Manipulation of ascorbate biosynthesis in Solanum lycopersicum (cv Money maker)

Cronje, Christelle (2010-12)

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010.

Thesis

ENGLISH ABSTRACT: Vitamin C (ascorbate or AsA) is a secondary metabolite produced in many eukaryotes including yeasts, plants and animals. It plays essential roles as an anti-oxidant and enzyme cofactor, functions as an electron donor and -acceptor and is involved in various developmental processes. This study was initiated with the aim of increasing vitamin C production in tomato. Three genes, namely GDP-mannose pyrophosphorylase (GMPase) from Saccharomyces cerevisiae, arabinono-1,4-lactone oxidase (ALO) from Saccharomyces cerevisiae and myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana were ectopically expressed in the tomato cultivar Money Maker. GMPase converts D-mannose-6-P to GDP-D-mannose. This reaction forms part of the well characterized, “Smirnoff-Wheeler” pathway. ALO catalyzes the terminal step in erythroascorbate synthesis in yeast. In situ it also metabolizes the plant and animal substrates for ascorbate manufacture. Myo-inositol (MI) is converted into D-glucuronate by the activity of MIOX. D-Glucuronate is a precursor to L-guluno-1,4-lactone synthesis which is the precursor to AsA in animals and thought to be present in plants. The genes were independently introduced with the aid of Agrobacterium tumefaciens mediated transformation and expressed under the control of the CaMV 35S promoter. Plants with increased GMPase activity consistently showed increased L-ascorbate levels in leaves and fruit of between 20- and 70% compared to the wild-type. Plants transcribing the ALO gene exhibited small increases in L-ascorbate in green fruit (p < 0.1). Leaf tissue from MIOX plants displayed significant activity increases (p < 0.05), and substantial decreases in MI. In green fruit two MIOX lines had increases in activity, cell wall uronic acids and AsA levels. Marginal increases in L-ascorbate would not warrant industrial application, but follow-up research with over-expression of other enzymes of the “Smirnoff-Wheeler” pathway should be explored.

AFRIKAANSE OPSOMMING: Vitamien C (askorbiensuur of AsA) is ʼn sekondêre metaboliet wat in baie eukariote, insluitend gis, plante en diere geproduseer word. Dit speel ʼn noodsaaklike rol as ʼn anti-oksidant en ensiem kofaktor, funktioneer as ʼn elekronskenker en aanvaarder en is betrokke in verskillende ontwikkelings prosesse. Hierdie studie was geїnisieer met die doel om vitamien C produksie in tamatie te vermeerder. Drie gene, naamlik GDP-mannose pirofosforilase (GMPase) van Saccharomyces cerevisiae, arabinono-1,4-laktoon oksidase (ALO) van Saccharomyces cerevisiae en mio-inositol oksigenase 2 (MIOX2) van Arabidopsis thaliana was ektopies uitgedruk in the tamatie kultivar, Money Maker. GMPase skakel D-mannose-6-P om na GDP-D-mannose. Hierdie reaksie is deel van die goed gekenmerkde “Smirnoff Wheeler” baan. ALO kataliseer the terminale stap in eritroaskorbiensuur sintese in gis. In situ metaboliseer dit ook die plant en dier substrate om askorbiensuur te vervaardig. Mio-inositol (MI) is omgeskakel na D-glukuronsuur deur die aktiwiteit van MIOX. D-glukuronsuur is ʼn voorloper in L-guluno-1,4-laktoon sintese wat dan ʼn voorloper is van AsA in diere en word ook verdink om in plante teenwoordig te wees. Die gene was onafhanklik ingestel met die hulp van Agrobakterium tumefaciens gemedїeerde transformasie en uitgedruk onder die beheer van die CaMV 35S promotor. Plante met verhoogde GMPase aktiwiteit het in blare en vrugte konsekwente toename in L-askorbiensuur vlakke met tussen 20 – 70% gewys in vergelyking met wilde-tipe. Plante wat ALO getranskribeer het, het klein stygings in L-askorbiensuur in groen vrugte gewys (p < 0.1). Blaarweefsel van MIOX plante wat verhoogde aktiwiteit vertoon het, (p < 0.05), het ook aansienlike dalings in MI gehad. In groen vrugte van MIOX het twee lyne verhoogte aktiwiteit, selwand uronsuur en AsA vlakke gehad. Klein toename in L-askorbiensuur is nie gepas vir industriële toepassing nie, maar opvolg navorsing moet ondersoek word met die oor-uitdrukking van ander “Smirnoff-Wheeler” baan ensieme.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/5291
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