In order to understand and manipulate carbon flux to sucrose one needs to consider not only its biosynthetic pathways, but also the competing sinks for carbon in various parts of the plant and at different stages of development. The cell wall and sucrose is known to be the major sinks for carbon in young and mature tissues of sugarcane. UDP-Glucose is a central metabolite in the synthesis of both sucrose and most of the cell wall polysaccharides (including cellulose, hemicellulose and pectic polymers) and manipulation of the flux into either of the cell wall components could therefore cause an increase of flux toward one or more of the competing sinks. In the present study UDP-Glucose dehydrogenase (UGD) activity was chosen for down regulation as it catalyzes the rate limiting step in the biosynthesis of the precursors of both hemicellulose and pectin, a major competing sink for assimilated carbon. Transgenic sugarcane lines with repressed UGD activity showed significantly increased sucrose accumulation in all internodes which was highly correlated with reduced UGD activity. Sucrose phosphate synthase had increased activation which suggests an alteration in carbon flux toward sucrose. The reduction of carbon flux through UGD was compensated for by an increase in the activity of the myo-inositol oxygenation pathway (MIOP), an alternative pathway for the synthesis of cell wall matrix precursors. The increased activity of the MIOP resulted in increased total uronic acids and pentoses in the cell wall. Total cell wall glucose was also increased which is a further indication of altered carbon metabolism.

Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2005. / The current understanding of the regulation of sucrose accumulation is still incomplete even though many scientists have investigated this subject. Components of trehalose metabolism have been implicated in the regulation of carbon flux in bacteria, yeast and more recently in plants. With a view to placing trehalose metabolism in the context of cytosolic sugarcane sucrose metabolism and carbon partitioning we have investigated the metabolites, transcripts and enzymes involved in this branch of carbohydrate metabolism in sugarcane internodal tissues. Sugarcane internodal trehalose levels varied between 0.31 ± 0.09 and 3.91 ± 0.99 nmol.g-1 fresh weight (FW). From statistical analysis of the metabolite profile it would appear that trehalose does not directly affect sucrose accumulation, although this does not preclude involvement of trehalose- 6-phosphate in the regulation of carbon partitioning. The metabolite data generated in this study demanded further investigation into the enzymes (and their transcripts) responsible for trehalose metabolism. Trehalose is synthesised in a two step process by the enzymes trehalose-6-phosphate synthase (EC 2.4.1.15, TPS) and trehalose-6-phosphate phosphatase (EC 3.1.3.12, TPP), and degraded by trehalase (EC 3.2.1.28). Two novel sugarcane partial cDNAs that coded for trehalase (tre) and actin (required for normalisation in profiling experiments) were isolated and used along with partial transcripts for TPS and TPP to determine transcript levels in different tissue- and genotypes. A putative full-length SugTPS cDNA was isolated and characterised. Enzyme activities for TPS, TPP and trehalase were measured at levels of 2.7 nmol.min-1.mg-1protein, 8.5 nmol.min-1.mg-1protein and 6.2 nmol.min-1.mg-1protein respectively, from young internodal protein extracts of sugarcane, variety N19. TPP enzyme activity and transcript levels were higher in S. spontaneum than Saccharum interspecific hybrids. Kinetic analysis of TPP and trehalase activities were performed with the purpose of providing parameters for an in silico kinetic model of trehalose and sucrose metabolism. Three isoforms of TPP were identified and desingated TPPAI, TPPAII and TPPB. Both TPPA isoforms had pH optima of 6.0, and TPPB of pH 6.5. Apparent Km values were determined as 0.447 ± 0.007 mM for TPPAI, 13.82 ± 1.98 mM for TPPAII and 1.387 ± 0.18 mM for TPPB. Partial purification and characterisation of trehalase demonstrated dual pH optima of 3.5 and 6.0, with Km values between 0.345 and 0.375 mM. These data were used as the basis for a kinetic model of trehalose metabolism. A previously described kinetic model of cytosolic sucrose metabolism has been expanded to include the trehalose pathway (TPS, TPP and trehalase). The aim was to supplement the available information on cytosolic metabolism in sugarcane storage parenchyma, identify points of control between sucrose and trehalose metabolism, and provide a platform from which further experimental and in silico modelling can be launched. The model predicted trehalose in the same order of magnitude as those determined in the metabolite profiling experiments. The majority of control of flux over the trehalose pathway resided in the TPS step, with flux control coefficients > 70% of the total pathway. Incorporation of the trehalose branch into the original sucrose model showed that reactions from the original model significantly affected the steady-state attributes of the trehalose pathway. Due to the relatively low flux through the trehalose branch of the expanded model, complete recycling of trehalose, and the lack of allosteric regulation by trehalose-6-phosphate or trehalose on any of the reactions from the original sucrose model, incorporation of the trehalose branch had no significant effect on either steady-state cytosolic sucrose concentration or flux of sucrose into the vacuole. The expanded model affords a basis from which to further investigate trehalose metabolism in the context of plant sucrose accumulation.

Dissertation (PhD) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Sugarcane (Saccharum spp. hybrids) is a commercial crop plant capable of storing up to 20% sucrose on a fresh mass basis in the culm. Knowledge about gene expression during sugarcane growth and maturation is limited. The aim of this study was to assess whether an Expressed Sequence Tag (EST)-based approach towards analysis of sugarcane would reveal new information about gene expression and metabolic processes associated with sugarcane growth and development. The specific objectives were two-fold: firstly, to develop an EST database for sugarcane and secondly, to identify and analyse genes that are expressed in different sugarcane tissue types and developmental stages, with a specific focus on leaf and culm. An EST database for sugarcane was initiated to obtain information on sugarcane gene sequences. A total cDNA library was constructed from sugarcane immature leaf (leaf roll: meristematic region) tissue and 250 clones randomly selected and subjected to single-pass DNA sequence analysis. Sugarcane ESTs were identified by sequence similarity searches against gene sequences in international databases. Of the 250 leaf roll clones, 26% exhibited similarity to known plant genes, 50% to non-plant genes while 24% represented new gene sequences. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes. A significant proportion of genes identified in the leaf roll were involved in processes related to protein synthesis and protein modification, as would be expected in meristematic tissues. Submission of 495 sugarcane gene sequences to the dbEST database represented the first sugarcane ESTs released into the public domain. Two subtracted cDNA libraries were constructed by reciprocal subtractive hybridisation between sugarcane immature and maturing internodal tissue. To explore gene expression during sugarcane culm maturation, partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries was performed. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases while 111 matched uncharacterised plant ESTs only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. The expression patterns of sugarcane genes were examined in different tissue sources and developmental stages to identify differentially expressed genes. cDNA arrays containing 1000 random clones from immature leaf and maturing culm cDNA libraries were hybridised with poly (At RNA from immature leaf, mature leaf, immature culm and maturing culm. All cDNAs examined hybridised to all four probes, but differences in signal intensity were observed for individual cDNAs between hybridisation events. No cDNAs displaying tissue- or developmental-stage specific expression were detected. Comparisons between hybridisation patterns identified 61 cDNAs that were more abundantly expressed in immature and mature leaf than the culm. Likewise, 25 cDNAs preferentially expressed in immature and maturing culm were detected. ESTs established for the differentially expressed cDNAs revealed sequence homology to a diverse collection of genes in both the leaf and the culm. These included genes associated with general cellular metabolism, transport, regulation and a variety of stress responses. None of the differentially expressed genes identified in the culm were homologous to genes known to be associated with sucrose accumulation. To examme differences at the level of gene transcription between low sucroseaccumulating and high sucrose-accumulating tissues, subtracted cDNA libraries were utilised. To isolate cDNAs differentially expressed during culm maturation, cDNA arrays containing 400 random clones (200 from each library) were screened with total cDNA probes prepared from immature and maturing culm poly (At RNA. Results indicated that 36% and 30% of the total number of cDNAs analysed were preferentially expressed in the immature and maturing culm, respectively. Northern analysis of selected clones confirmed culm developmental stage-preferential expression for most of the clones tested. ESTs generated for the 132 differentially expressed clones isolated exhibited homology to genes associated with cell wall metabolism, carbohydrate metabolism, stress responses and regulation, where the specific ESTs identified in the immature and maturing culm were distinct from each other. No developmentally regulated ESTs directly associated with sucrose metabolism were detected. These results suggest that growth and maturation of the sugarcane culm is associated with the expression of genes for a wide variety of metabolic processes. In addition, genes encoding enzymes directly involved with sucrose accumulation do not appear to be abundantly expressed in the culm. / AFRIKAANSE OPSOMMING: Kommersiële suikerriet variëteite (Saccharum spp. hibriede) is in staat om tot 20% sukrose op 'n vars massa basis in die stingel op te berg. Kennis oor geenuitdrukking tydens groei en rypwording is beperk. Die doel van die huidige studie was om vas te stelof 'n grootskaalse karatersisering van die geenvolgordes wat uitgedruk word "Expressed Sequence Tag (EST)-based approach" tot nuwe inligting aangaande die aard en omvang van metabolisme tydens groei en ontwikkeling van suikerriet sal lei. 'n Tweeledige benadering is in hierdie studie gevolg. Eerstens is 'n data basis oor die gene wat uitgedruk word "EST" databasis opgestel. Tweedens is gene geïdentifiseer en gekarakteriseer wat spesifiek op verskillende stadiums van ontwikkeling en in spesifiek weefsel uitgedruk word. Vir die opstel van die EST-databasis is 250 klone uit 'n totale cDNA biblioteek vanaf RNA uit suikerrietblaarweefsel (blaarrol:meristematiese streek) op 'n lukraak basis gekies en aan 'n enkel eenrigting DNA volgorde analise onderwerp. Suikerrriet EST's is geïdentifiseer deur middel van homologie soektogte teen geenvolgordes in internasionale databasisse. Uit die 250 blaarrol klone het 26% ooreenkomste met bekende plant gene en, 50% met nie-plant gene getoon. Ongeveer 24% het nuwe geenvolgordes verteenwoordig. Analise van die geïdentifeseerde klone het ooreenkomste met 'n breë diversiteit van gene getoon. 'n Betekenisvolle gedeelte van gene wat in die blaarrol geïdentifiseer is, is by proteïensintese en proteïenmodifikasies betrokke. Dit is in ooreenstemming met wat van meristematiese weefsel verwag kan word. Die 495 suikerriet geenvolgordes wat in die internasionale dbEST databasis gestort is, is die eerste sodanige inligting in die publieke domein. Twee spesifieke cDNA biblioteke (subtraction libraries) wat volgordes spesifiek aan onvolwasse suikerriet en rypwordende internodale weefsel bevat is voorberei. Geenuitdrukking gedurende die rypwordingsproses van die suikerrietstingel is bestudeer deur geenvolgorde analises van onwillekeurige geselekteerde klone van die twee eDNA biblioteke te doen. Van die 337 geenvolgordes wat geanaliseer is het 167 homologie met bekende gene en net 111ooreenkomste met ongekarakteriseerde plant gene getoon. Die oorblywende geenvolgordes het geen ooreenkomste met bekende gene getoon nie en daar kan dus aanvaar word dat hulle nuwe gene verteenwoordig. Die meerderheid ESTs het ooreenkomste met verskeie gene wat met sellulêre metabolisme geassosieer word getoon. ESTs wat homoloog was aan verskeie spannings geassosieerde gene was ook goed verteenwoordig. Die analise het gene wat by voorkeur tydens stringelrypwording uitgedruk word geidentifiseer. Die geenuitdrukkingspatrone van suikerriet in weefsels van verskillende oorsprong en ontwikkelingstadia is ondersoek om differensieel uitgedrukte gene te identifiseer. Reekse wat 1000 lukrake eDNA klone van onvolwasse en rypwordende stingel eDNA biblioteke is met poli-(A)-RNA van onvolwasse blaar, volwasse blaar, onvolwasse stingel en volwasse stingel gehibridiseer. Al die eDNA klone wat ondersoek is het met al vier die peilers gehibridiseer. Die intensiteit van die seine het egter grootliks gevarieer. Die analise het gelei tot die identifisering van 61 eDNA klone wat teen hoër vlakke in onvolwasse en volwasse blaar as in die stingel uitgedruk word. Daar is ook 25 eDNA klone wat by voorkeur in onvolwasse en rypwordende stingel uitgedruk word gevind. Gene wat geassosieer word met gewone sel metabolisme, vervoer prosesse, regulering en verskeie spannings-geassosieerde reaksies, is in die twee groepe teenwoordig. Geeneen van die volgordes wat selektief uitgedruk word kan met gene wat direk met sukrose akkumulering verband hou geassosieer word nie. Ten einde eDNA klone wat differensieel tydens rypwording van die stingel uitgedruk word te isoleer, is 400 eDNA klone (200 van elke biblioteek) lukraak geselekteer en met totale eDNA peilers, wat uit onvolwasse en rypwordende stingel poli-(A)-RNA voorberei is, gesif. Resultate het aangetoon dat 36% en 30% van die totale getal eDNA klonewat geanaliseer is, by voorkeur in die onvolwasse en rypwordende stingel uitgedruk word. RNA kladanalises van geselekteerde klone het getoon dat die meeste ontwikkelingstadium spesifieke uirtdrukkingspatrone het. Daar is gevind dat 132 van die EST klone homologie met gene geassosieerd met selwand- en koolhidraatmetabolisme, spannings geassosieerde- en reguleringsreaksies, toon. Die spesifieke ESTs wat in die onvolwasse en rypwordende stingel geïdentifiseer is het van mekaar verskil. Nie een van die ESTs wat geïdentifiseer is kan direk met sukrose metabolisme geassosieer word nie. Hierdie werk toon baie duidelik aan dat groei en rypwording van die suikerrietstingel met die uitdrukking van gene geassosieerd is wat by 'n hele aantal metaboliese prosesse betrokke is. Die resultate toon ook dat die gene wat vir ensieme kodeer wat direk by sukrose akkumulering betrokke is, nie teen hoë vlakke in die stingel uitgedruk word nie.

Thesis (PhD)--Stellenbosch University, 2012. / Salinity stress is one of the major environmental factors that lead to poor crop yield. This is due to overproduction of reactive oxygen species (ROS) which consequently lead to oxidative stress. Although these ROS may be required for normal physiological functions, their accumulation acts as a double edge sword, as they also cause oxidative damage to nucleic acids, lipids and proteins of plant cell membranes. Plants have evolved with an efficient antioxidant defensive system in order to protect and detoxify harmful effects of ROS. Ascorbate peroxidase (APX) is regarded as one of the major scavengers of H2O2. Although some studies have described the role of nitric oxide (NO) in diverse physiological processes in plants, there is still much to know as regards to modulation of APX activity by nitric oxide in salinity-induced stressed plants. For the purposes of this study, the effect of salt and exogenously applied NO on APX, dehydroascorbate reductase and antioxidant metabolite content was determined. This study investigated the use of NO donor 2,2'-(hydroxynitrosohydrazono) bis-ethanimine (DETA/NO) and diethylenetriamine (DETA) on soybean. The data obtained from this study shows that application of DETA/NO resulted in an increase of NO nodular content and also regulated APX activity. The NO-induced changes in APX enzymatic activity were coupled to altered nodule H2O2 content. Further analysis of APX enzymatic activity identified three APX isoforms for which augmented enzymatic activity occurred in response to NO. By supplementing salinity-induced stress soybeans with NO, this study shows that tolerance to salt stress is improved. The underlying mechanism of the NO-mediated tolerance to salt is shown to be its role in modulating the plant antioxidant defense system thus maintaining redox status under salinity-induced stress. Here, although there was increased APX activity in salt stressed plant, supplementing the salinity-induce stressed plants with NO resulted to even higher APX activity which was sufficient to detoxify ROS. Furthermore, this study shows that the NO-mediated effect is not limited in antioxidant enzymes but also involves regulating antioxidant metabolite ratio through modulating the antioxidant enzymes that are involved in the ascorbate -glutathione cycle.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: This project involved the analysis of genes in Solanaceae species that have previously been shown to be involved in the phosphorylation of starch or its subsequent dephosphorylation. Both these processes are essential for normal starch mobilization. A tomato conditional mutant lacking the starch phosphorylating enzyme glucan water dikinase was analyzed. It is known that starch accumulates transiently in tomato fruit and is degraded throughout the ripening process. The study aimed to determine the effect of inhibited starch degradation on fruit development. Unfortunately no effect on starch mobilisation was found in the fruit of the mutant. Immunoblot analysis revealed expression of Glucan Water Dikinase (GWD) within the fruit of the tomato mutant indicating that the conditionality of the mutation was compromised. The second set of experiments analyzed the roles of Starch Excess4 (SEX4), Like Sex Four-1 and Like Sex Four-2 (LSF1 and LSF2) in starch degradation in potato and Nicotiana benthamiana. These enzymes have, thus far, only been studied in Arabidopsis, with the proposed role for SEX4 and LSF2 being that they are involved in dephosphorylation of the C-6 and C-3 positions of starch breakdown products. The role of LSF1 is unclear, although it is not thought to be a phosphatase. SEX4, LSF1 and LSF2 were repressed individually while the expression of SEX4 and LSF2 were also inhibited simultaneously. Using a transient repression system in N. benthamiana it was shown that all of the genes play a role in leaf starch degradation. The SEX4 and LSF2 enzymes were shown to influence the proportion of phosphate located on the starch which contained an altered ratio of C-3/C-6 phosphate. Stably transformed potato plants were produced where SEX4 and LSF2 were successfully repressed in potato leaves and tubers. Although AtLSF2 had been shown not to be essential for normal starch degradation on its own, in potato plants when LSF2 was repressed, the plants developed a starch-excess phenotype. Taken together with the N. benthamiana data this indicates that LSF2 plays a bigger role in leaf starch degradation in Solanaceae than in Arabidopsis. The ratio of C-3/C-6 phosphate was also altered in tuber starch from some of the silenced plants. Starch from SEX4 repressed potato plants contained increased amounts of glucose-6-phosphate and increased glucose-3-phosphate in the tuber when compared to the WT. An increase in the proportion of C-6 or C-3 phosphate is not surprising with SEX4 being characterized as a phosphatase specific for C-6 position and LSF2 for the C-3 position in Arabidopsis, however the combined increase in C-3 and C-6 amounts in StSEX4 silenced plants is interesting. The differences seen in the phosphate alteration in both N. benthamiana leaves and potato tubers indicates that in Solanaceae species these proteins may have a slightly altered specificity when compared with Arabidopsis, although they are undoubtedly involved in starch degradation. The effect of silencing SEX4 or LSF2 on cold-induced sweetening was also investigated, with no effect being found. This may be because of functional redundancy between the proteins and a better approach in terms of blocking cold sweetening would be to simultaneously repress SEX4 and LSF2. Overall, these enzymes seem to play similar roles in leaves of Solanum species as has been described in Arabidopsis. The starch from the engineered plants did have an altered phosphate ratio and further analysis is needed to determine if this leads to improved or additional functionality. / AFRIKAANSE OPSOMMING: Die projek omhels die ontleding van gene van die Solanaceae spesie wat voorheengetoon het dat hulle deel neem in fosforilering of defosforilering van stysel. Altwee van hierdie reaksies is belangrik vir normale stysel metabolisme. ‘n Tamatie konditionele mutant was geanaliseer waarin die stysel fosforilering ensiem glucan water dikinase nie teenwoordig was nie. Die doel van die studie was om te ondersoek watter effek het n gebrek in stysel afbraak op die rypwording en ontwokkeling vrugte. Ongelukkig was geen effek op stysel metabolism in die munant se vrugte gesien. Immunoklad analise het getoon dat GWD protein wel uitdruk word in die vrugte en dus die mutant nie heeltemal effektief was nie. Die tweede stel van experimente het in aartappels en tabak die rol van SEX4, LSF1 en LSF2 in stysel afbraak ondersoek. Hierdie ensieme was huidiglik nog net deeglik in Arabidopsis bestudeer, waar daar gewys was dat SEX4 and LSF2 in die defosforilering van stysel by die C-6 en C-3 posisie deel neem. Die rol van LSF1 is nog onbekend, maar daar word huiglik gelgo dat dit is nie ‘n fosfatase nie. SEX4, LSF1, en LSF2 was onderdruk op sy eie, waar SEX4 en LSF2 gelyktydig onderdruk was. Met behulp van n verbygaande onderdrukking in tabak, was dit getoon dat al die bogenoemde gene n gedeeltelike rol speel in die afbraak van stysel. Dit was getoon dat SEX4 and LSF2 ensiemedie verhouding van waar fosfaat op stysel gelee is beinvloed en het n verandering in die C-3/C-6 phosphaat verhouding ook gehad. Aardappels was stabiel getransformeer en daar was suksesfol plante waar SEX4 en LSF2 onderdruk was in blare en knolle geproduseer. Alhoewel daar getoon was dat AtLSF2 op sy eie nie n groot rol speel in stysel katabolisme nie was daar wel gesien dat in aardappel wanner hierdie geen afgeskakel was dat daar n stysel oorskot fenotiepe ontwikkel. As die tabak resultate saamgevat word met die aardappel wil dit voorkom asof LSF2 n groter rol binne die stysel katabolisme in Solanaceae speel as in Arabidopsis. Daar was gevind dat die verhouding van C-3/C-6 fosfaat was in die knolle verander in perty van die lyne waar geen afskakeling wel plaasgevind het. Die verhouding van C-3/C-6 fosfaat was verander in knolle stysel van sommige stilgemaak plante. Sysel van SEX4 stilgemaak plante het hoër vlakke glukose-6-fosfaat en glukose-3-fosfaat in die knolle gehad wanner dit met die WT vergelyk was. n Toename in die persentasie van C-6 fosfaat is nie verbasend nie, SEX4 word gekenmerk as die spesifieke fosfatase verantwoordelik vir die fosfaat by die C-6 posisie en LSF2 spesifiek vir die C-3 posisie in Arabidopsis. Die gekombineerde toename in beide C-6 en C-3 bedrae in StSEX4 stilgemaak plante is wel heel interesant. Verandering in beide tabak blare and aartapple knolle dui daarop dat in solanacea spesie hierdie proteiene, n effens verandering in spesifisiteit kan hê as dit met Arabidopsis vergelyk word. Daar kan wel nie getwyfel word dat hulle wel n rol speel in stysel afbraak nie. Die effect watSEX4 of LSF2 op koue-geinduseerde soetheid het is ook ondersoek maar daar was geen effek gevind nie. Dit mag wees asgevolg van die funksionele onslag tussen die twee proteien en better benadering on die koue-soetheids effek te verhoed sou wees om beide protein op die selfde stadium aft e skakel. As daar in gegeheel gekyk word lyk dit asof hierdie protein die selfde rolle het in die Solanum spesies as in Arabidopsis.Die stysel van hierdie die ontwerpte plante het ‘n veranderde fosfaat verhouding getoon en veder analise is nodig om te bepaal of dit lei tot verbeterde einskappe of bykommende funksies.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2007. / The photosynthetic protist Euglena gracilis synthesizes a storage carbohydrate named paramylon, a glucan consisting only of β-(1-3)-glycosidic linkages. The enzyme that produces paramylon is a glycosyltransferase commonly known as paramylon synthase (EC 2.4.1.34; UDP-glucose: 1,3-β-D-glucan 3-β-D-glucosyl transferase). This enzyme uses UDP-glucose as its main substrate. In 2001, Bäumer et al. isolated and partially purified paramylon synthase, but never presented any sequence information. Hence, the main aim of this project was to isolate and characterize the gene(s) coding for the paramylon synthase. Different approaches were taken in order to isolate and characterize the gene(s). In the first part of the study molecular techniques were used to try and identify the gene. The two methods used were library screening and PCR amplification. Different libraries were screened using either functional staining or an affinity probe. The second method concentrated on the use of degenerate oligonucleotides, based on the amino acid sequences of conserved regions from known β-(1-3)-glucan synthase genes from various organisms, to PCR amplify the gene sequence from Euglena. These approaches were not successful in the isolation of the gene(s). In the second part of the study protein purification techniques were used in an attempt to obtain de novo protein sequence from the purified paramylon synthase enzyme. Several protein purification techniques were tried with the most successful being preparative ultra centrifugation followed either by sucrose density centrifugation or product entrapment (a type of affinity purification). These resulted in partial purification of the paramylon synthase protein. The partially purified proteins were separated using polyacrylamide gel electrophoresis, and the polypeptides able to bind the precursor, UDP-glucose, were identified using a radiolabeled isotope of UDP-glucose. These polypeptides were subjected to LC-MS-MS in order to obtain sequence information from them. One tryptic fragment showed high homology to β-(1,3)-glucan synthase genes from different yeasts.

Thesis (MSc)--University of Stellenbosch, 2004. / 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. / AFRIKAANSE OPSOMMING: Die proses van suikerakkumulering en -vervoer in suikerriet word steeds baie vaag verstaan. ‘n Deeglike begrip van die prosessewat betrokke is in die vervoer van sukrose is baie belangrik omdat transmembraan vervoer moontlik een van die belangrike beheerpunte in metabolisme mag wees. Die doelwitte van die studie was om ‘n beter begrip te bekom van die meganisme wat betrokke is by die vervoer en berging van sukrose in suikerriet. Die projek is in ‘n fisiologiese en ‘n molekulêre afdeling verdeel. In die fisiologiese afdeling is stingelweefsel van ‘n Saccharum hybried (variëteit N19) van verskillende stadiums van ontwikkeling (internodes 2-4, internode 8 en internode 15) gebruik. Opname van radioaktiewe (14C) sukrose, glukose en fruktose is as analise metode gebruik vir die suikeropname eksperimente. Die invloed van pH, suiker konsentrasie en inhibitore soos PCMBS (pchloromercuriphenylsulfonic acid) en TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) op die tempo van suikeropname is ondersoek. Die molekulêre deel fokus hoofsaaklik op die identifisering, isolering en karakterisering van nuwe sukrose vervoerproteine in suikerriet, met behulp van PCR en heteroloë uitdrukking in Xenopus laevis oösiete. Die 14C - opname eksperimente het tot die volgende gevolgtrekkings gelei: Heksoses speel die belangrikste rol in die vervoer van suiker in die riet as daar min of geen sukrose teenwoordig is nie. Sodra daar sukrose in groot mate teenwoordig is soos in die geval van ontwikkelde, ryp internodes, is die rol van sukrose egter belangriker. Sukrose is die maklikste opgeneem by pH 5, wat naby die pH optimum van die meeste sukrose vervoerproteïene is. TRIS en PCMBS het beide ‘n inhiberende effek op sukrose opname gehad, maar die invloed was groter by die laer sukrose konsentrasies. Tot onlangs was daar baie min inligting oor sukrose vervoer in suikerriet op DNA en RNA vlak. Die eerste sukrose vervoerprotein uit suikerriet, ShSUT1 (Saccharum Hibried Sukrose Transporter) is eers onlangs uit ‘n stingel - cDNA biblioteek geïsoleer (Rae et al., 2004) en die funksionering daarvan is in ‘n gisras (SEY6210) getoets. In my pogings om sukrose vervoer te verstaan is ‘n gedeeltelike cDNA, naamlik ScSUT(p) (partial Sugarcane Sucrose Transporter) van 1258 nukleotiede, uit cDNA afkomstig van suikerrietbotsel geïsoleer. Die nukleotiedvolgorde stem 94% ooreen met ShSUT1 en kodeer vir ‘n moontlike oopleesraam van 419 aminosure. Southern analises het aangedui dat ScSUT(p) ‘n lae kopie getal het, in ooreenstemming met wat vir ander sukrose vervoerproteïene gevind is. Northern analises het getoon dat die uitdrukking van ScSUT(p) konstitutatief is in sukrose akkumulerende sowel as sukrose bergingsweefsel. Jong weefsel (internode 2 en 3) het baie lae uitdrukking getoon, met die hoogste uitdrukking in blaarweefsel. Uit die resultate is afgelei dat ScSUT(p) ‘n rol in floeëmlading en -ontlading mag speel. Xenopus laevis oösiete, is as ‘n heteroloë uitdrukking sisteem gebruik om te bevestig dat ShSUT1 as ‘n sukrose vervoerproteïen funksioneer. Elektrofisiologie het nie daarin geslaag om ShSUT1 se spesifieke werkingsmeganisme te identifiseer nie. Aanduidings is egter gevind dat ShSUT1 moontlik nie as ‘n H+/sukrose simportsisteem werk nie, maar by gefasilliteerde vervoer van sukrose betrokke mag wees. Verdere navorsing is noodsaaklik om die meganisme van ShSUT1 se werking te verstaan.

Thesis (MSc)--University of Stellenbosch, 2005. / 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 / AFRIKAANSE OPSOMMING: Die doel van hierdie projek was tweeledig: eerstens om weefselvoorbereiding en in situhibridisasie- protokolle vir die stingelweefsel van suikerriet te ontwikkel; en tweedens om die ontwikkelde tegnieke te gebruik om die uitdrukkingspatrone van drie invertaseisovorme in die suikerriet-internodes van verskeie ontwikkelingstadia te ondersoek. Suikerriet-invertases is al vir jare lank die fokus van intense navorsing, maar baie min is bekend oor hulle weefselspesifieke verspreiding. Die idee was om meer kennis oor suikerriet-invertases se funksies en betrokkenheid by sukrose-akkumulasie te verkry deur in situ-hibridisasie te gebruik om hulle uitdrukkingspatrone te karakteriseer. Alhoewel in situ-hibridisasie deesdae gereeld gebruik word om geenuitdrukking in plante te bestudeer, is daar tot op datum slegs een publikasie wat die gebruik daarvan in onvolwasse suikerrietweefsel beskryf. Hierdie tegniek moes dus verder ontwikkel word, en dit is gedoen deur verskillende weefselvoorbereidingsmetodes te vergelyk en sistematies die verskillende parameters wat op elke metode van toepassing is te toets. Die in situ-hibridisasie-tegniek is ook ontwikkel deur die toetsing en vergelyking van 'n aantal sleutelparameters. Daar is gevind dat suikerrietweefsel voldoende gepreserveer word deur die intakte gemonteerde weefsel vir 48 uur te fikseer. Hoë hibridisasietemperature en hoë peilerkonsentrasies het die beste sein gegee; die insluiting van voorbehandeling met HCl en Pronase was noodsaaklik om die weefsel meer gevoelig vir die peiler te maak. Osmotiese invloede en die tyd nodig vir seindeteksie is verminder deur die viskositeit van die buffer te verminder. In die tweede deel van die studie is die ontwikkelde metode gebruik om die uitdrukkingspatrone van die drie invertase-isovorme in jong, ontwikkelende en volwasse internodes te ondersoek en die resultate is deur 'n noordelike oordraganalise gekomplementeer. Transkripte van al drie isovorme is in die stoorparenchiem en floëemweefsel gevind. Transkripvlakke van al drie isovorme het afgeneem in ontwikkelende weefsel, met oplosbare suurinvertase wat skerp afgeneem en tot onder die deteksie-limiet gedaal het in ontwikkelende en volwasse weefsel. Transkripvlakke van selwandinvertase en neutrale invertase het slegs geleidelik afgeneem en merkbare vlakke van albei was teenwoording in ontwikkelende en volwasse weefsel. Daar word voorgestel dat suurinvertase hoofsaaklik betrokke is by internodeverlenging, terwyl selwandinvertase skynbaar 'n belangrike rol in floëem-ontlading en turgor-beheer speel. Daar word voorgestel dat neutrale invertase betrokke is óf by die sukrose-sirkulering óf by die onderhoud van heksose-poele; die funksie van hierdie ensiem is egter steeds nie duidelik nie. Hierdie studie het die waarde van in situ-hibridisasie gedemonstreer maar terselfdetyd ook die beperkinge daarvan uitgewys, veral as meer tradisionele biochemiese tegnieke nie gebruik word om die resultate aan te vul nie. Alhoewel daar onsekerheid is oor die presiese funksies van die invertase-isovorme in suikerriet, het die studie die weg gebaan vir weefselspesifieke promotorontwerp en toekomstige in situ-studies van suikerrietinvertases.

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. 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.