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Characterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens.
Thesis (PhDAgric)--Stellenbosch University, 2020.
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| Language: | en_ZA en_ZA |
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Stellenbosch : Stellenbosch University
2020
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| _version_ | 1867613795238543360 |
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| access_status_str | Open Access |
| author | Wiese, Anna Johanna |
| author2 | Lloyd, James Richard |
| author_browse | Lloyd, James Richard Wiese, Anna Johanna |
| author_facet | Lloyd, James Richard Wiese, Anna Johanna |
| author_sort | Wiese, Anna Johanna |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhDAgric)--Stellenbosch University, 2020. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/109082 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA en_ZA |
| last_indexed | 2026-06-10T12:41:46.856Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/109082 Characterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens. Wiese, Anna Johanna Lloyd, James Richard Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology. Trehalose Sucrose -- Synthesis Sucrose synthase Sugar -- Analysis Plants -- Effect of sugars on Physcomitrella patens -- Genetics UCTD Thesis (PhDAgric)--Stellenbosch University, 2020. ENGLISH ABSTRACT: Plant sugars have dual functionality, in that they play a role in primary metabolism and partake in signal transduction pathways. As it relates to their signaling function, they relay information to the nucleus regarding energy status, allowing the plant to adapt accordingly. Most of what is known about these functions of sugars have come from research in vascular plants, leaving aspects thereof unaddressed in non-vascular plants. Certain bryophytes (non-vascular plants) have been developed over into model plants, with Physcomitrella patens representing one such model. This plant has become popular in studies of evolutionary development and non-vascular plant biology. In this study, I characterized two gene families in P. patens, namely sucrose synthases (SUS) and trehalose 6-phosphate synthases (TPS), whose homologs in higher plants are implicated in sugar metabolism and signaling. Sucrose, the end-product of photosynthesis, is central to primary carbon metabolism. Its synthesis and degradation are tightly controlled to balance out supply and demand throughout the plant. Sucrose synthases are implicated in phloem loading and sink strength in vascular plants, where its cleavage products can enter primary metabolism or be used in the synthesis of complex carbohydrates. Little is known about SUS function in non-vascular plants, and in this study, I report the characterization of four putative SUS homologs in P. patens. Phylogenetic classification of land plant SUS sequences revealed the existence of 5 clades, one of which contained only bryophyte-sequences including all those from P. patens. Analysis of the amino acid sequences revealed that residues involved in SUS regulation in higher plants were conserved in PpSUS proteins. I was able to demonstrate SUS activity in crude protein extracts, however, detailed kinetic characterization was hindered by protein expression in E. coli. Localization studies revealed that all PpSUS proteins were cytosolic, while expression analyses indicated that PpSUS genes have overlapping and unique expression patterns. Another sugar which is implicated in signaling is trehalose 6-phosphate (Tre6P), an intermediate in the trehalose biosynthesis pathway. Levels of this sugar phosphate change in parallel to that of sucrose, with researchers proposing that it plays a role in communicating sucrose availability. The second part of this study focussed on the proteins involved in Tre6P synthesis, namely trehalose 6-phosphate synthases (TPSs), which are divided into two classes, with class I proteins containing catalytically active polypeptides (Leyman et al., 2001; Lunn, 2007). Very little is known about the class II proteins, and in this study, I characterized members of this class in P. patens. Physcomitrella contains six TPS genes in its genome, four of which encode class II proteins. Phylogenetic classification differentiated TPS sequences from land plants into 2 clades (I and II) consisting of 7 sub-clades (IA-B and IIA-E), suggesting the existence of 1 ancestral TPS gene. Functional complementation revealed weak TPS catalytic activity for one of the class II TPS proteins, a first for any plant class II protein studied to date. Subcellular localization experiments conducted on three of the class II proteins revealed that they were cytosolic, while yeast two hybrid analyses indicated that these proteins do not form complexes with each other or the class I proteins. Finally, expression analyses indicated that class II genes have overlapping expression patterns. This study provides novel insights into the evolution of SUS and TPS genes in P. patens and, will serve as a platform for the design of future experiments related to these gene families in non-vascular plants. AFRIKAANSE OPSOMMING: Plantsuikers het tweeledige funksionering deurdat dit 'n rol speel in primêre metabolisme en deelneem aan seintransduksieweë. Met betrekking tot hul seinfunksie, stuur suikers inligting oor die energiestatus na die kern, wat die plant in staat stel om aanpassings te maak. Meeste van wat ons weet oor hierdie funksies van suikers, kom uit navorsing in vaatplante, wat aspekte daarvan in nie-vaatplante onbekend laat. Sekere bryofiete (nie-vaatplante) is ontwikkel tot modelplante, waarvan Physcomitrella patens een sulke model verteenwoordig. Hierdie plant het gewild geword in studies oor evolusionêre ontwikkeling en nie-vaskulêre plantbiologie. In hierdie studie het ek twee geenfamilies in P. patens gekenmerk, naamlik sukrose sintases (SUS) en trehalose 6-fosfaat sintases (TPS), die homoloë waarvan in hoër plante betrokke is by suikermetabolisme en seine. Sukrose, die eindproduk van fotosintese, is sentraal tot die primêre koolstof metabolisme. Die sintese en afbraak daarvan word streng beheer om die vraag en aanbod in die hele plant te balanseer. Sukrose sintases word geïmpliseer in floeem lading en sink sterkte in vaatplante, waar die afbreek produkte daarvan primêre metabolisme kan binnegaan of in die sintese van komplekse koolhidrate gebruik kan word. Baie min is bekend oor SUS funksie in nie-vaatplante, en in hierdie studie rapporteer ek die karakterisering van vier vermeende SUS homoloë in P. patens. Filogenetiese klassifikasie van SUS sekwense van landplante het aan die lig gebring dat daar vyf klades bestaan, waarvan een slegs bryofiet-sekwense bevat, insluitend almal van P.patens. Analise van die aminosuur volgordes het aan die lig gebring dat residue wat by SUS regulering by hoër plante betrokke was, ook in PpSUS proteïene gekonserveer is. Ons kon SUS aktiwiteit in ruwe proteïen ekstrakte demonstreer, maar gedetailleerde kinetiese karakterisering is verhinder deur proteïen uitdrukking in E. coli. Lokalisasie studies het aan die lig gebring dat alle PpSUS proteïene sitosolies was, terwyl uitdrukkings analises aangedui het dat PpSUS gene oorvleuelende en unieke uitdrukkings patrone het. 'N Ander suiker wat by seintransduksie geïmplementeer word, is trehalose 6-fosfaat (Tre6P), 'n tussenproduk in die trehalose biosintese-weg. Die vlakke van hierdie suikerfosfaat verander in parallel met die van sukrose, en navorsers stel voor dat dit 'n rol speel in die kommunikasie van die beskikbaarheid van sukrose. Die tweede deel van hierdie studie het gefokus op die proteïene wat betrokke is by Tre6P-sintese, naamlik trehalose 6-fosfaat sintases (TPS's), wat in twee klasse verdeel is, met klas I proteïene wat katalities aktiewe proteïene bevat (Leyman et al., 2001; Lunn, 2007). Daar is baie min bekend oor die klas II-proteïene, en in hierdie studie het ek lede van hierdie klas in P. patens gekenmerk. Physcomitrella bevat ses TPS gene in sy genoom, waarvan vier vir klas II proteïene kodeer. Filogenetiese indeling het TPS sekwense van landplante in 2 klades (I en II) gedifferensieer, bestaande uit 7 subklaaie (IA-B en IIA-E), wat daarop dui dat daar 1 voorouerlike TPS geen bestaan. Funksionele komplementering het swak TPS katalitiese aktiwiteit getoon vir een van die klas II TPS proteïene, ń eerste vir enige plant klas II proteïen wat tot dusver bestudeer is. Sub-sellulêre lokaliserings eksperimente wat op drie van die klas II proteïene uitgevoer is, het aan die lig gebring dat hulle sitosolies was, terwyl interaksie analises aangedui het dat hierdie proteïene nie komplekse met mekaar of die klas I proteïene vorm nie. Laastens het uitdrukkings analises aangedui dat klasse II gene oorvleuelende uitdrukkings patrone het. Hierdie studie bied nuwe insigte in die evolusie van SUS en TPS gene in P. patens en sal dien as 'n platform vir die ontwerp van toekomstige eksperimente wat verband hou met hierdie geenfamilies in nie-vaatplante. Doctoral 2020-08-25T14:32:56Z 2021-01-31T19:34:36Z 2020-08-25T14:32:56Z 2021-01-31T19:34:36Z 2020-12 Thesis http://hdl.handle.net/10019.1/109082 en_ZA en_ZA Stellenbosch University xiv, 132 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Trehalose Sucrose -- Synthesis Sucrose synthase Sugar -- Analysis Plants -- Effect of sugars on Physcomitrella patens -- Genetics UCTD Wiese, Anna Johanna Characterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens. |
| title | Characterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens. |
| title_full | Characterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens. |
| title_fullStr | Characterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens. |
| title_full_unstemmed | Characterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens. |
| title_short | Characterization of the Sucrose Synthase and class II Trehalose 6-Phosphate Synthase gene families in the moss Physcomitrella patens. |
| title_sort | characterization of the sucrose synthase and class ii trehalose 6 phosphate synthase gene families in the moss physcomitrella patens |
| topic | Trehalose Sucrose -- Synthesis Sucrose synthase Sugar -- Analysis Plants -- Effect of sugars on Physcomitrella patens -- Genetics UCTD |
| url | http://hdl.handle.net/10019.1/109082 |
| work_keys_str_mv | AT wieseannajohanna characterizationofthesucrosesynthaseandclassiitrehalose6phosphatesynthasegenefamiliesinthemossphyscomitrellapatens |