores de álcool do mundo, e por isso a cana-de-açúcar é considerada uma das principais culturas atuais. A ausência de informações sobre a sua sequência genômica levou à criação do programa SUCEST no final de 1990, onde foram disponibilizadas aproximadamente 240,000 sequências denominadas ESTs (Expression Sequence Tags), uma cobertura de quase 90% do genoma expresso da cana-de-açúcar. Desta forma, foi possível desenvolver uma plataforma de microarranjo Agilent de oligonucleotídeos com componentes do SUCEST. Por meio do programa de melhoramento da RIDESA e a análise por microarranjos, foi possível a identificação de vias metabólicas que podem estar relacionadas com a regulação do acúmulo de sacarose em cana-de-açúcar, principalmente aquelas que envolvem os fitormônios auxina e etileno. A obtenção de dados agrotecnológicos e de fisiologia permitiu a observação de um trade off metabólico, onde o acúmulo de sacarose parece ocorrer em detrimento do acúmulo de fibra. A obtenção de plantas silenciadas e superexpressando uma quinase da família da SnRK1 levou, através da análise de microrarranjos, a identificação de genes diferencialmente expressos envolvidos no estresse por seca como uma PP2C e deidrina. Nas plantas superexpressando uma SnRK1, há um aumento do conteúdo de sacarose na planta 88, que talvez indique que a superexpressão dessa quinase leve a um aumento do conteúdo de sacarose em cana. Com a obtenção de sequências genômicas acima do sítio de início da transcrição dessa mesma SnRK1, e de uma subunidade regulatória Akinβγ, foi possível identificar por análise computacional sequências conservadas envolvidas na regulação hormonal, resposta à seca e reações de luz, indicando que a transcrição desse gene pode resultar de diferentes respostas da planta. Esse trabalho permitiu novas diretrizes no estudo do acúmulo de sacarose no colmo, indicando que vias de transdução de sinais conservadas, mediadas por hormônios e fosforilação, podem ser as principais responsáveis por esse fenômeno em cana-de-açúcar. / The sugarcane is a C4 grass of the Poaceae family. Its main feature is the ability to store high sucrose concentrations in the culm. Due to the elevated photosynthetic activity, it can convert a great portion of solar radiation into biomass. Thus, it can be considered one of the best models for studies of source-sink relationship. Brazil is one of the largest alcohol producers and exporters in the world, and sugarcane is considered one of the main current cultivars. The absence of information about its genome sequence led to the creation of the SUCEST program in late 1990, from which approximately 240,000 sequences called ESTs (Expression Sequence Tags) were made available, with a coverage of almost 90% of the sugarcane expressed genome. Thus, it was possible to develop a microarray platform with Agilent oligonucleotide with SUCEST components. Through the RIDESA improvement program and microarray analysis, it was possible to identify metabolic pathways that may be related to the regulation of sugarcane sucrose accumulation, especially those involving the plant hormones auxin and ethylene. The agro-technological and physiology data allowed the observation of a metabolic trade-off, where the sucrose accumulation appears to occur at the expense of fiber accumulation. The production of plants that are silenced or overexpressing a kinase from SnRK1 family, led by microrarray analysis, allowed the identification of differentially expressed genes that are involved in drought stress, such as a PP2C and dehydrin. In plants overexpressing a SnRK1, there is an increased sucrose content in the plant 88, which may indicate that overexpression of this kinase leads to an increase in leaf sucrose content. After obtaining the genomic sequence above the transcription start site of the same SnRK1, and a regulatory subunit Akinβγ, it was possible to identify by computer analysis conserved sequences involved in regulating hormonal response to dry and light responses, indicating that the gene transcription may arise from different plant responses. This work allowed new guidelines in the study of sucrose accumulation in the culm, suggesting that conserved signal transduction pathways and hormone mediated phosphorylation may be the main reason for this phenomenon in sugarcane.

Delivery of photoassimilate, mainly sucrose (Suc) from photoautotrophic source leaves provides the substrate for the growth and maintenance of sink tissues such as roots, storage tissues, flowers and fruits, juvenile organs, and seeds. Phloem loading is the energized process of accumulating solute in the sieve element/companion cell complex of source leaf phloem to generate the hydrostatic pressure that drives long-distance transport. In many plants this is catalyzed by Suc/Proton (H+) symporters (SUTs) which are energized by the proton motive force (PMF). Overexpression of SUTs was tested as means to enhance phloem transport and plant productivity. Phloem specific overexpression of AtSUC2 in wild type (WT) tobacco resulted in enhanced Suc loading and transport, but against the hypothesis, plants were stunted and accumulated carbohydrates in the leaves, possibly due to lack of sufficient energy to support enhanced phloem transport. The energy for SUT mediated phloem loading is provided from the PMF, which is ultimately supplied by the oxidation of a small proportion of the loaded photoassimilates. It was previously shown that inorganic pyrophosphate (PPi) is necessary for this oxidation and overexpressing a proton-pumping pyrophosphatase (AVP1) enhanced both shoot and root growth, and augmented several energized processes like nutrient acquisition and stress responses. We propose that AVP1 localizes to the PM of phloem cells and uses PMF to synthesize PPi rather than hydrolyze it, and in doing so, maintains PPi levels for efficient Suc oxidation and ATP production. Enhanced ATP production in turn strengthens the PMF via plasma membrane (PM) ATPase, increasing phloem energization and phloem transport. Phloem-specific and constitutive AVP1 overexpressing lines showed increased growth and more efficiently moved carbohydrates to sink organs compared to WT. This suggested changes in metabolic flux but diagnostic metabolites of central metabolism did not show changes in steady state levels. This research focuses on fundamental aspects of carbon utilization and transport, and has a strong applied component, since increased H+-PPase activity enhances plant biomass, nutrient up-take capacities, and stress tolerance for as yet not fully characterized reasons.

Call number: LD2668 .T4 1985 A32 / Master of Science

The acid-catalyzed inversion of sucrose is often taken as an example of a first order reaction. It is, however, influenced by many factors such as temperature, type of acid used, concentration of sucrose, and the concentration of acid. What has received little attention so far is the influence of addition, in particular, other salts to the reacting solution. In this study, the influence of different salt solutions on the kinetics of sucrose inversion rate was studied at 29 °C by use of optical rotation measurements. The salts chosen for this study are readily soluble in sucrose solution and they provide an opportunity to study the interaction of electrolytes in aqueous solution of sucrose as well as their effects on the inversion of sucrose kinetics. The rates are found to be influenced by the concentration of the salts. No significant differences was measured when the salt were dissolved either in the sucrose or in the acid solutions. The influence of added salts to saccharide solutions was determined by evaluating the difference between the rotation of pure saccharides solutions and the rotation of pure saccharide solutions with salts. The changes in optical rotation were compared to the Hofmeister series. The saccharide-salt systems containing acidic salts (Na2HPO4 or NaH2PO4) were found to be dependent on the pH. Changing the molar ratio of sucrose and salt added also had an influence of the change in optical rotation. / Chemistry / M. Sc. (Chemistry)

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: This study had three main goals: 1. to investigate the occurrence on the protein level of sucrose synthase (SuSy) isoforms in sugarcane sink tissue, 2. to determine the kinetic properties of these isoforms, 3. to establish the tissue localisation of SuSy in the sugarcane culm The results are summarised below: Three SuSy isoforms were obtained from leaf roll tissue. The SuSyA and SuSyB isoforms differed in terms of charge characteristics, with SuSyA not binding to an anion exchange column that bound SuSyB and SuSyC under the same conditions. Both SuSyB and SuSyC isoforms were eluted at 180 mM KCl. The SuSyA and SuSyB isoforms were present during autumn, but during winter only the SuSyC isoform could be isolated. Even though they eluted at the same salt concentration, SuSyB and SuSyC were different isoforms, because they had different kinetic parameters, as well as different immunological properties. SuSyB and SuSyC could not have been mixtures of the same isoforms, since a polyclonal antiserum against SuSyB, which inactivates native SuSyB, did not inactivate SuSyC. All three isoforms had significantly different kinetic parameters, with the SuSyA isoform also having a much lower sucrose breakdown/synthesis ratio than the other two isoforms. Therefore, at least three SuSy isoforms occur in sugarcane leaf roll tissue on the protein level. The SuSyC isoform was subsequently kinetically characterised in detail. Data showed that the enzyme employs an ordered ternary complex mechanism, with UDP binding first and UDP-glucose dissociating last. These experimentally obtained kinetic parameters were then used to extend a kinetic model of sucrose accumulation. Data show that when the experimentally determined SuSy kineticparameters were entered into the model, a 40 % increase in sucrose concentration and 7 times reduction in fructose concentration resulted. These data illustrate the pronounced physiological effects that may result from the presence of different SuSy isoforms. SuSy protein localisation data, obtained by an immunohistochemical approach, indicated that SuSy protein was present in both storage parenchyma and vascular tissue of young, intermediate, and mature internodes. SuSy enzyme activity in different parts of the internodes was similar, except for internode 3, which had much higher activity in the bottom part of the internode, possibly because growth is faster here, hence a higher demand for sucrose cleavage exists here. / AFRIKAANSE OPSOMMING: Hierdie studie het ten doel gehad: 1. om die teenwoordigheid van sukrose sintase (SuSy) isovorme in suikkerriet swelgweefsel te ondersoek 2. om die kinetiese eienskappe van hierdie isovorme te ondersoek 3. om die weefsellokalisering van SuSy in die suikerrietstingel te bepaal Die resultate word hieronder opgesom: Drie SuSy isovorme is gevind in blaarrol weefsel. Die SuSyA en SuSyB isovorme het verskil in terme van ladingseienskappe, met SuSyA wat nie aan ‘n anioonuitruilkolom gebind het nie waaraan SuSyB en SuSyC wel onder dieselfde kondisies gebind het. Beide SuSyB en SuSyC isovorme is geëlueer van die kolom teen 180 mM KCl. Die SuSyA en SuSyB isovorme was teenwoordig gedurende herfs, maar in die winter was slegs SuSyC teenwoordig. Ten spyte van die feit dat SuSyB en SuSyC teen dieselfde soutkonsentrasie geëlueer is, het hulle verskillende isovorme verteenwoordig, aangesien hulle kinetiese en immunologiese eienskappe verskil het. SuSyB en SuSyC kon nie mengsels van dieselfde isovorme gewees het nie, want ‘n poliklonale antiserum teen SuSyB, wat SuSyB geïnaktiveer het, het nie SuSyC geïnaktiveer nie. Al drie isovorme het betekenisvol verskil wat kinetiese eienskappe betref, met die SuSyA isovorm wat ook ‘n baie laer sukrose afbraak/sintese verhouding gehad het as die ander twee isovorme. Daar is dus ten minste drie SuSy isovorme teenwoordig op die proteïen vlak in suikerriet blaarrol weefsel. Die in-detail kinetiese analise van die SuSyC isovorm het getoon dat die ensiem ‘n geordende drietallige kompleks meganisme het, met UDP wat eerste bind en UDP-glukose wat laaste dissosieer. Die eksperimenteel bepaalde kinetiese parameters is toe gebruik om ‘n kinetiese model van sukrose akkumulering uit tebrei. Data het getoon dat wanneer die generiese SuSy kinetiese parameters in die oorspronklike model vervang word met die eksperimenteel bepaalde waardes, die berekende sukrose konsentrasie met ongeveer 40 % toeneem, terwyl die fruktose konsentrasie ongeveer 7 keer afneem. Hierdie resultaat toon die groot fisiologiese effek wat die uitdrukking van verskillende SuSy isovorme op suikermetabolisme kan hê. Die SuSy proteïen lokaliseringsdata, wat met ‘n immunohistochemiese benadering verkry is, het aangedui dat SuSy in beide bergingsparenchiemselle sowel as vaatweefsel teenwoordig is in jong, intermediêre en volwasse internodes. SuSy ensiemaktiwiteit in verskillende dele van die internodes was soortgelyk, behalwe in internode 3, wat baie hoër aktiwiteit gehad het in die onderste deel van die internode as bo, moontlik weens vinniger groei in hierdie deel van die internode, wat afhanklik is van afbraakprodukte van sukrose.

The thesis provides a background on crystallisation, the effects of applied fields and summarises the techniques used for characterisation and analysis. The study of applied magnetic fields was carried out on three crystallising systems (a) sucrose, (b) lactose and (c) cocoa butter. Both sucrose and lactose were crystallised from aqueous solutions in incubators at 50°C in applied magnetic fields and the resulting crystals compared to the those obtained under zero field conditions. The results for the sucrose study where the magnetic treatment was carried out under static, dynamic pumped and dynamic syphoned conditions domonstrated that changes in phase, crystallinity, morphology and microcrystallinity were a result of the applied magnetic fields and additional strongly bound water was found to be present within the sucrose crystals most likely to be sucrose hydrates. The resulting sucrose crystals were dependant on the type of field applied, the purity of the sucrose solution and the residence time within the applied field. The lactose study under static conditions provided similar results concluding that applied fields resulted in a more controlled crystallisation resulting in increased crystal size, increased crystallinity and changes in morphology. Crystallisation of cocoa butter from the melt, under normal production conditions in applied fields, resulted in changes in morphology and the time taken to reach optimum tempering which were dependant on the type of applied field and the residence time in the applied field.