Site-directed mutagenesis and functional analysis of Eucalyptus sucrose synthase 1 (Egsusy 1) in Arabidopsis Thaliana

Abstract

Conventional breeding technologies, which are being used for the domestication of tree species, struggle to meet the current demand for wood products such as paper, pulp and timber. Future targets of developing cellulosic bio-fuels to mitigate the global demand for renewable energy will contribute to the increased demand of wood products. The strides made by biotechnology present opportunities for faster and sustainable genetic improvement of trees by targeting specific traits with accuracy to deliver on current and future wood product demands. Tree biotechnology will enable breeders to target important traits in trees for improvement, such as wood fibre with high cellulose content. Desired improvements include the reduction of lignin in trees within physiological limits and changing its structural components for easy extraction and to increase of flux and partitioning of carbon into cellulose. These processes involve highly complex pathways with complex regulatory mechanisms, which are influenced directly and indirectly by internal and external stimuli. Therefore, there is a vital need to discover and functionally validate these mechanism and the components involved to unravel the complexities of wood development. However, a fundamental constraint is to increase cellulose deposition and simultaneously ensure that the balance of carbon allocation between other carbon utilising pathways is maintained under normal tree growth conditions. Therefore, understanding the assimilation of carbon in autotrophic (source) tissues, the partitioning of carbon into heterotrophic (sink) tissues and deciphering the pathways and genes involved is required to develop molecular strategies that will sustain this balance. This dissertation focuses on sucrose synthase (SuSy) involvement in sucrose carbon allocation towards plants growth and development and more specifically, towards cellulose deposition. Chapter One reviews sucrose carbon metabolising enzymes and their regulatory mechanisms related to their potential role in increasing cellulose deposition. Focus was placed on the N-terminal serine phosphorylation domain of the sucrose synthase enzyme and its potential involvement in regulating SuSy sub-cellular localization to support cellulose biosynthesis. Chapter Two of this dissertation reports on the site-directed mutagenesis of the N-terminal non-catalytic serine phosphorylation domain of Eucalyptus grandis sucrose synthase 1 (EgSuSy1) gene. The unmodified and modified EgSuSy1-S11A were functionally characterized as potential candidates for allocation of sucrose carbon towards cellulose biosynthesis by overexpression in transgenic Arabidopsis plants. The objective of this research is to sub-localize sucrose synthase activity in the membrane fraction thereby allocating sucrose carbon towards cellulose biosynthesis. Our observation indicated that the EgSuSy1 lines had more fibre development with slightly smaller secondary cell wall thickness, whereas EgSuSy1-S11A lines seem to maintain fibre development and secondary cell wall compared to the Col-0 control. No significant changes were observed in the percentage content of secondary cell wall sugars between Col-0, EgSuSy1 and EgSuSy1- S11A. The research findings presented in this dissertation represent the outcomes of the study undertaken from January 2007 to July 2009 in the Department of Genetics, University of Pretoria under the supervision of Prof. A.A. Myburg and Dr. S. Naidoo. Chapter Two is prepared in the format of a manuscript for a peer-reviewed research journal (e.g. Tree Physiology). Therefore, there is some degree of repetition between Chapter One and Chapter Two. The work from this study has not been presented orally or as a poster in any formal scientific meeting due to IP restrictions.