Acknowledgement Unraveling the function of trehalose biosynthesis genes in lateral root development Celine DE MAESSCHALCK Master‟s dissertation submitted to obtain the degree of Master of Biochemistry and Biotechnology Major Plant Biotechnology Academic year 2009-2010 Promoter(s): Prof. Dr. Tom Beeckman Scientific supervisor: Drs. Lorena López Department Plant Biotechnology and Genetics VIB - Department Plant Systems Biology II Acknowledgement ACKNOWLEDGEMENTS First I would like to thank Prof. Dr. T. Beeckman for the opportunities to make my Master thesis possible in his Lab. También me gustaría agradecer a Lorena por haber sido tan buena consejera y por la preocupación que siempre ha mostrado. Ella me ha apoyado durante todo el proceso de componer y analizar los informes. I also need to thank the other people in the lab, Gert, Dominique, Boris, Giel, Marlies, Leen, Wei and Maria, who helps me when I had some questions or problems during the pratical work and to motivate me during writing. Daarnaast moet ik mijn medestudenten bedanken: Evy, Isabel, Morgane, Silke, Lynda, Brecht en Wolf, voor de leuke vijf jaar als student. Ook een dankwoordje voor de klimmers, Bram, Kristof en Thomas die steeds luisterden naar mijn avonturen over epjes wegen en zijwortels tellen. Bram wil ik nog eens extra bedanken voor het nalezen en verbeteren van mijn thesis en steeds klaar te staan met nuttige tips and tricks. Thomas zorgde dan weer voor de muzikale noot gedurende mijn thesis. Ook de brugse vrienden en vriendinnen moet ik bedanken: Loesje voor de Girls-talk en er gewoon te zijn wanneer het nodig was, Charlotte voor het leren van mijn eerste zinnetjes spaans, de scouts,…. Dan hebben we natuurlijk ook de oppeppers van de muffe PC-klas: Annelies, Fien, Silke, Lieven en Brecht. Bedankt voor de gezellige pizza avonden, de vele lachmomenten met onze over-uitbundige Silke, het oplossen van de rebussen op het oppep-bord, de Rihanna muziek op de late uurtjes, het klagen/zagen/raadgeven en vooral het samen schrijven. En als laatste mag ik ook niet mijn ouders en zusjes vergeten te bedanken, voor hun onvoorwaardelijke steun gedurende mijn hele studie en deze thesis. Het is niet altijd gemakkelijk geweest, maar ze bleven toch steeds in mij geloven. Thank all of you!! Celine Table of contents TABLE OF CONTENTS Acknowledgements .................................................................................................................................................. I List of abbreviations .............................................................................................................................................. IV Samenvatting ......................................................................................................................................................... VI Summary .............................................................................................................................................................. VII 1 Introduction ................................................................................................................................................... 1 1.1 Trehalose ............................................................................................................................................... 2 1.1.1 The synthesis of trehalose ............................................................................................................. 2 1.1.2 The breakdown of trehalose .......................................................................................................... 3 1.2 Trehalose pathway enzymes .................................................................................................................. 4 1.2.1 Trehalose phosphate synthases (TPS) in plants ............................................................................ 5 1.2.2 Trehalose phosphate phosphatases (TPP) in plants ....................................................................... 6 1.2.3 trehalase ........................................................................................................................................ 7 1.2.4 The relationship of the enzymes ................................................................................................... 7 1.3 The modulation of the trehalose pathway in plants ............................................................................... 9 1.3.1 Trehalose-6-phosphate (T6P) ........................................................................................................ 9 1.3.2 TPS1 ............................................................................................................................................. 9 1.3.3 The inflorescence arcitecture ...................................................................................................... 10 1.3.4 Starvation .................................................................................................................................... 11 1.3.5 Sucrose ........................................................................................................................................ 12 1.3.6 Abiotic stress............................................................................................................................... 12 1.3.7 Nitrogen ...................................................................................................................................... 13 1.4 Lateral root development ..................................................................................................................... 14 1.4.1 Lateral root development ............................................................................................................ 15 1.4.2 Lateral root initation (LRI) ......................................................................................................... 15 1.4.3 The developmental stages of the lateral root ............................................................................... 19 2 Aim .............................................................................................................................................................. 20 3 Results ......................................................................................................................................................... 21 3.1 Expression pattern of TPP genes ......................................................................................................... 21 3.1.1 Expression during lateral root development ............................................................................... 22 3.1.2 Expression in the root tissue ....................................................................................................... 24 3.1.3 TPP genes expression pattern ..................................................................................................... 25 I Table of contents 3.2 Effects of knockout and overexpression TPPB lines in the development of A.Thaliana..................... 26 3.3 Effects of day length on the root growth of TPP knockout and overexpression lines ............................. ............................................................................................................................................................. 28 3.4 The effect of trehalose on the plant growth of knockout and overexpression TPP lines ......................... 31 3.5 Effect of sucrose on plant growth in different TPP knockout and overexpression lines ......................... 33 3.6 Effect of drought on the growth of the TPP mutant lines .................................................................... 34 3.7 The effects of nitrogen starvation on root/shoot biomass in knockout and overexpression lines ........ 36 4 Discussion ................................................................................................................................................... 39 4.1 Expression pattern of TPP genes ......................................................................................................... 39 4.2 Effects of TPPB knockout and overexpression in the development of A.Thaliana ............................. 40 4.3 Effects of day length on the root growth of TPP knockout and overexpression lines ............................. 41 4.4 The effect of trehalose on the plant growth of knockout and overexpression TPP lines ......................... 41 4.5 Effect of sucrose on plant growth in different TPP knockout and overexpression lines ......................... 42 4.6 Effect of drought on the growth of the TPP mutant lines .................................................................... 42 4.7 The effects of nitrogen starvation on root/shoot biomass in knockout and overexpression lines ........ 43 4.8 Conclusion ........................................................................................................................................... 43 5 Material and methods .................................................................................................................................. 44 5.1 TPP-GUS/GFP markerlines ................................................................................................................. 44 5.2 TPP knock-out and overexpression lines ............................................................................................. 44 5.3 Plant materials and general growth conditions ...................................................................................