Roles of disproportionating enzymes in the moss Physcomitrella patens

Stander, Emily Amor (2015-12)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/97992
This item appears in the following collections: