Analysis of aspects of starch metabolism in Physcomitrella patens
| dc.contributor.advisor | Lloyd, James Richard | en_ZA |
| dc.contributor.advisor | Kossmann, Jens | en_ZA |
| dc.contributor.author | Mdodana, Ntombizanele Thobela | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology. | en_ZA |
| dc.date.accessioned | 2021-10-28T12:43:41Z | |
| dc.date.accessioned | 2021-12-22T14:34:25Z | |
| dc.date.available | 2021-10-28T12:43:41Z | |
| dc.date.available | 2021-12-22T14:34:25Z | |
| dc.date.issued | 2021-12 | |
| dc.description | Thesis (PhDAgric)--Stellenbosch University, 2021. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: Starch is an important polysaccharide produced by plants and is widely used in industry mainly as a food thickener, but also in other important processes, such as the development of textiles and p aper manufacture. This polyglucan consists of two glucose polymers, amylose and amylopectin. As the major storage carbohydrate, starch is synthesised during the day before being catabolised at night to sustain plant growth and metabolism. Starch metabolism is well studied in vascular plants such as Arabidopsis thaliana and Solanum tuberosum, however, information about these processes are less well understood in non-vascular plants. The bryophyte Physcomitrella patens is an excellent plant model system for many reasons; for example its fully sequenced genome and the ability to produce knockout mutants using homologous recombination. Attempts to gain a better understanding of the function and regulation of some of the key metabolic enzymes involved in starch metabolism in non-vascular plants have recently emerged (Stander, 2015; Jacobs, 2018; Mdodana et., al 2019). This project focused on two aspects of starch metabolism to determine whether some of the pathways and mechanisms involved during these processes are conserved between Physcomitrella patens and vascular plants. The first part of this dissertation examines the roles of glucan water dikinase enzymes (GWD) in P. patens. In angiosperms these polypeptides are involved in starch degradation through catalysing starch phosphorylation. Five isoforms, PpGWDa-e, were identified in and phylogenetic analysis demonstrated the two (PpGWDa and PpGWDb) were most similar to Arabidopsis GWD1 or GWD2, while another two (PpGWDd and PpGWDe) were most similar to GWD3/PWD. The final isoform (PpGWDc) was likely to be inactive as it lacks the essential catalytic histidine. Both PpGWDa and PpGWDb targeted to chloroplasts. Using homologous recombination, knockout mutant lines were successfully generated for PpGWDa and PpGWDb each isoform both as individually and together in double mutants. Inserts in either gene resulted in reduced amounts of starch phosphate compared to the control, with Ppgwda mutant lines interestingly containing less glucose 6-phosphate in starch than Ppgwdb lines. Double mutant (Ppgwda/Ppgwdb; DM) lines contained even less glucose 6-phosphate in starch than Ppgwda single mutants. When plants were grown over a diurnal cycle it was shown that, like vascular plants, starch accumulated in the light period and was degraded at night. Both Ppgwd1a and DM lines accumulated significantly higher amounts of starch compared to Ppgwd1b and the control lines at almost all time points. Soluble sugars on the other hand were significantly reduced in Ppgwd1a and the DM lines compared with Ppgwd1b and the control lines. The Ppgwd1a and DM mutant lines also demonstrated a n observable morphological phenotypic alteration characterized by lack of gametophore development which could be reversed by growing the plant on media supplemented with glucose. In the second experimental chapter, two isoamylase (ISA) like starch debranching enzymes, ISA1 and ISA2, were examined. Mutations in these genes in vascular plants can result in the production of the water-soluble polysaccharide (WSP), phytoglycogen at the expense of starch. Both single and double mutants showed loss of a debranching enzyme activity band in activity gels indicating that ISA1 and ISA2 exist as a heterocomplex in Physcomitrella patens. Analysis of purified recombinant protein demonstrated, surprisingly that both PpISA1 and PpISA2 peptides were catalytically active. Analysis of single and double mutant plants demonstrated that all contained increased amounts of WSP. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Stysel is 'n belangrike polisakkaried wat deur plante geproduseer word en word wyd gebruik in die industrie, hoofsaaklik as voedselverdikker, maar ook in ander belangrike prosesse, soos die ontwikkeling van tekstiele en papiervervaardiging. Hierdie poli-glukan bestaan uit twee glukose- polimere, amylose en amylopektien. As die belangrikste opbergkoolhidraat, word stysel gedurende die dag voordat dit in die nag gekataliseer word, gesintetiseer om plantgroei en metabolisme te handhaaf. Styselmetabolisme word goed bestudeer in vaskulêre plante soos Arabidopsis thaliana en Solanum tuberosum, maar inligting oor hierdie prosesse word minder goed verstaan by nie-vaskulêre plante. Die bryofiet Physcomitrella patens is om baie redes 'n uitstekende plantmodelstelsel; byvoorbeeld die volledig volgorde genoom en die vermoë om uitklopmutante te produseer met behulp van homoloë rekombinasie. Pogings om 'n beter begrip te kry van die funksie en regulering van sommige van die belangrikste metaboliese ensieme wat betrokke is by styselmetabolisme by nie-vaskulêre plante, het onlangs na vore gekom (Stander, 2015; Jacobs, 2018; Mdodana et., al 2019). Hierdie projek het op twee aspekte van styselmetabolisme gefokus om te bepaal of sommige van die paaie en meganismes wat tydens hierdie prosesse betrokke is, tussen Physcomitrella patens en vaskulêre plante bewaar word. Die eerste deel van hierdie proefskrif ondersoek die rolle van glukanwater-dikinase-ensieme (GWD) in P. patens. In angiosperme is hierdie polipeptiede betrokke by styselafbraak deur styselfosforylering te kataliseer. Vyf isoforme, PpGWDa-e, is in Physcomitrella patens geïdentifiseer. Filogenetiese analise het getoon dat die twee (PpGWDa en PpGWDb) die meeste ooreenstem met Arabidopsis GWD1 of GWD2, terwyl nog twee (PpGWDd en PpGWDe) die meeste ooreenstem met GWD3 / PWD. Die finale isoform (PpGWDc) was waarskynlik onaktief omdat dit nie die essensiële katalitiese histidien het nie. Beide PpGWDa en PpGWDb is gemik op chloroplaste. Met behulp van homoloë rekombinasie is uitklopmutante lyne suksesvol gegenereer vir PpGWDa en PpGWDb elk isoform, sowel as individueel en saam in dubbele mutante. Insetsels in een van die gene het gelei tot verminderde hoeveelhede styselfosfaat in vergelyking met die kontrole, met Ppgwda-mutantlyne wat interessant genoeg glukose 6-fosfaat in stysel bevat as Ppgwdb-lyne. Dubbelmutante (Ppgwda / Ppgwdb; DM) lyne bevat nog minder glukose 6-fosfaat in stysel as Ppgwda enkelmutante. Toe plante oor 'n dagsiklus gekweek is, is dit aangetoon dat stysel, soos vaskulêre plante, in die ligperiode opgehoop het en snags afgebreek is. Beide Ppgwd1a- en DM-lyne het aansienlik hoër hoeveelhede stysel opgehoop in vergelyking met Ppgwd1b en die beheerlyne byna te alle tye. Aan die ander kant is oplosbare suikers aansienlik verminder in Ppgwd1a en die DM-lyne in vergelyking met Ppgwd1b en die beheerlyne. Die Ppgwd1a- en DM-mutantlyne het ook 'n waarneembare fenotipiese effek getoon, gekenmerk deur 'n gebrek aan gametofore-ontwikkeling wanneer dit op BCD-medium gekweek word. Dit kan omgekeer word deur die aanleg op media te laat groei, aangevul met glukose. In die tweede eksperimentele hoofstuk is twee isoamylase (ISA), soos stysel-ontlas-ensieme, ISA1 en ISA2, ondersoek. Mutasies in hierdie gene in vaskulêre plante kan lei tot die produksie van die wateroplosbare polisakkaried (WSP), fitoglykogeen ten koste van stysel. Beide enkel- en dubbele mutante het die verlies van 'n debranching-ensiemaktiwiteitsband in aktiwiteitsgels getoon, wat daarop dui dat ISA1 en ISA2 as 'n heterokompleks in Physcomitrella patens bestaan. Analise van gesuiwerde rekombinante proteïen het getoon, verbasend dat beide PpISA1 en PpISA2 peptiede katalities aktief was. Analise van enkel- en dubbelmutante plante het getoon dat almal groter hoeveelhede WSP bevat. | af_ZA |
| dc.description.version | Doctoral | en_ZA |
| dc.format.extent | xii, 146 pages : illustrations (some color) | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/124006 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Physcomitrella patens | en_ZA |
| dc.subject | Isoamylase | en_ZA |
| dc.subject | Starch -- Synthesis | en_ZA |
| dc.subject | Glucan water dikinase | en_ZA |
| dc.subject | Glucans -- Metabolism | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | Analysis of aspects of starch metabolism in Physcomitrella patens | en_ZA |
| dc.type | Thesis | en_ZA |