Le Mexique se caractérise par la présence sur son territoire de nombreuses espèces d’agave qui peuvent être cultivées ou non. En particulier l’Agave tequilana Weber var. azul a une grande importance économique, car elle constitue la principale matière première pour l’élaboration de la tequila. Les agaves durant leur développement, qui dure plusieurs années, accumulent des réserves de sucres constitués par des fructanes. Actuellement, l’optimisation de l’hydrolyse des fructanes d’agave est surtout importante pour l’industrie de la tequila. Elle permettra d’améliorer les rendements d’extraction des sucres. La méthode classique d’hydrolyse des fructanes est constituée principalement d’un procédé de cuisson des agaves crus. L’utilisation d’enzymes spécifiques pour réaliser ce même procédé d’hydrolyse suscite un récent intérêt industriel, parce qu’il permettrait une réduction de la consommation d’énergie. Les fructanes d’agave présentent des structures complexes, les résidus de fructose sont reliés par des liaisons osidiques de type β (2→1) et β (2→6), et la structure est fortement branchée. Il est nécessaire de comprendre les changements de structure des fructanes en fonction de l’étape de croissance des plantes, pour connaître la variabilité naturelle du substrat utilisé pour l’hydrolyse. D’autre part, il est important de découvrir de nouvelles enzymes susceptibles d’hydrolyser de manière spécifique les fructanes d’agave, et les caractériser biochimiquement, pour arriver à une meilleure connaissance de l’intéraction enzyme-substrat qui permettra le développement de nouvelles applications industrielles possibles pour les fructanes d’Agave tequilana. Dans ce travail, la première partie est consacrée à la détermination de la composition en sucres solubles et à la caractérisation de la structure des fructanes d’Agave tequilana présents dans des plantes d’âges différents. Puis, dans la deuxième partie, la purification et la caractérisation biochimique d’une fructanase isolée d’une souche de levure Kluyveromyces marxianus obtenue à partir du procédé de fermentation du mezcal (boisson d’agave distillée) a été étudiée. L’activité de cette enzyme a été comparée à un cocktail enzymatique commercial le fructozyme®. Finalement, dans une troisième partie, des levures isolées de la fermentation de différents types de mezcal ont été criblées et ont permis la sélection de souches capables de dégrader spécifiquement les fructanes d’Agave / Mexico has a high diversity of Agave plants, which could be cultivated or not. The most economically important is Agave tequilana Weber var. azul, because it is the raw material for the tequila elaboration process. As agaves grow, they accumulate reserve sugars as fructans. Actually, optimizing the A. tequilana fructans hydrolysis, in order to increase the sugar yield, is important to the tequila industry. Traditionally, agaves are cooked to hydrolyze the fructans. However, using enzymes for hydrolysis may reduce energy consumption and increase sugar yields.The fructans of A. tequilana have a complex structure, composed of fructose chains with β (2→1) and β (2→6) linkages with branching points. It is important to understand how the structure of these molecules changes as a function of plant growth, in order to know the natural variability of the substrate that must be hydrolysed. It is also necessary to find new enzymes for the efficient hydrolysis of A. tequilana fructans, and to characterize them biochemically for a better understanding of the enzyme-substrate interaction.The present work has three parts that focuses separately on each of these needs: First, characterizing the water soluble carbohydrates and the structure of the A. tequilana fructans as a function of the plant’s growth (age). Second, purifying and biochemically characterizing a fructanase from Kluyveromyces marxianus yeast isolated from the fermentation of mezcal, and comparing it to a commercial cocktail (Fructozyme®). Third, a screening of enzymes from yeasts used to ferment mezcal, in order to determine their ability to hydrolyze A. tequilana fructans

Sugarcane holds great potential as a biofactory for the tailored production of novel products of commercial value. In many cases however, the accumulation of an alien product within the cytoplasm interferes with essential cell metabolism. To avoid potential interference, targeting the accumulation of biofactory products into vacuoles may be beneficial. Vacuoles represent one endpoint of the plant endomembrane system where proteins destined for inclusion must contain appropriate targeting peptide signals. However, targeting peptide signals used previously to direct heterologous proteins to the vacuole have not yet been shown to function efficiently in sugarcane. The emphasis of the work described in this thesis was first to characterise the diversity of vacuole types in selected sugarcane tissues, and second to identify and test the function of putative vacuolar targeting signals identified in vacuolar proteins of sugarcane. In order to investigate vacuole diversity in sugarcane cells, a series of membrane-permeable fluorescent probes were used to assess both the acidity and proteolytic properties of vacuolar compartments. It is clear that even from early in development, large vacuoles filled most of the volume of storage parenchyma cells within the developing sugarcane stem. These vacuoles were intensely acidic and contained active aminopeptidases. In leaf cells, vacuoles labelled by chromogenic indicators and enzyme substrates appear much more diverse in pH and proteolytic intensity owing to the multiple functions that leaf cells participate in. As the predominant sugarcane vacuole in vegetative tissues appears to be proteolytic, sugarcane sequences showing homology to proteases and protease inhibitors in other plant species were aligned and compared to identify potential vacuolar targeting signals. This analysis revealed the presence of several candidate vacuolar targeting motifs which displayed high conservation across plant homologues. One such motif, represented by the sequence IRLPS, was identified in the N-terminal region of a legumain protein from sugarcane, which was homologous to known vacuolar processing enzymes in other species. To test the efficacy of the legumain targeting signal and to compare with other motifs, a series of GFP reporter constructs was synthesised and expressed in sugarcane. The sugarcane legumain vacuole targeting signal was particularly efficient at directing an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP fusion proteins in the vacuole appeared to be dependent on cell type, suggesting that the vacuolar environment can vary in its ability to degrade introduced proteins. The legumain vacuole targeting signal was further tested for its ability to direct an avidin protein and a fructosyltransferase enzyme into the lytic vacuole of transgenic sugarcane plants. Avidin, derived initially from chicken egg white, is a glycoprotein that displays a high affinity to the vitamin biotin. For this reason it has been investigated for use in sugarcane as a biocontrol agent against cane grub species. For the production of avidin in planta careful targeting to an appropriate subcellular location is required to avoid a detrimental depletion of available plant cell biotin reserves. When the legumain targeting signal was fused to avidin and expressed as a stably integrated transgene, the avidin protein was detected by immunoblotting but appeared to be proteolytically cleaved within the lytic vacuole in all sugarcane tissues analysed. These plants were phenotypically indistinguishable from controls, indicating that avidin did not appreciably deplete cellular biotin reserves while in transit through the endomembrane system. In contrast, when avidin was designed for either retention in the endoplasmic reticulum or for transit to a different type of vacuole using a heterologous targeting signal, stably transformed plants exhibited a biotin deficient phenotype. This suggests that the legumain vacuole targeting signal was efficient at directing heterologous proteins to a lytic type vacuole where they can be degraded and inactivated if susceptible to proteolysis. When the fructosyltransferase (ftf) gene from Streptococcus salivarius was stably transformed into sugarcane and directed into the lytic vacuole using the legumain vacuole targeting signal, no fructan product could be detected. The low pH and proteolytic environment of this vacuole together with low expression of this bacterial transgene most likely resulted in minimal Ftf activity. Taken together, evidence is presented that the legumain vacuolar targeting signal functions efficiently in directing transgene products such as GFP, avidin and a fructosyltransferase enzyme into a lytic type vacuole. This vacuole has been demonstrated to be both acidic and proteolytic and therefore strategies to improve the stability of heterologous proteins targeted to this vacuolar environment are required and may be specific to the product in question.

Dissertation (PhD)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Under terminal water deficit, the impact of stem carbohydrate remobilization has greater significance because post-anthesis assimilation is limited, and grain growth depends on translocation of carbohydrate reserves. The working hypothesis of this thesis is that increases in stem carbohydrates facilitate tolerance to terminal drought in wheat. The goals of this thesis are to examine this hypothesis using physiological and genetic tools; identify genes that are related to QTL for stem carbohydrate; work with wheat and barley breeders to integrate findings into the breeding program of the Department of Agricultural and Food Western Australia. The physiological data of three drought experiments (two years in a glasshouse and one year in the field) suggested the maximum level of stem water soluble carbohydrate (WSC) is not consistently related to grain weight, especially, under water deficit. The patterns of WSC accumulation after anthesis differed depending on variety and suggested that WSC degradation and translocation have different genetic determinants. Most of the carbohydrates in stem WSC in wheat are fructans. Because 1-FEH gene was an important gene in fructan degradation, the three copies of this gene (1-FEH w1, 1-FEH w2 and 1-FEH w3) were isolated from the respective genomes of bread wheat. In addition, the genes were mapped to chromosome locations and coincided with QTL for grain weight. The results of gene expression studies show that 1-FEH w3 had significantly higher levels in the stem and sheath which negatively corresponded to the level of stem WSC in two wheat varieties in both water-deficit and well-watered treatments. Strikingly, the 1-FEH w3 appeared to be activated by water deficit in Westonia but not in Kauz. The results suggest that stem WSC level is not, on its own, a reliable criterion to identify potential grain yield in wheat exposed to water deficit during grain filling. The expression of 1-FEH w3 may provide a better indicator when linked to instantaneous water use efficiency, osmotic potential and green leaf retention, and this requires validation in field grown plants. In view of the location of the contribution to grain filling of stem WSC, this is a potential candidate gene contributing to grain filling. The numerous differences of intron sequences of 1-FEH genes would provide more opportunities to find markers associated with the QTL. A new FEH gene was partially isolated from Chinese Spring and the sequence was closely related to 1-FEH genes. This gene, FEH w4, was mapped to 6AS using Chinese Spring deletion bin lines. The polymorphism of this gene was found between different bread varieties using PCRs and RFLPs, and this allowed the gene to be mapped to two populations of Hanxuan 10 × Lumai 14 and Cranbrook × Halberd. In the population of Hanxuan 10 × Lumai 14, it was close to SSR marker xgwm334 and wmc297 where the QTL of thousand grain weight and grain filling efficiency were located. This result indicated this gene might be another possible candidate gene for grain weight and grain filling in wheat.

Fructans are polysaccharides that are made up mainly of fructose. They are non-digestible carbohydrates and act as prebiotics to selectively promote the growth of colonic bifidobacteria, thereby improving human gut health. Fructans are present in the grain of wheat (Triticum aestivum L.), a staple food crop. Until now, there has been no research on genetic improvement of the concentration of fructans in wheat grain, partly because it has been difficult to accurately measure. One aim of this research project was to develop a simple and effective method to measure the fructan concentration in wheat grain. This was achieved by modifying a method that involves extraction of fructans from wheat grain followed by enzymatic hydrolysis to break down fructans into monosaccharides and quantification by anion-exchange liquid chromatography coupled with pulsed amperometric detection. The modified procedure is reliable and allows the handling of large numbers of flour samples at a relatively low cost, and can therefore be useful for assessing large numbers of wheat breeding lines. Using this method, grain samples taken from a diverse set of 117 wheat cultivars and breeding lines, including parents of mapping populations, were analysed for grain fructan concentration. There was significant genotypic variation among these materials, with grain fructan concentration ranging from 0.3 to 2.3% of grain dry weight. There was no evidence of strong genotype-byenvironment interaction; the fructan concentrations of the same genotypes were positively correlated over different environments in Australia. Genetic mapping was carried out to detect and map loci affecting grain fructan concentration in wheat using a doubled haploid population derived from a cross between Berkut (high fructan) and Krichauff (low fructan). Grain samples were obtained from two field sites in South Australia and one in Kazakhstan. Fructan concentration varied widely within the population (0.6-2.6% of grain dry weight), with heritability estimated as h² = 0.71. A linkage map of 528 molecular markers covering 21 wheat chromosomes was used for locating quantitative trait loci (QTL). Genetic mapping identified two major QTLs on chromosomes 6D and 7A, with the (high fructan concentration) alleles contributed from Berkut, contributing to a 30-40% increase in wheat grain fructan compared to the Krichauff alleles. Effects of these chromosome regions were validated in additional environments and in another mapping population, Sokoll/Krichauff, with the favourable alleles contributed from Sokoll. The major QTL on chromosome 7A was in the same region with a reported fructosyltransferase orthologue (AB029888), while the major QTL on chromosome 6D seemed to be co-located with a reported gene encoding for a fructan-degrading enzyme 1-exohydrolase (1-FEHw2). It is concluded that grain fructan concentration of wheat can be improved by breeding and that molecular markers could be used to select effectively for favourable alleles in two regions of the wheat genome. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Fructans are polysaccharides that are made up mainly of fructose. They are non-digestible carbohydrates and act as prebiotics to selectively promote the growth of colonic bifidobacteria, thereby improving human gut health. Fructans are present in the grain of wheat (Triticum aestivum L.), a staple food crop. Until now, there has been no research on genetic improvement of the concentration of fructans in wheat grain, partly because it has been difficult to accurately measure. One aim of this research project was to develop a simple and effective method to measure the fructan concentration in wheat grain. This was achieved by modifying a method that involves extraction of fructans from wheat grain followed by enzymatic hydrolysis to break down fructans into monosaccharides and quantification by anion-exchange liquid chromatography coupled with pulsed amperometric detection. The modified procedure is reliable and allows the handling of large numbers of flour samples at a relatively low cost, and can therefore be useful for assessing large numbers of wheat breeding lines. Using this method, grain samples taken from a diverse set of 117 wheat cultivars and breeding lines, including parents of mapping populations, were analysed for grain fructan concentration. There was significant genotypic variation among these materials, with grain fructan concentration ranging from 0.3 to 2.3% of grain dry weight. There was no evidence of strong genotype-byenvironment interaction; the fructan concentrations of the same genotypes were positively correlated over different environments in Australia. Genetic mapping was carried out to detect and map loci affecting grain fructan concentration in wheat using a doubled haploid population derived from a cross between Berkut (high fructan) and Krichauff (low fructan). Grain samples were obtained from two field sites in South Australia and one in Kazakhstan. Fructan concentration varied widely within the population (0.6-2.6% of grain dry weight), with heritability estimated as h² = 0.71. A linkage map of 528 molecular markers covering 21 wheat chromosomes was used for locating quantitative trait loci (QTL). Genetic mapping identified two major QTLs on chromosomes 6D and 7A, with the (high fructan concentration) alleles contributed from Berkut, contributing to a 30-40% increase in wheat grain fructan compared to the Krichauff alleles. Effects of these chromosome regions were validated in additional environments and in another mapping population, Sokoll/Krichauff, with the favourable alleles contributed from Sokoll. The major QTL on chromosome 7A was in the same region with a reported fructosyltransferase orthologue (AB029888), while the major QTL on chromosome 6D seemed to be co-located with a reported gene encoding for a fructan-degrading enzyme 1-exohydrolase (1-FEHw2). It is concluded that grain fructan concentration of wheat can be improved by breeding and that molecular markers could be used to select effectively for favourable alleles in two regions of the wheat genome. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Orientador: Ranulfo Monte Alegre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T22:59:40Z (GMT). No. of bitstreams: 1 Meirelles_RenataMiterhof_M.pdf: 815729 bytes, checksum: 9edb1e9645d9423ee9870568496b2ffd (MD5) Previous issue date: 2010 / Resumo: O mercado consumidor da frutose tem aumentado significativamente nos últimos anos pela sua utilização cada vez maior em substituição à sacarose em virtude do seu poder edulcorante 70% superior e dos benefícios fisiológicos importantes, como metabolismo independente da insulina, sendo adequada para alimentos fabricados especificamente para diabéticos. Sua maior aplicação tecnológica encontra-se no uso de xaropes enriquecidos com frutose em vários segmentos industriais como alimentício, farmacêutico e químico. Comercialmente, a obtenção de frutose envolve um processo de alto custo sendo interessante o desenvolvimento de um processo que combine a produção por via enzimática e a utilização da sacarose como substrato para obtenção de frutose de alto grau de pureza. O objetivo geral deste trabalho foi estudar e otimizar a hidrólise da levana para obtenção de frutose livre. Para tal, levana foi previamente obtida pela levanassacarase durante fermentação da cepa mutante de Zymomonas mobilis CCT 4494 em substrato a base de sacarose. Kluyveromyces marxianus NRRL Y-8281 foi selecionada dentre três linhagens da espécie Kluyveromyces marxianus (CCT4294, NRRL Y-8281 e NRRL Y-610) devido à sua maior produção de frutana ß-frutosidase. Foram realizados delineamentos experimentais tendo como variáveis temperatura, pHinicial, concentrações iniciais de levana, extrato de levedura e peptona. A enzima agiu exohidroliticamente obtendo apenas frutose como produto, e não foi observado inibição da reação pelo produto. A frutana ß-frutosidase de Kluyveromyces marxianus NRRL Y-8281 foi caracterizada parcialmente quanto ao pH e temperatura ótimos (4,4 e 50 ºC), estabilidade térmica e pH de pré incubação, além dos parâmetros cinéticos da Equação de Michaelis-Mentem, Km e Vmáx (61,5 µmol/mL e 0,0112 µmol/mL.min, respectivamente) para o substrato levana / Abstract: The market for fructose consumption has increased significantly in the last years by increasing its use in place of sucrose, because of its sweetening power 70% higher than the sucrose and the physiological benefits like independent metabolism of insulin, which is, therefore, suitable for food made specifically for diabetics. His greatest technological application is the use of enriched fructose syrups in various industries like food, pharmaceutical and chemical ones. Commercially, the obtainment of fructose involves a high cost, being interesting to develop a process that combines the production of fructose by an enzyme, using sucrose as substrate to obtain fructose of high purity. Therefore, the objective of this work was to study and optimize the hydrolysis of levan to obtain free fructose. To accomplish this, the levan was previously obtained by levansucrase during fermentation of mutant strain of Zymomonas mobilis CCT 4494 in sucrose substrate. Kluyveromyces marxianus NRRL Y-8281 was selected among three strains of the species Kluyveromyces marxianus (CCT4294, NRRL Y-8281 and NRRL Y-610) by the increased production of fructan ß-frutosidase. Experimental designs were performed having as variables temperature, initial pH, initial concentrations of levan, yeast extract and peptone. The enzyme acted in a exohydrolytically fashion, getting only fructose as released product, and it was not observed inhibition by product reaction. The fructan exo-ß-frutosidase of Kluyveromyces marxianus NRRL Y-8281 was partially characterized for optimum pH and temperature (4.4 and 50 °C), thermal and pH stability, besides the kinetic parameters of the Michaelis-Mentem equation, Km and Vmáx (61,5 µmol/mL and 0,0112 µmol/mL.min, respectively) to the substrate levan / Mestrado / Mestre em Engenharia de Alimentos

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Biopolymers exhibit the required material properties to replace conventional, non-biodegradable, petroleum-based polymer products. They have a closed carbon cycle, making them carbon neutral and environmentally friendly. Biopolymers are produced from non-toxic substrates during in vivo enzymatic reactions. Biosynthesis of the most commercially important biopolymers is too complex to be reproduced in in vitro reactions. Identification of the genes responsible for their biosynthesis has been under investigation, with some pathways already elucidated. The genes involved in the biosynthesis of these polymers have been targeted for genetic manipulation to increase productivity, as well as create tailor-made polymers. Novel biopolymers and the genes responsible for their synthesis are of interest for their potential commercial applications. Bacteria produce a wide range of biopolymers and are being implemented as the bio-factories for biopolymer production. They are capable of utilising easily accessible and renewable carbon sources such as sucrose for polymer biosynthesis. Bacteria thus allow for economical production of these environmentally beneficial polymers. In this study, the gene responsible for the production of an unknown biopolymer from an unknown bacterium was identified. The biopolymer producing bacteria were grown on media enriched with sucrose as carbon source, during an expression library screening in a previous study. Expression library technology was used to search for the gene and it was identified as a 424 amino acid levansucrase which had a 100% homology to Leuconostoc mesenteroides M1FT levansucrase (AAT81165.1). Biopolymer analysis revealed that the biopolymer was a levan, a polysaccharide consisting of only fructose molecules with a molecular weight of ± 5 kDa. Analysis of a 516 bp fragment of the 16S rRNA determined that the unknown bacteria were a Pseudomonas species. / AFRIKAANSE OPSOMMING: Bio-polimere besit noodsaaklike materiële eienskappe wat toelaat dat dit konvensionele, nie bio-afbreekbare, petroleum-gebasseerde polimeer produkte kan vervang. Hulle het n geslote koolstof kringloop en is dus koolstof neutraal en omgewingsvriendelik. Bio-polimere word vervaardig van nie-toksiese substrate, gedurende ensiematiese reaksies in vivo. Die belangrikste kommersiële bio-polimere se ensiematiese produksie is te kompleks om in ʼn in vitro reaksie te herproduseer. Ondersoeke tot die identifikasie van die gene wat verantwoordelik is vir die produksie van die polimere is onderweg, en sommige produksie paaie is reeds bekend. Die bekende gene word geteiken vir genetiese manipulasie om hulle produktiwiteit te vermeerder en om unieke polimere te produseer. Unieke bio-polimere en die gene wat vir hul produksie verantwoordelik is, is van belang vir hulle potentiële implimentering in komersiële toepassings. Bakteria produseer ʼn verskeidenheid bio-polimere en word as die bio-fabrieke vir polimeerproduksie geimplimenteer. Hulle kan maklik bekombare koolstofbronne, soos sukrose, gebruik om bio-polimere te produseer. Bakteria laat dus die ekonomiese produksie van hierdie omgewingsvriendelike polimere toe. In hierdie studie word die geen wat verantwoordelik is vir die produksie van ʼn onbekende bio-polimeer van ʼn onbekende bakteria, geidentifiseer. Die bakteria was gevind op media, wat verryk was met sukrose as koolstofbron, tydens ʼn vorige studie, waartydens ʼn uitdrukkingsbiblioteek gesif was op hierdie media. Uitdrukkingsbiblioteek tegnologie was gebruik om die geen te vind. Die geen was geidentifiseer as ʼn 424 aminosuur, homo-fruktose-polimeer produseerende geen, ʼn “levansucrase”. Die geen het ʼn 100% homologie met die M1FT “levansucrase” geen (AAT81165.1) van Leuconostoc mesenteroides gehad. Analise van die bio-polimeer het bepaal dat die polimeer ʼn polisakkaried was, wat slegs uit fruktose molekules bestaan het. Die molekulêre gewig van die polimeer was ± 5 kDa. Analise van ʼn 516 bp fragment van die 16S rRNS het bepaal dat die bakteria van die Pseudomonas spesie afkomstig was.

The main aim of the work presented in this dissertation was to explore the possibility to genetically manipulate the sucrose storing crops, sugarcane and sweet sorghum, to convert their sucrose reserves into higher-value alternatives. For the purpose of this study we focussed on fructans as alternative sucrose-based high-value carbohydrates, since these fructose polymers are of significant commercial interest. To investigate the technical feasibility of transforming sugarcane and sweet sorghum to produce this novel carbohydrate, we proposed to transfer the fructosyltransferase genes from Cynara scolymus into these plants by means of particle bombardment. In order to apply this technology to sweet sorghum, an in vitro culture system suitable for transformation had to be established. For this purpose an extensive screening process with different combinations of variables were conducted. Though the relationships between these variables proved to be complex, it was concluded that immature zygotic embryos could be used to initiate a genotype-independent totipotent regeneration system with a 65% callus induction rate, provided that initiation takes place during summer. Stable transformation and regeneration of these calli were however not successful and will have to be optimised to allow future applications. By introducing fructosyltransferase genes into sugarcane, we succeeded in transforming sugarcane into a crop that produces a variety of fructans of the inulintype. Low molecular weight (LMW) inulins were found to accumulate in the mature internodes of 42% of the transgenic sugarcane plants expressing the sucrose:sucrose 1-fructosyltransferase (1-SST) gene, and in 77% of the plants that incorporated both 1-SST and fructan:fructan 1-fructosyltransferase (1-FFT), while only 8% of these plants accumulated high molecular weight (HMW) inulins. Our results demonstrated that sugarcane could be manipulated to synthesise and accumulate fructans without the induction of phenotypical irregularities. Inulins with a degree of polymerisation up to 60 were found in sugarcane storage tissue. In these HMW inulin-producing plants, up to 78% of the endogenous sucrose in the mature sugarcane culm was converted to inulin. This enabled inulin accumulation up to 165.3 mg g-1 fresh weight (FW), which is comparable to that found in native plants. These transgenic sugarcane plants, therefore exhibit great potential as a future industrial inulin source. Fructan production was detected in all the sugarcane plant tissue tested, predominantly as 1-kestose. In contrast with the fact that fructan accumulation in leaves did not affect the endogenous sucrose concentrations in these organs, the sucrose content of mature internodes that accumulated high levels of 1-kestose was severely reduced. However, increases in total sugar content, in some instances up to 63% higher than control plants, were observed. This phenomenon was investigated with the use of radio-labelled-isotopes. An increase in the allocation of incoming carbon towards sucrose storage, resulting in higher carbon partitioning into both 1- kestose and sucrose, were detected in the culms of transgenic compared to control lines. This modification therefore established an extra carbohydrate sink in the vacuoles that affected photosynthate partitioning and increased total soluble sugar content. The data suggests that sucrose sensing is the main regulatory mechanism responsible for adapting carbon flow in the cells to maintain sucrose concentration.