Ribulose 1,5-biphosphate carboxylase/oxygenase is activated by incubation with CO₂ and Mg²⁺. Several compounds are known to mediate this CO₂ dependent activation, including several chloroplast metabolites. A search, based on structural similarities to known activators, encountered more than twenty previously unreported effectors. Activators appear to share several common characteristics, including two anionic groups which usually occupy terminal positions on the molecule. Terminal groups on activators include carboxylate, phosphate, phosphonate, and sulfonate. Activators which do not have at least one phosphate or phosphonate terminal group have a hydroxyl or carboxyl containing side chain. Positive effectors change the response of the carboxylase by allowing activation at lower levels of CO₂ and/or Mg²⁺, and at lower pH values. Higher CO₂ concentrations also allow activation at lower pH values. The ratio between the carboxylase and oxygenase functions of this enzyme at air levels of CO₂ and O₂ does not change with effector induced changes in enzyme activity, suggesting that they share the same active site. There is an apparent difference in the response of the enzyme before and after purification to incubation in phosphate, and to inhibition by the substrate ribulose 1,5-biphosphate. These observations suggest that the isolated protein is different from the native enzyme. Several carboxylase activators are taken up by isolated chloroplasts in an exchange for phosphate catalyzed by the phosphate translocator. Included in this group is phosphonopropionate, a non-metabolized analogue of phosphoglycerate. Addition of this effector to chloroplasts under conditions of photosynthesis results in increased carboxylase activity, but a decrease in both ribulose 1,5-biphosphate levels and CO₂ fixation. There is a slow leakage of phosphate from isolated chloroplasts at room temperature. This leakage does not occur during illumination, nor at ice temperature, but increases with increasing hydroxide ion over physiological pH values. This slow leakage is probably an artifact of chloroplast isolation procedures. In isolated chloroplasts, phosphate levels appear to control the partition of photosynthate between starch formation and sugar phosphate export. During periods of net accumulation of starch in the chloroplast, there is concomitant degradation occurring, such that the accumulation of label into the starch fraction may not reflect the actual rate of starch synthesis.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Starch is a polyglucan made up of the two glucose polymers, amylose and amylopectin. Plants use starch to store excess carbohydrates from photosynthesis which get used for growth during the night. Starch metabolism is well undertood in higher plants such as A. thaliana thaliana and Solanum tuberosum with well-established pathways worked out for the enzymes involved in its synthesis and degradation. The bryophyte Physcomitrella patens has emerged as a popular choice for studying gene function in lower plants both because its genome has been sequenced and because of the ease of establishing knockout mutants via homologous recombination. Many metabolic functions have been studied in P. patens but, until now, little has been done in examining starch metabolism in moss. This study focused on two enzymes that have been found to be involved in starch degradation in higher plants, Disproportionating enzyme 1 (DPE1) and Disproportionating enzyme 2 (DPE2). DPE1 isoforms have been found to break down malto-oligosaccharides, which are products of starch degradation, into glucose within the chloroplast. On the other hand DPE2 catabolizes maltose to glucose in the cytosol. Higher plants that were silenced in these two genes were unable to degrade starch effectively, which lead to an increase in starch, malto-oligosaccharides or maltose and reduced growth. Three orthologs were identified for DPE1 in P. patens (PpDPE1A, B and C) and one for DPE2 (PpDPE2). Only PpDPE1B and PpDPE1C were found to be expressed in P. patens at the beginning of the light period but further investigation would be necessary at different time points as these genes were shown to be optimally expressed at the end of the light period. Targeted gene knockouts were made for each in P. patens which showed a reduced growth phenotype for all, indicating that these genes do play a role in starch catabolism that influences growth. There was, however, no significant change in starch content between the mutant lines and wild type (Wt). GFP fusion proteins showed PpDPE2 to be localized in cytosol, in close proximity to the chloroplast membrane. Similar findings have been found for DPE2 in A. thaliana and S. tuberosum. We hypothesize that PpDPE2 may play a role in cold tolerance in moss as an increase in starch breakdown has been witnessed in cold treated moss as well as increased transcript levels of starch metabolism genes and a maltose transporter. This opens a door to the further study of these generated mutant lines under cold stress. / AFRIKAANSE OPSOMMING: Stysel is ‘n poliglukaan wat bestaan uit die twee glukose polimere: amilose en amilopektien. Plante gebruik stysel om oortollige koolhidrate van fotosintese wat vir groei gebruik word gedurende die nag te berg. Styselmetabolisme in hoër plante soos A. thaliana thaliana en Solanum tuberosum word goed verstaan, met gevestigde paaie uitgewerk vir die ensieme wat betrokke is by die sintese en afbreek daarvan. Die briofiet Physcomitrella patens is ‘n populêre keuse vir die bestudering van geenfunksie in laer plante, omdat die genoomvolgorde bepaal is en as gevolg van die gemak waarmee ‘uitklop’-mutante via homoloë rekombinasie gevorm kan word. Baie metaboliese funksies is bestudeer in P. patens maar tot nou is min gedoen om die styselmetabolisme in mos te ondersoek. Hierdie studie het gefokus op twee ensieme, DPE1 and DPE2, wat gevind is om betrokke is afbreek van stysel in hoër plante. Dit is voorheen bevind dat DPE1 isoforme malto-oligosakkariedes (wat produkte is van styselafbraak) afbreek na glukose in the chloroplast. Aan die ander kant kataboliseer DPE2 maltose na glukose in die sitosol. Hoër plante waarin hierdie gene stilgemaak is, is nie instaat daartoe om stysel effektief af te breek nie. Dit lei tot ‘n verhoging in stysel, malto-oligosakkariede of maltose en verminderde groei. Drie ortoloë is geïdentifiseer vir DPE1 in P. patens (PpDPE1A, B en C) en een vir DPE2 (PpDPE2). Slegs PpDPE1B en PpDPE1C word uitgedruk in P. patens aan die begin van die ligperiode, maar verder ondersoek sal nodig wees op verskillende tydpunte, omdat dit bewys is dat hierdie gene optimaal uitgedruk word tydens die einde van die ligperiode. Geteikende geen uiklop-mutante is gemaak vir elk in P. patens wat ‘n verminderde-groei fenotipe vertoon het vir almal, wat aandui dat hierdie gene ‘n rol speel in styselkatobolisme wat groei beïnvloed. Daar was egter geen beduidende verskil in styselinhoud van die mutante lyne en die wilde tipe nie. GFP-fusieproteïne het gewys dat PpDPE2 gelokaliseer is in die sitosol, naby aan die chloroplast membraan. Soorgelyke bevindinge is ook gemaak in DPE2 in A. thaliana en S. tuberosum. Dit word gestel dat PpDPE2 moontlik ‘n rol speel in kouetoleransie in moss, omdat ‘n verhoging in styselafbraak opgemerk is in koue-behandelde moss sowel as verhoogde transkripsievlakke van styselmetabolisme gene en ‘n maltose transporter. Dit maak ‘n deur oop vir verdere studie van hierdie gegenereerde mutant-lyne onder kouestres.

Phosphorous in its inorganic form, orthophosphate (Pi), is found in every compartment of the plant cell and serves as a substrate, product or effector for a wide range of metabolic processes. Several Pi transporters exist in plants and these help regulate Pi homeostasis within different cellular compartments. The PHT4 family of organellar Pi transporters consists of six members in the model plant Arabidopsis thaliana, and five of these are localized to plastids. I used gene expression analyses and reverse genetics to demonstrate functional specialization for the PHT4 family members with a focus on PHT4;1 and PHT4;2. The PHT4;1 Pi transporter is localized to chloroplast thylakoid membranes and it is expressed in a circadian manner. Plants that lack a functional copy of the PHT4;1 gene have reduced rosette size and altered responses to photooxidative stress. The PHT4;2 transporter is localized to heterotrophic plastids in roots and other sink organs and pht4;2 mutants exhibit decreased starch accumulation, which is consistent with a defect in Pi export, and increased rosette size, which is caused by increased cell proliferation. These results confirm that PHT4;1 and PHT4;2 have specialized functions and that plastidic Pi homeostasis influences broad aspects of plant metabolism, including abiotic stress response and control of lateral organ growth.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Starch is widely used in industries around the word, some of these are food, oil drilling, paper milling and cosmetics. It is a polymer which has two components, amylose and amylopectin. The production and degradation if starch in plants is fairly well studied and a sizable number of enzymes have been identified which play critical roles in its metabolism. There are still remaining questions, namely if there are more unidentified enzymes that play roles and how the enzymes interact with each another. To study the effect on starch metabolism possible novel starch metabolic genes were studied by analysing Arabidopsis T-DNA insertion mutants for two genes, designated SP1 (At5g39790) and CBD1 (At5g01260). cDNAs for these two was used to produce recombinant protein and investigated potential activities. The cbd1 mutant plants had a starch excess phenotype with iodine staining but this could not be confirmed with quantitative starch measurements. The sp1 mutants did not have a significant difference in all the lines and time points when compared to the Wt plants. No link could be established between the SP1 kinase domain and glucan phosphorylation. From my data a clear involvement of these two genes could not yet be elucidated. To study the interactions of starch metabolic proteins (BEI, BEII, GWD and ISA2) chimeric RNAi constructs was built and transformed into potato. Only StBEI and StBEII lines could be analysed and it was found that the G6P content was increased in both StBEI and StBEII. The BEII leaves and tubers had increased amylose contents. Intriguingly it would appear that starch isolated from both the tubers and leaves of StBEI lines demonstrated a reduction in amylose, with the leaves showing a much bigger decease than the tubers. This needs to be confirmed and the remaining lines need to be analysed. Gaining knowledge about starch metabolism is critical in producing engineered crops that can produce more starch in a smaller agricultural area. With the population growing beyond 8 billion individuals it will be one of the best routes to enhance cop yields through biotechnology. / AFRIKAANSE OPSOMMING: Stysel word reg oor die wereld benut in ‘n verskyndenheid van industiee. Dit is divers en sluit die voedsel, oliebooring, papiermeule en die kosmetiese bedryf in. Dit is ‘n polimeer wat uit twee komponete: amylose en amylopektien bestaan. Stysel metabolisme, wat die vervaardiging en afbreek van dit insluit, is al baie goed bestudeer. Die ensieme wat ‘n kritiese rol speel is al gevind, maar daar bly nogsteeds ‘n paar vrae wat moet beantwoord word. Is daar nog ensieme wat ‘n rol speel wat nog nie geidentifiseer is nie? Wat is die manier hoe die bekende ensieme met mekaar ‘n interaksie het? Om die invloed van twee moonlike nuwe stysel metabolisme gene te bestudeer, is T-DNA insersie mutante ondersoek. Hulle word na verwys in die studie as SP1 (At5g39790) en CBD1 (At5g01260). cDNAs vir hierdie twee was gemaak vir die vervaardeging van rekombonante proteine. Hierdie rekombinante proteine was dan ondersoek vir moonlike aktiwiteite. ‘n Oormaat stysel was wel gevind in die cbd1 mutant plante wanner n jodium vlek tegniek gebruik was. Ongelukkig kon hierdie oormaat die bevestig word wanner n kwantitatiewe metode gebruik was nie. Daar was nie ‘n beduidende verskil in stysel wanner die sp1 mutante plante vergelyk was met die wilde tiepe nie. Daar kon ook geen verbintenis gevind word tussen die kinase area en die fosforilasie van stysel nie. Volgens hierdie data kon daar die n duidelike verbintenis gevind word tussen die twee gene en stysel metabolisme nie. Om die interaksies tussen bekende stysel metabolisme proteine (BEI, BEII, GWD en ISA2) te bestudeer was chimeriese RNAi konstrukte gebou en toe in aartappels in getransformeer. Slegs die StBEI and StBEII kon geanalisser word en daar was bevind dat die G6P hoeveelheid in beide hoër was. Amilose was in groter hoeveelheide teenwoordig in beide BEII blare en knolle. ‘n Onverwagse obserwasie was gemaak toe die BEI lyne ondersoek was. Daar was gevind dat in die blare en knolle daar ‘n laer hoeveelheid amilose was. Die blare het wel baie laer amilose gehad as die knolle. Die obserwasie moet bevestig word met n ander tegniek en die orige RNAi lyne moet nog bestudeer word. Om al die fasette van stysel metabolisme te ken is uiters belangrik vir die vervaardiging van gewasse wat groter opbrengste lewer in n kleiner area. Met die wereld bevolking wat al verby 8 biljoen individue gestyg het is dit moontlik al hoe almal voor gesorg kan word in terme van voeding.

Thesis (PhD (Plant Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Virus Induced Gene Silencing (VIGS) was optimized to allow for the study of starch metabolism. The plastidial inorganic pyrophosphatase gene, for which a mutant has never been identified, was studied using VIGS and it was found to have a broad role in this subcellular compartment. The accumulation of inorganic pyrophosphate limited the production of starch, carotenoids, chlorophyll, and increased the plants susceptibility to drought stress. These effects highlight the importance of this enzyme in maintaining a low intraplastidial concentration of PPi providing an environment which facilitates these anabolic processes. Several genes involved in starch synthesis and degradation were also targeted with the aim of establishing a system of multiple gene silencing for the study of metabolic pathways. One, two and three genes were successfully silenced using this system which was validated based on previously published data. Interestingly, simultaneous silencing of the two isoforms of disproportionating enzyme led to a novel phenotype as a large reduction in starch instead of the expected increase was observed. / No Afrikaans abstract available

Starch is a water-insoluble glucose biopolymer used as an energy cache in plants and is synthesized and degraded in a diurnal cycle. Reversible phosphorylation of starch granules regulates the solubility and, consequentially, the bioavailability of starch glucans to degradative enzymes. Glucan phosphatases release phosphate from starch glucans and their activity is essential to the proper diurnal metabolism of starch. Previously, the structural basis of glucan phosphatase activity was entirely unknown. The work in this dissertation outlines the structural mechanism of activity of two plant glucan phosphatases called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). The crystal structures of SEX4 and LSF2 were determined with and without phosphoglucan ligands bound, revealing the basis of their interaction with an endogenous substrate. The data show that SEX4 and LSF2 interact with starch glucans via distinctive mechanisms. SEX4 binds glucan chains via an aromatic-rich pocket spanning its Carbohydrate Binding Module (CBM) and catalytic Dual Specificity Phosphatase (DSP) domains. Conversely, LSF2 lacks a CBM and, instead, binds glucans at two non-catalytic surface-binding sites that are located distally from its active site. In addition, it was previously reported that SEX4 and LSF2 act upon distinct phospho-glucan substrates: SEX4 preferentially dephosphorylates the C6-position of starch glucans and LSF2 exclusively dephosphorylates the C3- position. The data herein reveal that SEX4 and LSF2 contain differences in their active site topology that serve to position the glucan chain in opposite orientations, therefore accounting for the differences in substrate specificity. Using these insights, SEX4 was engineered with reversed substrate specificity, i.e. preferential C3-specific activity. Previous work has established the interaction between phosphatases and protein, lipid, and nucleic acids; however, the current study represents the first insights into phosphatase interaction with carbohydrate substrates. In addition, the insights gained provide a model that will be used in future studies with the mammalian glucan phosphatase laforin, which is linked to neurodegeneration and the fatal epileptic disorder Laforaʼs Disease.

Unter geeigneten Wachstumsbedingungen weisen Algenkulturen oft eine größere Produktivität der Zellen auf, als sie bei höheren Pflanzen zu beobachten ist. Chlamydomonas reinhardtii-Zellen sind vergleichsweise klein. So beträgt das Zellvolumen während des vegetativen Zellzyklus etwa 50–3500 µm³. Im Vergleich zu höheren Pflanzen ist in einer Algensuspension die Konzentration der Biomasse allerdings gering. So enthält beispielsweise 1 ml einer üblichen Konzentration zwischen 10E6 und 10E7 Algenzellen. Quantifizierungen von Metaboliten oder Makromolekülen, die zur Modellierung von zellulären Prozessen genutzt werden, werden meist im Zellensemble vorgenommen. Tatsächlich unterliegt jedoch jede Algenzelle einer individuellen Entwicklung, die die Identifizierung charakteristischer allgemeingültiger Systemparameter erschwert. Ziel dieser Arbeit war es, biochemisch relevante Messgrößen in-vivo und in-vitro mit Hilfe optischer Verfahren zu identifizieren und zu quantifizieren. Im ersten Teil der Arbeit wurde ein Puls-Amplituden-Modulation(PAM)-Fluorimetriemessplatz zur Messung der durch äußere Einflüsse bedingten veränderlichen Chlorophyllfluoreszenz an einzelnen Zellen vorgestellt. Die Verwendung eines kommerziellen Mikroskops, die Implementierung empfindlicher Nachweiselektronik und einer geeignete Immobilisierungsmethode ermöglichten es, ein Signal-zu-Rauschverhältnis zu erreichen, mit dem Fluoreszenzsignale einzelner lebender Chlamydomonas-Zellen gemessen werden konnten. Insbesondere wurden das Zellvolumen und der als Maß für die Effizienz des Photosyntheseapparats bzw. die Zellfitness geltende Chlorophyllfluoreszenzparameter Fv/Fm ermittelt und ein hohes Maß an Heterogenität dieser zellulären Parameter in verschiedenen Entwicklungsstadien der synchronisierten Chlamydomonas-Zellen festgestellt. Im zweiten Teil der Arbeit wurden die bildgebende Laser-Scanning-Mikroskopie und anschließende Bilddatenanalyse zur quantitativen Erfassung der wachstumsabhängigen zellulären Parameter angewandt. Ein kommerzielles konfokales Mikroskop wurde um die Möglichkeit der nichtlinearen Mikroskopie erweitert. Diese hat den Vorteil einer lokalisierten Anregung, damit verbunden einer höheren Ortsauflösung und insgesamt geringeren Probenbelastung. Weiterhin besteht neben der Signalgewinnung durch Fluoreszenzanregung die Möglichkeit der Erzeugung der Zweiten Harmonischen (SHG) an biophotonischen Strukturen, wie der zellulären Stärke. Anhand der Verteilungsfunktionen war es möglich mit Hilfe von modelltheoretischen Ansätzen zelluläre Parameter zu ermitteln, die messtechnisch nicht unmittelbar zugänglich sind. Die morphologischen Informationen der Bilddaten ermöglichten die Bestimmung der Zellvolumina und die Volumina subzellularer Strukturen, wie Nuclei, extranucleäre DNA oder Stärkegranula. Weiterhin konnte die Anzahl subzellulärer Strukturen innerhalb einer Zelle bzw. eines Zellverbunds ermittelt werden. Die Analyse der in den Bilddaten enthaltenen Signalintensitäten war Grundlage einer relativen Konzentrationsbestimmung von zellulären Komponenten, wie DNA bzw. Stärke. Mit dem hier vorgestellten Verfahren der nichtlinearen Mikroskopie und nachfolgender Bilddatenanalyse konnte erstmalig die Verteilung des zellulären Stärkegehalts in einer Chlamydomonas-Population während des Wachstums bzw. nach induziertem Stärkeabbau verfolgt werden. Im weiteren Verlauf wurde diese Methode auch auf Gefrierschnitte höherer Pflanzen, wie Arabidopsis thaliana, angewendet. Im Ergebnis wurde gezeigt, dass viele zelluläre Parameter, wie das Volumen, der zelluläre DNA- und Stärkegehalt bzw. die Anzahl der Stärkegranula durch eine Lognormalverteilung, mit wachstumsabhängiger Parametrisierung, beschrieben werden. Zelluläre Parameter, wie Stoffkonzentration und zelluläres Volumen, zeigen keine signifikanten Korrelationen zueinander, woraus geschlussfolgert werden muss, dass es ein hohes Maß an Heterogenität der zellulären Parameter innerhalb der synchronisierten Chlamydomonas-Populationen gibt. Diese Aussage gilt sowohl für die als homogenste Form geltenden Synchronkulturen von Chlamydomonas reinhardtii als auch für die gemessenen zellulären Parameter im intakten Zellverbund höherer Pflanzen. Dieses Ergebnis ist insbesondere für modelltheoretische Betrachtungen von Relevanz, die sich auf empirische Daten bzw. zelluläre Parameter stützen welche im Zellensemble gemessen wurden und somit nicht notwendigerweise den zellulären Status einer einzelnen Zelle repräsentieren. / Under appropriate growth conditions cells of algae cultures often show a greater productivity than it is observed for cells in higher plants. The cells of Chlamydomonas reinhardtii are relatively small. The cell volume during the vegetative cell cycle ranges only between 50-3500 µm³. Compared to higher plants the concentration of biomass in an algal suspension is small. Thus, 1 ml of a suspension with a standard concentration contains between 10E6 and 10E7 algal cells. Quantification of metabolites or macromolecules, which are used for modeling of cellular processes, is usually carried out in the cell ensemble. However, every single algal cell undergoes an individual development, which makes the identification of characteristic universal system parameters far more complicated. The aim of this work was to identify and quantify relevant biochemical parameters, which were measured in vivo and in vitro using optical methods. In the first part, a Pulse Amplitude Modulation (PAM) measuring station was introduced to measure the variable chlorophyll fluorescence of individual cells. A commercial microscope was combined with sensitive detection electronics and the application of suitable immobilization methods. This allowed the achievement of a signal-to-noise ratio which made it possible to measure the fluorescence signals of individual living Chlamydomonas cells. In particular, cell volume and the chlorophyll fluorescence parameter Fv/Fm as a measure of the photosynthetic apparatus efficiency and cell fitness were determined. A high degree of cellular heterogeneity of these parameters in different development stages of synchronized Chlamydomonas cells was determined. In the second part, the imaging laser scanning microscopy and subsequent image analysis for quantitative detection of the growth-dependent cellular parameters were applied. A commercial confocal microscope was extended by the possibility of non-linear microscopy. Hereby, a more localized excitation of the samples was possible. Hence, a higher spatial resolution and lower overall sample stressing were achieved. Besides signal generation through fluorescence excitation, second harmonic generation (SHG) on biophotonic structures, such as cellular starch, was applied. Based on distribution functions cellular parameters were determined by using theoretical model approaches. This allowed the characterization of parameters that were not directly accessible by measurement. The morphological information of the image data enabled the determination of cell volume and volumes of sub-cellular structures such as nuclei, extra-nuclear DNA, and starch granules. Furthermore, the number of sub-cellular structures within a cell or a cell compound was determined. Analysis of signal intensities constituted the basis of relative quantification of cellular components such as DNA and starch. For the first time, the method of non-linear microscopy and subsequent image analysis enabled the characterization of the cellular starch distribution of a Chlamydomonas population during cell growth, and after induced starch degradation, respectively. Subsequently, this method was additionally applied to frozen sections of higher plants like Arabidopsis thaliana. As a result it was shown that many cellular parameters like volume, cellular DNA content, and number of starch granules are described by means of a log-normal distribution with growth-related parameterization. Cellular parameters, such as concentration and cellular volume, showed no significant correlations among each other. Therefore, it was concluded that there is a high degree of cellular parameter heterogeneity within synchronized Chlamydomonas populations. This applies not only to synchronized cultures of Chlamydomonas reinhardtii, which are currently considered as the most homogeneous form, but also to measured cellular parameters of intact cell assemblies in higher plants. The result is especially important for model-theoretic considerations, which are based on empirical data, and cellular parameters obtained from cell ensembles, respectively.