Browsing Masters Degrees (Genetics) by browse.metadata.advisor "Botha, F. C."
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- ItemCarbon turnover and sucrose metabolism in the culm of transgenic sugarcane producing 1-Kestose(Stellenbosch : University of Stellenbosch, 2007-12) Nicholson, Tarryn Louise; Botha, F. C.; Huckett, B. I.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology.Carbon partitioning was investigated in sugarcane (Saccharum spp. hybrids) that was genetically modified with sucrose: sucrose 1-fructosyltransferase (1-SST; EC 18.104.22.168) 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 22.214.171.124) 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.
- ItemThe characterization of vacuolar pyrophosphatase expression in sugarcane(Stellenbosch : University of Stellenbosch, 2005-03) Swart, Johannes Cornelius; Groenewald, J-H.; Botha, F. C.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB)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.
- ItemDevelopment of in situ hybridisation to examine tissue-specific expression patterns of the invertase genes in sugarcane culm(Stellenbosch : University of Stellenbosch, 2005-04) Turner, Gabrielle M.; Cramer, M. D.; Botha, F. C.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).ENGLISH ABSTRACT: The goals of this project were firstly to develop the tissue preparation and in situ hybridisation protocols for sugarcane culm tissue, and secondly to use the developed techniques to examine the expression patterns of three invertase isoforms in sugarcane internodes of various developmental stages. Sugarcane invertases have been the focus of intense research for many years, yet almost nothing is known of their tissue-specific distribution. It was thought that by characterising their expression patterns using in situ hybridisation, more knowledge of their functions and involvement in sucrose accumulation would be gained. Although in situ hybridisation is now regularly used to study gene expression in plants, there is to date only a single publication describing its use on immature sugarcane tissue. Therefore this technique needed further development, and this was achieved by comparing different tissue preparation methods, as well as by systematically testing the various parameters pertaining to each method. The in situ hybridization technique was also developed by testing and comparing a number of key parameters. It was found that fixing whole mount tissue for 48 h preserved sugarcane tissue adequately. High hybridization temperatures and probe concentrations provided the best signal, and including pre-treatment with HCl and Pronase was essential in sensitizing the tissue to the probe. A less viscous detection buffer reduced both osmotic effects and time required for signal detection. In the second part of this study, the developed method was used to examine the expression patterns of the three invertase isoforms in young, maturing and mature internodes of sugarcane, and the results were complemented with Northern blot analysis. Transcript of all three isoforms was found to be present in the storage parenchyma and in the phloem tissue. Transcript levels of all three isoforms declined in maturing tissue, with soluble acid invertase declining sharply and dropping below detection in maturing and mature tissue. Transcript levels of cell wall invertase and neutral invertase declined only gradually, and appreciable levels of both were still present in mature tissue. Acid invertase is suggested to be mainly involved in internode elongation, while cell wall invertase would appear to play important roles in phloem unloading and turgor control. Neutral invertase is suggested to be involved in either sucrose cycling or maintenance of hexose pools, however the function of this enzyme remains unclear. This study has demonstrated the value of in situ hybridization, yet at the same time has shown its limitations, especially when more traditional biochemical techniques are not employed to complement the results. Although the precise functions of the invertase isoforms in sugarcane remain inconclusive, this study has opened up the way for tissuespecific promoter design and future in situ studies of sugarcane invertases
- ItemDifferential gene expression during berry ripening in Vitis vinifera (cv Chardonnay) : isolation of specific sequences through subtractive cloning(Stellenbosch : Stellenbosch University, 2002-12) Olivier, Abraham Jacobus; Botha, F. C.; Groenewald, S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).ENGLISH ABSTRACT: Grapevine is worldwide an agronomically important crop. Traditionally selective breeding has been used to improve existing cultivars. In the last ten years, however, the advent of biotechnology has shortened these breeding programmes by producing transgenic grapevine. Because this new technology is aimed at the possible genetic manipulation of the ripening process in grape berries, it is important to elucidate all the mechanisms that may be involved in ripening. The aim of the present study was the identification of genes that play an important role during the ripening process in grape berries. This was achieved by investigation of putative differentially expressed genes in ripening Chardonnay berries isolated through subtractive hybridisation. Two subtraction libraries, representing early and late ripening stages were constructed. Four of the ten genes analysed exhibited expression during berry ripening. One of the four genes was expressed in a tissue and stage specific manner. Further characterisation of eight of the DNA and protein sequences revealed that the putative translation products of these clones had homologues that are involved in amongst others cell wall structure in other species. These included UDP-glucose dehydrogenase, which is involved in the synthesis of hemicellulose precursors. The remaining seven clones encoded putative stress response proteins. These included two heat shock proteins, a vacuolar pyrophosphatase and a protein involved in cell division. It is suggested that specific grape mRNAs accumulate in response to stresses such as the storage of high concentrations of sugars and rapid cell expansion. These processes occur rapidly during the ripening of berries. Accumulation of specific mRNAs can be attributed to part of the normal ripening developmental programme.
- ItemEnzyme profiling of a range of sugarcane tissue types with different levels of sucrose(Stellenbosch : University of Stellenbosch, 2005-12) Orendo-Smith, R.; Botha, F. C.; Groenewald, J-H.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.
- ItemExpression behaviour of primary carbon metabolism genes during sugarcane culm development(Stellenbosch : University of Stellenbosch, 2004-04) McCormick, Alistair James; Watt, D.; Huckett, B. I.; Botha, F. C.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology.ENGLISH ABSTRACT: Despite numerous attempts involving a variety of target genes, the successful transgenic manipulation of sucrose accumulation in sugarcane remains elusive. It is becoming increasingly apparent that enhancing sucrose storage in the culm by molecular means may depend on the modification of the activity of a novel gene target. One possible approach to identify target genes playing crucial coarse regulatory roles in sucrose accumulation is to assess gene expression during the developmental transition of the culm from active growth to maturation. This study has resulted in the successful optimisation of a mRNA hybridisation technique to characterise the expression of 90 carbohydrate metabolism-related genes in three developmentally distinct regions of sugarcane culm. A further goal of this work was to extend the limited knowledge of the regulation of sucrose metabolism in sugarcane, as well as to complement existing data from physiological and biochemical studies. Three mRNA populations derived from the different culm regions were assayed and their hybridisation intensities to the immobilised gene sequences statistically evaluated. The relative mRNA transcript abundance of 74 genes from three differing regions of culm maturity was documented. Genes exhibiting high relative expression in the culm included aldolase, hexokinase, cellulase, alcohol dehydrogenase and soluble acid invertase. Several genes (15) were demonstrated to have significantly different expression levels in the culm regions assessed. These included UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which were down-regulated between immature and mature internodes. Conversely, sucrose phosphate synthase, sucrose synthase and neutral invertase exhibited up-regulation in maturing internodal tissue. A variety of sugar transporters were also found to be up-regulated in mature culm, indicating a possible control point of flux into mature stem sink tissues. Combined with knowledge of the levels of key metabolites and metabolic intermediates this gene expression data will contribute to identifying key control points of sucrose accumulation in sugarcane and assist in the identification of gene targets for future manipulation by transgenic approaches.
- ItemThe influence of genetic manipulation of cytosolic aldolase (ALDc) on respiration in sugarcane(Stellenbosch : University of Stellenbosch, 2005-03) Scheepers, Ilana; Botha, F. C.; Potier, B. A. M.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB)Previous studies indicated that cytosolic aldolase (ALDc) could be a rate limiting step in glycolysis and thus play a role in the regulation of carbon partitioning in sink tissues. In this study the role of ALDc in sugarcane was studied. Expression patterns of both ALDc transcript and protein were examined. In contrast to the leaves where ALDc expression is very low, the enzyme (transcript and protein) levels were high in all internodal tissues at all stages of maturity. In the leaves the plastidic isoform was prevalent as found previously in other C4 plants. The similar pattern of expression in transcript and protein abundance illustrate that there are no activators or inhibitors of ALDc activity present in sugarcane. The control on ALDc activity in sugarcane is therefore regulation of gene expression. To investigate the possibility that ALDc could be regulating carbon partitioning in sugarcane a series of transgenic sugarcane plants in the varieties NCo310 and N19 were produced. The presence and expression of the transgene and resultant effect on ALDc levels were determined for all the transgenic lines. The degree of ALDc reduction varied, with the biggest suppression of aldolase being 90% of that of the control plants. Alteration of ALDc activity caused no obvious phenotype. In both the varieties large decreases in ALDc tended to to lead to higher sucrose levels than that of the the control plants. 14C radiolabelling studies were conducted to investigate the effect of reduced ALDc levels on respiration and carbon partitioning. No differences in carbon metabolism could be found between the transgenic and control plants. Even in the line exhibiting a more than 90% decrease, the residual ALDc was sufficient for plants to grow normally under favourable glasshouse conditions. This would suggest that ALDc does not play a role in the regulation of flux through glycolysis, carbon partitioning and sucrose accumulation.
- ItemInfluence of hexose-phosphates and carbon cycling on sucrose accumulation in sugarcane spp.(Stellenbosch : University of Stellenbosch, 2005-12) Van der Merwe, Margaretha Johanna; Botha, F. C.; Groenewald, J-H.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.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 126.96.36.199) 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.
- ItemIsolation and characterisation of a culm-specific promoter element from sugarcane(Stellenbosch : University of Stellenbosch, 2005-03) Goshu Abraha, Tsion; Botha, F. C.; Groenewald, J-H.; University of Stellenbosch. Faculty of Science. Dept of Botany and Zoology. Institute of Plant Biotechnology.Sugarcane (Saccharum spp) is an important crop worldwide and is cultivated for the high level of sucrose in its mature internodes. Because of the exhaustion of the genetic potential in the commercial sugarcane germplasm conventional breeding has not lately been able to enhance sucrose content. Currently there is a concerted effort to improve culm sucrose content by genetic engineering which will require appropriate transgenes and promoters. One of the major constraints to genetic engineering of sugarcane is the lack of stable promoters required to drive tissue- or organ-specific expression of transgenes. Tissue and developmental stage specific promoters allow targeting of transgene activity and in doing so reduce the impact on non-target tissues. These promoters could also be advantageous to manipulate certain aspects of sucrose metabolism specifically in mature culm tissue. In addition, no promoters are currently freely available to the South African Sugar Industry for use in their transgenic program. The primary goal of this project was therefore to isolate a mature tissue-specific promoter for use in transgenic sugarcane plants. The approach followed was firstly, to identify an endogenous gene expressed in the desired pattern, and then to isolate the corresponding promoter from the sugarcane genome. cDNA macroarrays were initially used to identify differentially expressed sequences. The tissue specificity of potential clones was confirmed using RNA blot analysis. Two clones (c23-a and c22-a) were isolated and confirmed to be mature culm specific. Clone c22-a (putative dirigent-like protein) was selected for promoter isolation based on its culm tissue specific expression pattern and its proximity to the 5’ end of the gene. Furthermore, to confirm the activity of this promoter in the storage parenchyma cells, the exact cellular localisation of the transcript in the mature tissue was determined through in situ hybridisation. In situ hybridisation results confirmed the presence of the transcript in the parenchyma cells of mature culm tissue only. Moreover, the transcript is present in high concentrations in the parenchyma tissues surrounding the vascular bundles and parenchyma cells of the vascular complex. The selected dirigent-like gene was sequenced to allow the design of primers that could be used for the isolation of the corresponding promoter region using a long-range inverse PCR (LR-iPCR) method. Using these we have successfully isolated two highly homologous promoter regions of the dirigent like gene of respectively 1151 and 985 base pairs. In silico analyses confirmed the presence of various transcription motifs, including a TATA-box. However, experimental verification is needed to fully assess the functionality of these promoter regions. Verifying the activity of the isolated promoters through transient expression analysis proved to be problematic because of their highly mature culm specificity. Both constructs are therefore being used to obtain stable transformants in which promoter activity can be evaluated in mature internodal tissues.
- ItemThe manipulation of fructose 2,6-bisphosphate levels in sugarcane(Stellenbosch : University of Stellenbosch, 2006-03) Hiten, Nicholas Fletcher; Botha, F. C.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB)Fructose 2,6-bisphosphate (Fru 2,6-P2) is an important regulatory molecule in plant carbohydrate metabolism. There were three main objectives in this study. Firstly, to determine whether the recombinant rat 6-phosphofructo 2-kinase (6PF2K, EC 188.8.131.52) and fructose 2,6-bisphosphatase (FBPase2, EC 184.108.40.206) enzymes, which catalyse the synthesis and degradation of Fru 2,6-P2 respectively, showed any catalytic activity as fusion proteins. Secondly, to alter the levels of Fru 2,6-P2 in sugarcane, an important agricultural crop due to its ability to store large quantities of sucrose, by expressing the recombinant genes. Thirdly, to investigate whether sugar metabolism in photosynthetic- (leaves) and non-photosynthetic tissue (internodes) were subsequently influenced. Activity tests performed on the bacterially expressed glutathione-S-transferase (GST) fusion 6PF2K and FBPase2 enzymes showed that they were catalytically active. In addition antibodies were raised against the bacterially expressed proteins. Methods for extracting and measuring Fru 2,6-P2 from sugarcane tissues had to be optimised because it is known that the extraction efficiencies of Fru 2,6-P2 could vary significantly between different plant species and also within tissues from the same species. A chloroform/methanol extraction method was established that provided Fru 2,6-P2 recoveries of 93% and 85% from sugarcane leaves and internodes respectively. Diurnal changes in the levels of Fru 2,6-P2, sucrose and starch were measured and the results suggested a role for Fru 2,6-P2 in photosynthetic sucrose metabolism and in the partitioning of carbon between sucrose and starch in sugarcane leaves. Transgenic sugarcane plants expressing either a recombinant rat FBPase2 (ODe lines) or 6PF2K (OCe lines) were generated. The ODe lines contained decreased leaf Fru 2,6-P2 levels but increased internodal Fru 2,6-P2 levels compared to the control plants. Higher leaf sucrose and reducing sugars (glucose and fructose) were measured in the transgenic plants than the control plants. The transgenic lines contained decreased internodal sucrose and increased reducing sugars compared to the control plants. Opposite trends were observed for Fru 2,6-P2 and sucrose when leaves, internodes 3+4 or internodes 7+8 of the different plant lines were compared. In contrast, no consistent trends between Fru 2,6-P2 and sucrose were evident in the OCe transgenic lines.
- ItemResponses of sugarcane to aluminium toxicity(Stellenbosch : Stellenbosch University, 2006-03) Rowland, Ronelle; Cramer, M. D.; Botha, F. C.; Watt, D. A.; Stellenbosch University. Faculty of Agriscience. Dept. of Genetics.ENGLISH ABSTRACT: The aims of this study were (1) to determine whether N03- orNH4+ nutrition could influence the effect of Al on N12 and N19 sugarcane plants grown in sand or hydroponic medium and (2) to assess whether the different root environments of sand and hydroponically cultured plants could influence the effect of Al on Nl2 and N19 sugarcane plants supplied with N03- or NH/ nutrition. N12 and N19 sugarcane was grown in sand and hydroponic culture with and without Al and with either N03- or NH4+as N source. Biomass accumulation, tissue N, P and Al and root assimilation of 14C02 supplied to the root system were measured. Both N12 and N19 sugarcane plants were found to be relatively Al tolerant (tolerating up to 1 mM Al). This lack of effect of Al on plant growth might be due to amelioration of Al-toxicity in sugarcane by the adequate supply of carbon skeletons from the C4 photosynthetic pathways of sugarcane. The supply of carbon skeletons may enable both cultivars to exude large amounts of organic acids into the rhizosphere, which confers a dual advantage to these plants. Organic acids can form soluble complexes with Al thus preventing its entry into the roots and can form soluble complexes with nutrients (cations), which makes some nutrients (e.g. P) more available for plant uptake. The availability of carbon in the root system is dependent on the N source. Increased growth of Al treated plants supplied with NH4 +, relative to those grown on N03-, might be due to the capacity of C4 photosynthesis to meet the needs for both the assimilation of NH4 + into amino acids and the synthesis/excretion of organic acids for Al-detoxification. The fact that growth was improved with NNH4 + and Al may indicate that NH4 + and Al cations compete for access to the root tissue resulting in an increase in root activity, organic acid exudation and nutrient uptake. It was postulated that sand-grown plants would be less influenced by Al than hydroponic plants because organic acids can form a protective sheath that shields the root apex from the toxic Al cations in sand due to the relative lack of mobility of the soil solution. However, Al increased the growth of NH4 +-fed hydroponically grown plants more than that of NH4 +-fed sand grown plants. Thus we did not find evidence to support our expectation that the roots of the hydroponically grown plants would be more exposed to Al due to nutrients and organic acids being uniformly distributed in the growth solution compared to sand grown plants.
- ItemSucrose transporters and sucrose uptake mechanisms in sugarcane(Stellenbosch : University of Stellenbosch, 2004-12) Titus, Charlene H. A. (Charlene Helecyn Agatha); Cramer, M. D.; Botha, F. C.; University of Stellenbosch. Faculty of Science. Dept. of Botany and Zoology. Institute for Plant Biotechnology.ENGLISH ABSTRACT: The process of sugar accumulation and transport in sugarcane is still poorly understood. Understanding the processes involved in sucrose transport are important, since membrane transport might be important control points in this pathway. The goals of this project were to unravel the mechanisms of sugar transport in sugarcane culm tissue by using 14C-sugar analysis as well as molecular techniques to identify possible sucrose transporters. Developing (internode 2 and 4) and maturing (internode 8 and 15) culm tissue of sugarcane (Saccharum hybrid) commercial variety N19 was used for all tissue disc experiments. Tissue discs from internodes of different developmental stages were cut from field grown sugarcane plants (cv. N19) and the uptake of 14C-labelled glucose, fructose and sucrose measured. The uptake rates were measured at varying pH, temperature and concentrations of sugars. Hexoses were found to be the major sugar taken up and sucrose was only important when little hexose was available, as was found in the mature ripe internodes. Sucrose uptake differs between tissues and our study showed that sucrose was taken up rapidly at pH 5, similar to the pH optimum of most sucrose transporters Inhibition studies with TRIS (2-amino-2- (hydroxymethyl)-1,3-propanediol) and PCMBS (p-chloromercuribenzenesulphonic acid) indicated that more than one sucrose transporter activity may be present in the sugarcane system at different sucrose concentrations. To date work on sugarcane sucrose transporter expression on DNA and RNA level has been limited. Only recently a sucrose transporter from Saccharum hybrid sugarcane stem cDNA libray, ShSUT1 (Saccharum hybrid Sucrose Transporter ) was isolated and functionally characterized in the yeast strain SEY 6210 (Rae et al., 2004). In an effort to understand sucrose transport in sugarcane culm tissue, a partial sucrose transporter cDNA, ScSUT1(p) from Saccharum hybrid sugarcane a bud cDNA library was isolated, and cloned from a bud cDNA library. The clone was designated ScSUT(p) as a partial Sugarcane Sucrose Transporter. The ScSUT1(p) sequence showed 94% identity to ShSUT1 on nucleotide level over 1258 nucleotides and had an estimated open reading frame of 419 amino acids. Southern blot analysis indicated that the transporter had a low copy number and the ScSUT1(p) transcript expression was constitutive in sucrose accumulating and sucrose storing stem tissue, but was less abundant in immature tissue such as internodes 2 and 3 and in lateral buds. It was concluded that the primary function of ScSUT1(p), was not phloem unloading but that the transporter may be involved in phloem loading, as it is abundant in mature source leaves. ShSUT1 cDNA was obtained from Dr C Grof and the functionality of ShSUT1 as a sucrose transporter in Xenopus leavis oocytes was confirmed. However, electrophysiological measurements on the oocytes demonstrated no measurable current associated with sucrose challenge to the oocytes indicating that the transporter activity was either very low or possibly non-electrogenic. Further investigation is required to characterise the specific mechanism and kinetic properties of this transporter.