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Investigation of Glycosyltransferases from Oat

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Investigation of Glycosyltransferases from Oat Investigation of glycosyltransferases from oat Thomas LOUVEAU A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy University of East Anglia John Innes Centre Norwich, the United Kingdoms © October 2013 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there-from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. I Abstract Plants produce a diversity of secondary metabolites crucial for their survival into specific ecological niches. Many of these compounds are glycosides generated by the action of family one UDP-dependant glycosyltransferases (UGTs). Glycosylated products of UGTs are known to be essential for reproductive fitness, defence against pathogens, and signalling; UGTs also have a role in the detoxification of xenobiotics. To date, little is known about monocot UGTs compare to their dicot counterparts, despite their potential role in defence and modification of health-promoting component of cereals essential to human diet. This thesis focuses on identification and functional investigation of UGTs expressed in in the diploid oat species Avena strigosa. Chapters 1 and 2 consist of the General Introduction and Material and Methods, respectively. In chapter 3, a systematic analysis of root-expressed UGTs was carried out using transcriptomic and proteomic approaches. A subset of UGTs was then selected for biochemical analysis. Of particular interest were candidates for glycosylation of avenacin, an antimicrobial triterpenoid glycoside that protects oat against fungi infection. In chapter 4, the sugar donor specificity of the recombinant UGTs and their activity towards different triterpenoid acceptors was investigated. In chapter 5, a transient expression system was established in Nicotiana benthamiana in order to investigate UGT activity. Heterologous co-expression of UGTs with early avenacin biosynthetic pathway enzymes leads to biosynthesis of new-to-nature triterpenoid glycosides, so providing a powerful system for functional analysis of terpene glycosylation in planta. In chapter 6, the catalytic properties of the UGT collection towards different plant natural products was investigated, leading to the production of glycosides of interest. The contribution of this study to the understanding of the evolution and function of monocot UGTs and to their potential commercial exploitation is discussed in the chapter 7. II Acknowledgement I would like to express my deepest gratitude to my primary supervisor Professor Anne Osbourn, for giving me the opportunity to perform my PhD in her laboratory and for her precious advices and support. I would also like to address a special word of thanks to my secondary supervisor Professor Rob Field, for guiding me through the meanders of chemistry and for his patience and support when it comes to the writing. I am also addressing a great thank you to Dr P. O’Maille for the inspirational discussions and material support. I would also like to express all of my gratitude to Dr Martin Rejzek for invaluable technical support and teaching. I also would like to thank Professor G. Lomonossoff, R. Melton, Dr S. Mugford, Dr K. Geisler, Dr A. Owatworakit, Dr A. Leveau and Dr R. Thimmappa for their good advice and support. I want to express my gratitude to Professor B. Henrissat for hosting me in his lab. I am also addressing a great thank you to all of A. Osbourn Lab members past and present for the warm atmosphere making the lab a very pleasant place to work. Finally, I would like to thanks my family – Caroline, Alice and “little Ligusticum “– for their invaluable support and their infinite source of cheerfulness. I also want to thanks my parents, brother and stepfamily for their support throughout my studies and I have a deep thought to Pipik and Papy Jo at the time of submitting my thesis. Contents Chapter 1 – General introduction ............................................................................ 1 1.1 Plant secondary metabolism .......................................................................... 1 1.1.1 Introduction to plant secondary metabolism .................................................. 1 1.1.2 A large part of secondary metabolites are glycosylated ................................ 2 1.2 Glycosylation of plant secondary metabolites ............................................... 3 1.2.1 Glycosylation is a common feature of plant secondary metabolites.............. 3 1.2.2 Activity of plant UGTs .................................................................................. 5 1.2.3 Recognition of the sugar donor ...................................................................... 6 1.2.4 Acceptor recognition - Towards understanding regiospecificity of glycosylation by UGTs ................................................................................................ 8 1.2.5 Phylogeny of plant UGTs, evolution and function prediction ....................... 9 1.3 Terpenoids and triterpenoid glycosides ....................................................... 11 1.3.1 The plant terpenome .................................................................................... 11 1.3.2 Glycosylated triterpenes (saponins) ............................................................. 13 1.3.3 Glycosylation of triterpenes by UGTs ......................................................... 18 1.4 Avenacins .................................................................................................... 22 1.4.1 Role in plant defence ................................................................................... 22 1.4.2 Avenacin structure ....................................................................................... 23 1.4.3 Biosynthesis of avenacins ............................................................................ 24 1.5 PhD project overview .................................................................................. 30 Chapter 2 - Materials & methods ........................................................................... 31 2.1 Materials ...................................................................................................... 31 2.1.1 Biological materials ..................................................................................... 31 I 2.1.2 Bacterial strains ............................................................................................ 31 2.1.3 Primers ......................................................................................................... 32 2.1.4 Plasmids ....................................................................................................... 39 2.1.5 Chemicals ..................................................................................................... 39 2.2 Methods ....................................................................................................... 40 2.2.1 Growing conditions of A. strigosa and tissue collection ............................. 40 2.2.2 Genomic DNA isolation from A. strigosa ................................................... 40 2.2.3 RNA isolation .............................................................................................. 41 2.2.4 cDNA synthesis ........................................................................................... 41 2.2.5 DNA electrophoresis .................................................................................... 42 2.2.6 Sequencing reaction of DNA fragment ........................................................ 42 2.2.7 Transcript expression analysis ..................................................................... 43 2.2.8 Rapid amplification of cDNA 5’end (RACE PCR) ..................................... 44 2.2.9 Cloning UGT transcripts using a gateway strategy ..................................... 47 2.2.10 Cell transformation ...................................................................................... 51 2.2.11 Plasmid extraction and purification ............................................................. 51 2.2.12 Colony PCR ................................................................................................. 52 2.2.13 Purification of recombinant enzymes .......................................................... 53 2.2.14 Enzymatic assays ......................................................................................... 54 2.2.15 NMR analysis............................................................................................... 57 2.2.16 Protein extraction ......................................................................................... 57 2.2.17 Protein quantification by Bradford method ................................................. 57 2.2.18 Protein electrophoresis ................................................................................. 58 2.2.19 Western blot ................................................................................................. 58 2.2.20 Proteomic analysis (from G.Saalbach) ........................................................ 59 II 2.2.21 Phylogeny .................................................................................................... 59 2.2.22 Transient expression in Nicotiana benthamiana.......................................... 60 2.2.23 Analysis of metabolites from Nicotiana benthamiana leaves ....................
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