The subcellular localization of Eucalyptus grandis sucrose synthase 1 (EgSUSY1) fusion proteins expressed in Arabidopsis thaliana

Abstract

Sucrose is the major transported photoassimilate in plants and is degraded concurrently by two enzymes: invertases and sucrose synthase. Sucrose synthase catalyzes the reversible conversion of UDP and sucrose to form fructose and UDP-glucose, the latter being the activated substrate for many metabolic processes including cellulose biosynthesis. There is evidence that sucrose synthase is phosphorylated as a regulatory mechanism of carbon allocation at a conserved N-terminal serine residue. The phosphorylation or dephosphorylation at this specific site has also been found to shift the protein localization in a tissue and species specific manner. A literature study of the functional regulation of sucrose synthase in plants has highlighted several scientific questions: Is sucrose synthase cellular localization regulated by phosphorylation of an N-terminal conserved serine residue? What are the regulatory mechanisms underlying within and between species variation in sucrose synthase localization? Does sucrose synthase associate with the cellulose synthase enzyme complex? Can cellulose biosynthesis be increased by over-expression of the membrane-associated form of sucrose synthase? The aim of this M.Sc study was to determine the subcellular localization of Eucalyptus grandis sucrose synthase 1 (EgSUSY1) fusion proteins expressed in Arabidopsis thaliana plants. This was investigated through modifying the 11th serine residue of EgSUSY1 into either a non-polar alanine residue that cannot be phosphorylated (S11A), or into a negatively charged glutamic acid residue which may mimic phosphorylation at this site (S11E). The modified proteins were translationally fused to green fluorescent protein (GFP) and expressed in transgenic Arabidopsis thaliana. The proteins’ subcellular localization were analysed in planta using laser scanning confocal microscopy (LSCM). Findings in this study point to the peripheral localization of modified and unmodified GFPEgSUSY1 proteins with a prominent cytoplasmic component. No evidence was found for the localization of modified or unmodified GFP-EgSUSY1 proteins within the extracellular matrix. The current study did not establish nor negate plasma membrane association of any of the GFP-EgSUSY1 fusion proteins. It was concluded that alternative methodologies need to be explored to further address issues surrounding subcellular localization of sucrose synthase. These studies will not only aid in defining the role of this enzyme in carbon allocation, but also add to our expanding knowledge of cellulose biosynthesis and cell wall formation. Copyright 2011, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Sauer, J 2011, The subcellular localization of eucalyptus grandis sucrose synthase 1 (EgSUSY1) fusion proteins expressed in Arabidopsis thaliana, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02102012-102209 / > C12/4/111/gm