Analysis of aspects of the starch metabolic pathway in lower plants

Jacobs, Ingrid (2018-12)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Starch is an important plant storage polysaccharide that has been demonstrated to have a major influence on plant growth. Transitory starch is synthesized in the leaves of plants during the day as a product of photosynthesis and degraded at night to allow continued carbon allocation for growth and cellular processes. It is also produced and stored for longer periods of time in non-photosynthetic organs such as stems, tubers and seeds. The study of starch is important for several reasons – not only is it a vital part of the human diet, it is also utilised in many non-food applications such as the paper, textile, oil and pharmaceutical industries. The pathway of starch metabolism in higher plants has been studied for decades and of late, Arabidopsis has become the workhorse plant for many starch researchers due to the plethora of insertion mutants that are readily available. However, the predominant use of Arabidopsis as a model system has led to a narrow understanding of starch metabolism restricted to that of synthesis and degradation of leaf starch. There are ongoing attempts to translate the knowledge gleaned from Arabidopsis into studies on the storage organs of crop plants (e.g. rice and maize endosperm, potato tubers), as well as starch metabolism in lower plants (e.g. algae and mosses) to aid in elucidating the evolutionary development of starch metabolism in land plants. This study investigated two aspects of starch metabolism in lower plants to determine whether the pathway of starch metabolism observed in higher plants is conserved. Firstly, a previously uncharacterised starch synthase from the red alga Chondrus crispus was examined due to the reported differences in substrate preference between red algal and green plant starch synthases, and the deviation in compartmentalisation of starch synthesis and storage in members of the red algae. The C. crispus starch synthase was analysed by means of multiple sequence alignment, site-directed mutagenesis and recombinant protein expression and purification. Features unique to red algal starch synthases were identified, including a C-terminal glycogen binding domain and sequence variations in important residues involved with substrate binding. During recombinant expression, the C. crispus protein was insoluble and accumulated in inclusion bodies. Attempts to recover active protein through optimisation of expression, the use of alternative expression systems and protein refolding were unsuccessful and biochemical characterisation of the starch synthase could not be performed. Secondly, four putative orthologues of the Arabidopsis maltose excess (MEX) transporter were identified in the moss Physcomitrella patens and their functions examined through the generation of knockout mutant lines and complementation of Escherichia coli mutants defective for sugar transporters. Knockout mutants were successfully generated for the P. patens MEX1a gene, while complementation studies failed to produce active protein. Expression profiling in wild type P. patens suggest that the four PpMEX genes are differentially expressed depending on the developmental stage of the culture and may have specialised functions in various growth structures.

AFRIKAANSE OPSOMMING: Stysel is ‘n belangrike polisakkaried wat gestoor word in plante waar dit dien as ‘n noemenswaardige bron van koolstof vir plantegroei. Gedurende die dag word kortstondige stysel geproduseer in die blare van plante deur die proses van fotosintese en word dan deur die nag afgebreek om voortdurend koolstof te voorsien vir plantegroei en ander selprosesse. Dit word ook geproduseer en gestoor vir langer tydperke in plantorgane wat nie fotosintese ondergaan nie, soos stamme, knolle en sade. Stysel word bestudeer vir verskeie redes – nie net vorm dit ‘n belangrike komponent van die menslike diëet nie, maar word ook gebruik in die papier-, tekstiele- , olie- en artsenykunde-industriëe. Die roete van styselmetabolisme word al vir dekades ondersoek in hoër plante. Arabidopsis het onlangs die gekose sisteem geword van navorsers as gevolg van die oorvloed van mutante lyne wat vrylik beskikbaar is. Die oorheersende gebruik van Arabidopsis het egter ‘n noue begrip van styselmetabolisme tot gevolg gehad wat beperk is tot dié van stysel in blare. Daar word voortdurend pogings aangewend om die kennis wat deur studies op Arabidopsis verkry is, toe te pas op die stoororgane van gewasse (bv. die kiemwit van rys en koring, en ook aartappelknolle), sowel as styselmetabolisme in laer plante (bv. alge en mos) om die evolusionêre ontwikkeling daarvan in landplante te ontrafel. Hierdie studie het twee aspekte van styselmetabolism in laer plante ondersoek om te bepaal of die pad van styselmetabolisme wat in hoër plante waargeneem is, bewaar word. Eerstens was ‘n voorheen onbeskryfde stysel-sintase van die rooi alg Chondrus crispus bestudeer omdat daar vantevore berig is oor verskille in rooi alge en hoër plante met betrekking tot substraatvoorkeur en ligging van styselproduksie en -stoor in die sel. Die C. crispus stysel-sintase was ondersoek deur middel van ‘multiple sequence alignment’, ‘site-directed mutagenesis’ en rekombinante proteïenuitdrukking en –suiwering. Kenmerke wat uniek is tot die rooi alg sintase, soos ‘n C-terminaal glikogeen-bindingsgebied en verskille in aminosure wat ‘n rol speel in substraatbinding, was geïdentifiseer. Tydens rekombinant proteïenuitdrukking was die C. crispus proteïen onoplosbaar en het geaggregeer. Pogings om aktiewe proteïen te herkry deur optimalisering van uitdrukking, die gebruik van alternatiewe uitdrukkingsisteme en proteïen-hervouing was onsuksesvol en biochemiese karaktarisering van die proteïen kon dus nie uitgevoer word nie. Tweedens was vier putatiewe ortoloë van die Arabidopsis ‘maltose excess (MEX)’ vervoerder geïdentifiseer in die mos Physcomitrella patens. Die funksies van hierdie vier gene was ondersoek deur mutante mos lyne te genereer en Escherichia coli mutante, wat gebrekkig is vir sekere suikervervoerderproteïene, te komplementeer. Die generasie van ‘n mutante lyn vir die MEX1a geen was suksesvol, terwyl die komplementering van E. coli misluk het as gevolg van ‘n gebrek aan die uitdrukking van aktiewe rekombinante proteïen. Die uitdrukkingsprofiel van die PpMEX gene in wildetipe P. patens dui aan dat die gene moontlik differensieel uitgedruk word en afhangend is van die ontwikkelingsfase van die mos. Dus het die vier gene moontlik gespesialiseerde funksies in verskillende mosstrukture.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/104896
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