The biochemical basis for the regulation of sucrose accumulation is not fully understood. The present study was thus aimed at investigating aspects of 'coarse' (enzyme activity) and 'fine' (metabolite) control of glycolytic enzyme activity in relation to carbon partitioning in the developing stalk (internodes 3 to 10), and between varieties with significant differences in sucrose content. Particular emphasis was placed on studying pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), since this enzyme has been implicated in sucrose metabolism in other plant species. Within the developing stalk, internodal maturation was associated with a redirection carbon from the insoluble matter and total respiration (C02 production and biosynthesis) to sucrose storage. Between varieties, with significant variation in sucrose content, there was an inverse relationship between hexose monophosphate partitioning into respiration and sucrose. The reduction in carbon flux to respiration was not associated with a decline in the extractable specific activity of PK, PFK and PFP. There was also no alteration in the regulation of PK, PFK and FBPase by change in the mass action ratios. Hexose monophosphate concentration declined approximately two to three-fold from internodes 3 to 9 and Fru-6-P concentration was within the lower Km or 80.5 range (Fru-6-P) of PFP and PFK, respectively (as reported from the literature) . Within the developing stalk, substrate limitation might have contributed to the decline in carbon partitioning to respiration. In sugarcane, the levels of PFP activity were controlled in part by PFP protein expression. 8ugarcane PFP polypeptide(s) are resolved as a single protein with a molecular mass of approximately 72 kO. PFP catalysed a reaction close to equilibrium in all intemodes investigated, and the concentration of Fru-2,6-P2 was shown to be in excess of the requirement to stimulate PFP activity. Carbon flux from the triose-P to hexose monophosphate pool was apparent in sugarcane, suggesting that PFP activity was functional in vivo. The developmental profile of specific PFP activity was not positively correlated to the increasing rate of sucrose accumulation in the top ten internodes of the developing stalk. Between different sugarcane varieties, specific PFP activity was shown to be inversely correlated to sucrose content. / Thesis (Ph.D.)-University of Natal, Durban, 1997.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005. / Vacuolar Pyrophosphatase (V-PPase) has never been studied in sugarcane before and to date nothing is known about V-PPase in sugarcane, except for the sequences of a few expressed sequence tags (ESTs). The aim of this project was to characterize V-PPase expression in several hybrid sugarcane varieties that differ significantly in sucrose content, with the main objective of the study to assess whether V-PPase is correlated in any way to the sucrose storage phenotype. Therefore, the goals of this project were to (i) develop molecular tools for the detection and quantification of V-PPase on a DNA, RNA, protein and enzyme level and (ii) to use these tools to characterize the expression of V-PPase within the culm of the three hybrid varieties. The cDNA sequence of the catalytic subunit of the sugarcane V-PPase gene was cloned, expressed in a bacterial system and the V-PPase peptide was purified. This peptide was used for the immunization of mice and the production of polyclonal anti-VPPase antiserum. Anti-VPPase antiserum reacted specifically with a single polypeptide among vacuolar membrane proteins. Moreover, anti-VPPase antiserum recognized V-PPase from various monocotyledons and dicotyledons. The anti-VPPase antiserum was used for the establishment of an ELISA system to determine V-PPase protein content in vacuolar membrane preparations. This system proved to have several advantages over the protein blotting technique and shared a strong linear relation with V-PPase specific activity, showing that these two tests are compatible and reliable. The optimisation of sugarcane V-PPase zero-order kinetics was fundamental in order to measure V-PPase specific activity accurately. It had a relative broad pH optimum, retaining more than 90% of its maximum activity between pH 6.50 and 7.25. V-PPase required both Mg2+ and K+, in addition to PPi, for maximum activity in vitro. The reported kinetic variables are within range of previous data determined for other species, including mung bean, red beet and sugar beet. V-PPase protein level and specific activity within the sugarcane culm followed a similar trend , withoiofofoenaobserved for sucrose accumulation rates observed in sugarcane. Moreover, V-PPase protein contents and specific activity share the same general trend as total sucrose content in a specific tissue compared among the three varieties. No significant differences were observed in V-ATPase activity among the three varieties. Our findings suggest that V-PPase may play a role in sucrose accumulation in sugarcane.

Thesis (MSc (Genetics. Institute for Plant Biotechnology))--University of Stellenbosch, 2006. / The main goal of this project was to elucidate the apparent role of neutral invertase (NI) in sucrose accumulation in sugarcane. In the first part of the study putative transgenic cell lines (transformed with antisense NI constructs) were characterised to confirm the stable integration and expression of the transgene. Batch suspension cultures were used to initiate replicate cultures of several of these transgenic lines as well as a control, and the metabolism of the cultures during a 14 day growth cycle was examined. The transgenic lines had substantially reduced levels of NI activity. While the activities of the other invertases remained unchanged, the activity of sucrose synthase (SuSy) was significantly higher in the transgenic suspension cultures relative to the control. Throughout the growth cycle, sucrose concentrations in the transgenic lines were consistently higher, and glucose and fructose concentrations lower, than the control. The transgenic cultures also exhibited a decreased growth rate in comparison to the control. Labelling studies confirmed a decrease in the in vivo rate of invertase-mediated sucrose hydrolysis in the transgenic lines, as well as indicating a decline in the partitioning of carbon to respiratory pathways in these cultures. In the second part of the study, which focussed on greenhouse-grown transgenic plants, similar results were reported. NI activity was significantly decreased, and SuSy activity increased in all of the tissues sampled. The sucrose concentration and purity were also higher in the transgenic tissues, while the in vivo sucrose hydrolysis rate was lower. Allocation of carbon to respiration was lower in the transgenic plants, suggesting that a decrease in sucrose breakdown reduces the availability of hexoses for growth and respiration. Overall, the results suggest that NI plays a key role in the control of sucrose metabolism, and that changes in the activity of this enzyme have far-reaching effects on cellular metabolism. The fact that the trends reported in the whole-plant studies parallel those of the suspension cultures confirms that suspension cultures can be used as a model system in metabolic engineering research in sugarcane. Thus the possibility now exists to analyse large numbers of transgenic lines in a quicker time frame and at a reduced cost in comparison to conventional methods.

Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2005. / Sucrose accumulation, marked by a continuous cycle of synthesis and degradation, is characterised by a shift of carbon away from the insoluble matter and respiratory intermediates into sucrose. Despite this shift, a significant proportion of carbon is returned to these pools by hexose-phosphate: triose-phosphate cycling and/or sucrose cycling. Little is known about the magnitude and behaviour of these cycles in sugarcane. Contradictory reports on the relationship between these two cycles have led to the evaluation of the link between the hexose-phosphate: triose-phosphate- and sucrose cycle. In addition, it still needs to be tested whether these cycles could significantly influence carbon partitioning within sugarcane internodal tissue. In this work, a comprehensive metabolic profile was constructed for sugarcane internodal tissue by gas chromatography-mass spectrometry (GC-MS) in order to determine the steady state levels of a broad range of primary metabolites that are involved in these cycles. The power of GC-MS was illustrated by the detection of raffinose, maltose, ribose, xylitol, inositol, galactose, arabinose and quinic acid, which was quantified for the first time in sugarcane internodal tissue. Analyses were not solely based on the prevailing metabolite levels, but also on the interactions between these metabolites. Thus, in a complementary approach the metabolic flux between the two substrate cycles was assessed by 13C nuclear magnetic resonance (NMR). Analyses of transgenic sugarcane clones with 45-95% reduced cytosolic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) activity displayed no visual phenotypic change, but significant changes were evident in in vivo metabolite levels. Sucrose concentrations increased six and three-fold in young and maturing internodal tissue, respectively. Reduced PFP activity also resulted in an eight-fold increase in the hexose-phosphate: triose-phosphate ratio in the transgenic immature internodes. In addition, the hexose-phosphate: triose-phosphate cycling decreased in the immature internodes of the transgenic lines if compared to the immature control internode. However, there was no significant difference between the hexose-phosphate: triose-phosphate cycling in the mature internodal tissue of the transgenic and the control lines. This illustrated that PFP mediates hexose-phosphate: triose-phosphate cycling in immature sugarcane internodal tissue. Unpredictably, reduced PFP activity led to a ten-fold increase in sucrose cycling in the transgenic immature internodes. The combination of metabolite profiling and flux distribution measurements demonstrated that the fluxes through the sucrose and the hexose-phosphate pools were not co-regulated in sugarcane internodal tissue. From these observations a model was constructed that implicates higher sucrose cycling as a consequence of increased sucrose concentrations.

Thesisa (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2005. / The study had two main objectives: 1) to investigate specific enzyme activity profiles at various developmental stages and to determine possible implications for sucrose metabolism, 2) to incorporate enzyme activity data of different internodes to obtain a detailed model of every stage in the tissue maturation process. The most significant findings of the regulation of sucrose accumulation in this study are centred on three main point controls in sucrose metabolism pathway. Firstly, the maturation of sugarcane internodes coincided with an increase of SPS in most genotypes, and this underlines the key role of this enzyme in sucrose accumulation. Secondly, SuSy activity (cleavage reaction) correlated negatively with sucrose concentration and hence with tissue maturation process, in most of the varieties. This finding indicates that SuSy could well be implicated in sucrose metabolism. Thirdly, in vitro PFP activity was found to be negatively correlated to sucrose content in sugarcane varieties differing in amount of sucrose.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) is an important glycolytic enzyme and catalyses the reversible conversion of fructose-6-phosphate (Fr-6-P) and pyrophosphate (PPi) to fructose 1,6-bisphosphate (Fr-1,6-P2) and inorganic phosphate (Pi). Sugarcane PFP has been inversely correlated with sucrose content across segregating F1 varieties. The down-regulation of PFP in cultivar NCo310 in a previous study led to an increase in sucrose accumulation and fibre content in immature tissue. Several potential transgenic sugarcane lines from genotypes 88H0019 and N27, transformed with the untranslatable sense sugarcane PFP-β gene, were characterized in this study. Initial screening for transgenesis was determined by slot blot and Southern blot analysis to confirm the presence of the co-transformed selectable marker npt II transgene. Northern blot analysis confirmed expression of the 1.2 kb PFP-β transcript in 7 of 9 lines analyzed. Sugar analysis using standard South African Sugarcane Research Institute (SASRI) mill room practices and HPLC was performed on 12 month old pot grown stalks divided into immature and mature tissue sections. The analysis of wild type 88H0019 showed an average sucrose content of 17.84 and 30.76 g sucrose/stalk in immature and mature tissue, respectively. However, no significant difference between the putative transgenic plant values and wild type controls was seen. PFP specific activity was determined in these tissues using enzymatic assay analysis and although levels obtained in immature tissue were between 5-18 nmol/min/mg protein, they were less than values previously reported in sugarcane. The results indicated that no down-regulation of PFP in immature tissue occurred when comparing transgenic and wild type plants. A more discrete internodal tissue sampling method was used to overcome the difficulty of detecting small changes in PFP enzyme activity in bulked stalk tissue sections. Fine analysis of PFP was conducted on specific developmental tissues and single stalks were divided into immature (internodes 1-3), maturing (internodes 4-5) and mature (internodes 7-8) regions. Sucrose analysis was performed using HPLC and PFP activity was determined enzymatically on each tissue type. The analysis of discrete developmental tissues showed specific PFP activity of 60-80 nmol/min/mg protein in young tissue, an amount which falls in the range previously obtained for sugarcane. However there was no significant difference between PFP or sucrose in the transgenic lines when compared with the wild type controls in any of the three developmental tissues examined. Western blotting and densitometric analysis of the blots confirmed the lack of PFP down-regulation in immature tissue in all lines. A final analysis of PFP iv in immature stalk tissue on selected lines was performed using quantitative PCR, which became available near the end of the study. The fold change of each transgenic line indicated that there was a minor increase in PFP confirming the lack of effect of transgenesis. Although evidence for the expression of the PFP-β transgene was seen in the northern blot, no further evidence for transgenesis could be found to support the desired effect of down-regulation of PFP. Characterization of transgenic stalks in this study was hindered by a limited number of lines available for analysis and large variability between replicate samples. Sampling techniques employed in an attempt to make use of existing standard SASRI mill room practices for sugar analysis highlighted the need for a more precise sampling method, specifically when determining the effects of an enzyme manipulation such as PFP. A refined approach has been developed which will assist researchers in the choice of analytical techniques for screening and characterization of potential transgenic lines in the future.

Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. / Despite numerous attempts involving a variety of target genes, the identity of the key regulatory genes of sucrose metabolism in sugarcane is still illusive. To date, genomic research into sucrose accumulation in sugarcane has focused on genes that are expressed in association with stalk development/maturation, with the aim of identifying key regulatory steps in sucrose metabolism. The identification of possible controlling points, however, is complicated by the polyploid nature of sugarcane. Although these studies have yielded extensive annotated gene lists and correlative data, the identity of key regulatory genes remains elusive. A close relative of sugarcane, Sorghum bicolor, is diploid, has a small genome size and accumulates sucrose in the stalk parenchyma. The main aim of the work presented in this thesis was to use S. bicolor as a model to identify genes that are differentially expressed during sucrose accumulation in the stalk of low and high sucrose genotypes. In the first part of the study, a macroarray protocol for identification of differentially expressed genes during sorghum development was established. Firstly, the macroarray sensitivity of probe-target hybridisation was optimised with increasing amounts of target DNA i.e. 0.005-0.075 pmol. The hybridisation signal intensity increased as expected with increasing amounts of probe until the hybridisation signals reached maximum levels at 0.05 pmol. As a result, to ensure quantitative cDNA detection, probes were arrayed at 0.05 pmol when 1 μg target cDNA was used. Secondly, intra-array and inter-array membrane reproducibility was found to be high. In addition, the protocol was able to detect species of mRNA at the lowest detection limit tested (0.06%) and permits the detection of an eight-fold variation in transcript levels. The conclusion was therefore that the protocol was reproducible, robust and can reliably detect changes in mRNA levels. In the second part of the study, sugar accumulation levels in the immature and maturing internodal tissues of sorghum GH1 and SH2 genotypes were compared during the boot and softdough stages. Sugars (i.e. fructose, glucose and sucrose) accumulated differently in the immature and maturing internodes in both sorghum genotypes during the boot and softdough stages, with sucrose being the dominant sugar in both stages. Based on these differences in sugar accumulation patterns, immature and maturing internodal tissues of sorghum genotypes were compared for differentially expressed genes. A number of genes were found to be significantly differentially expressed during both stages. In order to validate the reliability of the macroarray analysis, fourteen genes were arbitrarily selected for semi-quantitative RT-PCR. Seven genes (50%) revealed a similar pattern of transcript expression, confirming the macroarray results. The other seven genes, however, showed a different expression trend compared with the macroarrays. In this study, ESTs from rice and sugarcane were used for probing sorghum. The probability of cross-hybridisation between the probes and various isoforms of the homologous sorghum sequences is thus high, potentially leading to the identification of false positives. In addition, variation in expression patterns could have been introduced by technical and biological variation. Lastly, to verify that changes in the levels of a transcript are also reflected in changes in enzyme activity, seven candidates were tested for enzyme activity. Only three i.e. soluble acid invertase (SAI), sucrose synthase (SuSy) and alcohol dehydrogenase (ADH), out of these seven genes showed enzyme activity levels reflective of the relative transcript expression. We concluded that changes in transcript levels may or may not immediately lead to similar changes in enzyme activity. In addition, enzyme activity may be controlled at transcriptional and at posttranscriptional levels. In conclusion, sugar accumulation in low (GH1) and high (SH2) sucrose sorghum genotypes is influenced by differences in gene expression. In addition, the power of macroarrays and confirmation with semi-quantitative RT-PCR for identification of differentially expressed genes in sorghum genotypes was demonstrated. Moreover, the transcript and enzyme activity patterns of SAI, SuSy and ADH genes showed expression patterns similar to those of sugarcane during sucrose accumulation. Therefore, using sorghum as a model promises to enhance and refine our understanding of sucrose accumulation in sugarcane.

Sugarcane plants (cultivar NCo376) were studied to assess the effects of temperature and season (spring and autumn equinox) on the morphological and physiological response of stalk components. Plants were grown from single-eyed setts for ca. five months and then placed into three temperature controlled glasshouses (22/12°C (C), 27/17°C (W) and 32/22°C (H) day/night temperatures). The plants were sampled twice weekly over a one month period., and intemodes 4, 6 and 10 of the primary haulms of each plant sampled for growth and sugar analysis. During spring, the leaf emergence rates were 0.0303, 0.1095 and 0.1682 leaves d(-1) at temperatures C, W and H, respectively; and 0.0327, 0.0824 and 0.113 leaves d(-1) in autumn. The phyllochron intervals were 114°Cd in spring and 147°Cd in autumn. Highest green leaf blade area of the primary haulms was achieved at H (438.0 and 511.7 cm2 in spring and autumn, respectively). The stalk extension rates were 1.22, 1.02, 0.38 cm d(-1) (spring) and 1.35, 0.98, 0.45 cm d(-1) (autumn), respectively, in descending order of temperature. Total biomass and stalk biomass per plant were not affected by temperature, despite the differences in stem elongation. Internodes of plants at C were shorter but thicker and heavier than the comparable internodes of plants at W and H. In autumn, the mature internode sucrose concentrations were 35.5, 29.2 and 25.5% at C, W and H, respectively; corresponding to mean RS% of 5.7, 9.8 and 13.3%, and fibre % of 58.8, 61.1 and 61.3%, at the respective ascending order of temperature. Sucrose % in the mature internodes in spring were 27.8, 20.9 and 19.9% at C, W and H, respectively; corresponding to RS% of 5.9, 9.76 and 10.9% and fibre % of 66.3, 69.4 and 69.2% at the respective ascending order of temperature. Temperature effect on the concentration of the stalk components of the immature internodes was in general not significant. Sucrose partitioning coefficients in the mature internodes were 0.25, 0.21 and 0.20 in spring and 0.50, 0.32 and 0.21 in autumn (at C, W and H, respectively). Data that resulted from this study, which is isolated to temperature and cultivar NCo376 can be used in models of sugarcane that simulate leaf appearance and senescence, assimilate partitioning between leaf and stalk and assimilate partitioning between the stalk components namely sucrose, reducing sugars and fibre. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Sugarcane is a crop that is farmed commercially due to the high amounts of sucrose that is stored within the mature internodes of the stem. Numerous studies have been done to understand sugar metabolism in this crop as well as to enhance sucrose yields. Until now sugarcane improvement strategies have been implemented through either breeding programs or transgenic manipulation. Public mistrust and regulatory hurdles, however, have made the commercialisation of transgenic crops difficult, expensive and timeconsuming. In this thesis two projects will address issues relating to the above. The first will address an effort to increase sucrose accumulation within the sugarcane culm. This was attempted via the expression of an Arabidopsis thaliana vacuolar pyrophosphatase (AtV-PPase) gene, linked to the maize ubiquitin promoter, in sugarcane callus. It was anticipated that increased activity of the tonoplast-bound AtV-PPase will result in increased sucrose accumulation in the vacuole. Transgenic sugarcane callus lines were tested for soluble sugar content which suggested no significant increase in sucrose content. However, this may change upon further assessment of sugarcane suspension cultures and glasshouse plants. The second project was concerned with the development of a novel sugarcane transformation technology that utilises only sugarcane sequences. This ‘cisgenic’ approach to sugarcane transformation will require a native sugarcane promoter, terminator, vector backbone and selection marker. It was attempted to first isolate a functional promoter as well as developing a selection system based on an endogenous selection marker. A promoter was amplified from sugarcane, using primers designed on a sorghum template, and its expression assessed using a GFP reporter gene. Unfortunately expression could not be confirmed in transgenic sugarcane callus. Currently, an alternative approach is followed by using short fragments of constitutively expressed genes to screen sugarcane Bacterial Artificial Chromosome (BAC) libraries to isolate their corresponding promoters. Lastly, it was attempted to develop a selection system for transgenic sugarcane based on resistance to the herbicide chlorosulfuron. A mutant acetolactate synthase (alsb) gene from tobacco, which has shown to confer resistance to the tobacco, was transformed into sugarcane callus. It was anticipated that this gene will confer chlorosulfuron resistance to transgenic sugarcane. If resistance is achieved, the corresponding sugarcane gene will be mutated via site-directed mutagenesis and checked if it also confers resistance to sugarcane. Results showed that although transgenic lines were generated, resistance development is still inconclusive. / AFRIKAANSE OPSOMMING: Suikerriet is ‘n kommersiële gewas wat verbou word as gevolg van die hoë hoeveelhede sukrose wat gestoor word in die volwasse tussenknope van die stam. Verskeie studies is al gedoen om suiker metabolisme in die gewas te ondersoek, sowel as om die sukrose opbrengs te verhoog. Huidige strategieë vir suikerriet verbetering word beywer deur middel van teel-programme of transgeniese manipulasie. Die kommersialiseëring van transgeniese gewasse word egter bemoeilik deur publieke wanpersepsies, sowel as regulatoriese uitdagings. Hierdie tesis beoog om boenoemde kwessies aan te spreek, deur middel van twee projekte. Die eerste projek poog om sukrose akkumulasie in sukerriet te verhoog. Dit was onderneem om die Arabidopsis thaliana vakuolere pirofosfatase (AtV-PPase) geen, wat verbind is met die mielie ubiquitien promoter, uit te druk in suikerriet kallus. Daar was verwag dat die verhoogde aktiwiteit van die tonoplast-gebonde AtV-PPase sal veroorsaak dat meer sukrose in die vakuool akkumuleer. Oplosbare suiker inhoud was getoets in transgeniese suikerriet kallus lyne, maar geen merkbare verhoging in sukrose inhoud was waargeneem nie. Hierdie mag egter verander met verdere ondersoeke in suikerriet suspensie-kulture en glashuis-plante. Die tweede projek het beywer om ‘n nuwe suikerriet transformasie tegnologie te ontwikkel, wat slegs van suikerriet genetiese materiaal gebruik maak. Hierdie ‘cisgeniese’ benadering tot suikerriet transformasie sal ‘n inheemse suikerriet promoter, terminator, vektor ruggraat en seleksie-merker, benodig. Dit was eers beoog om ‘n funksionele promoter te isoleer, sowel as om ‘n seleksie sisteem, gebasseer op ‘n inheemse seleksie merker, te ontwikkel. Deur gebruik te maak van primers wat op ‘n sorghum templaat gebasseer is, was ‘n promotor geisoleer vanuit suikerriet; die uitdrukking hiervan is bepaal deur gebruik te maak van ‘n GFP verklikker geen. Ongelukkig kon uitdrukking nie bevestig word in transgeniese suikerriet kallus nie. Tans word suikerriet Kunsmatige Bakterieële Chromosoom (KBC) biblioteke geskandeer, deur gebruik te maak van geen-fragmente van globaal-uitgedrukte gene, om ooreenstemmende suikerriet promoters te isoleer. Die tweede deel van die cisgeniese projek het beoog om ‘n seleksie sisteem vir transgeniese suikerriet te ontwikkel, wat gebasseer is op weerstand teen die plantdoder chlorosulfuron. Suikerriet kallus was getranformeer met ‘n mutante tabak geen – asektolaktaat sintase (alsb) – wat chlorosulfuron weerstand in tabak meebring. Daar was verwag dat die geen chlorosulfuron weerstand aan transgeniese suikerriet sou oordra. Indien weerstand ontwikkel, sal die ooreenstemende suikerriet geen deur gerigte mutagenese gemuteer word; dan sal dit kan bepaal word of weerstand ook oorgedra word aan suikerriet. Daar is bevind dat alhoewel transgeniese lyne gegenereer is, daar steeds nie ‘n konklusiewe bevestiging van weerstand ontwikkeling is nie.

Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. / Carbon partitioning was investigated in sugarcane (Saccharum spp. hybrids) that was genetically modified with sucrose: sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) from Cynara scolymus. This enzyme catalyses the transfer of a fructosyl moiety from one sucrose molecule to another to produce the trisaccharide 1-kestose. Molecular characterisation of four sugarcane lines, regenerated after transformation, confirmed that two lines (2153 and 2121) were transgenic, with at least one intact copy of 1-SST present in line 2153, and a minimum of five copies (or portions thereof) present in line 2121. The novel gene was successfully transcribed and translated in both lines, as confirmed by cDNA gel blot hybridisation and HPLC analysis respectively. Kestose production was stable under field resembling conditions and levels of this trisaccharide progressively increased with increasing internodal maturity from 7.94 ± 2.96 nmol.g-1 fresh mass (fm) in internode 6 to 112.01 ± 17.42 nmol.g-1 fm in internode 16 of 2153, and by 1.05 ± 0.93 nmol.g-1 fm from the youngest to the oldest internode in line 2121. Sugarcane line 2153 contained 100 times more 1-kestose than 2121 in the oldest sampled internode hence the lines were referred to as high- and low-1-kestose producers. The production of 1-kestose did not reduce sucrose levels in the transgenics, instead they contained significantly higher levels of sucrose than the control line NCo310 (p<0.01, N=72). The production of this alternative sugar in addition to elevated sucrose levels significantly increased the total sugar content in the transgenic lines (p<0.01, N=72). Moreover, the high-1-kestose producer had statistically more total sugar than the low-1-kestose producer (p<0.01, N=72). Soluble acid invertase (SAI) and neutral invertase (NI, β-fructofuranosidase EC 3.2.1.26) from non-transgenic sugarcane internodal tissues were separated and partially purified. Kinetic analysis of the purified invertases revealed two isoforms of SAI eluting at approximately 100 mM KCl in a linear gradient while NI eluted at approximately 500 mM KCl. The final specific activities of SAI and NI were 88.57 pkat.mg-1 protein and 92.31 pkat.mg-1 protein, respectively. This implied a 16- fold purification of SAI, and 4- fold purification of NI. The pH optimum for NI was 7.0 and that for soluble acid invertase less than 5.0. Due to the broad pH activities of the invertases, activities significantly overlapped between pH 4.5 and 7.0. The affinity of these invertases for 1-kestose hydrolysis was tested. The invertases displayed hyperbolic saturation kinetics for sucrose, and had low affinities for 1-kestose with Km values ranging from 50 - 247 mM. Furthermore, the presence of 200 mM 1-kestose had an inhibitory effect on SAI-mediated sucrose hydrolysis reducing activity to 51 % and 54 % for isoform 1 and 2 respectively. To determine whether carbon allocation had been altered by the expression and activity of 1-SST, 14C whole-plant radiolabelling experiments were conducted. Radiolabelled CO2 was fed to the leaf subtending internode 5 and the allocation of carbon to different parts of the culm was assessed. There was no significant difference in the distribution of total radiolabel down the culm of the three sugarcane lines (p>0.05, N=72). However, the percentage of total radiolabel in the water-soluble fraction per internode in the high-1-kestose producer was significantly higher than the other two lines (p<0.01, N=72). As a result, the percentage radiolabel in the waterinsoluble fraction in this transgenic was concomitantly lower than in the other lines. Carbon was therefore redirected from the water-insoluble fraction to the water-soluble fraction to account for the additive production of 1-kestose. The expression of 1-SST in sugarcane therefore established an additional carbohydrate sink by the flow of carbon from the sucrose pool into 1-kestose. This did not lead to a depletion of the sucrose pool, but rather stimulated carbon channelling into this pathway, thereby increasing the non-structural carbohydrate content of the plant in one of the transgenics. The work described in this study is the first to report on carbon partitioning in 1- kestose-producing sugarcane grown under field resembling conditions. It contributes significantly to an improved understanding of carbon partitioning in the culm, and demonstrates that an alternative sugar can be produced in sugarcane under field resembling conditions.