The role of a canonical Poly(A) Polymerase in organ-identity dependent size regulation in Arabidopsis thaliana Lang Son Vi A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy John Innes Centre Norwich October 2011 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. 1 Abstract Different organs of plants and animals grow to characteristic sizes and shaped that depend on their identity. We still know surprisingly little about how organ identity modified patterns of growth within the primordium to achieve the correct final size. Here, I report on a novel mutant in Arabidopsis thaliana that forms smaller leaves, but bigger flowers. Intriguingly, the effect of the mutation strictly depends on the identity of the organ, marking out this mutation as a promising entry point to studying the relation between organ identity and growth. The phenotypes are due to a partial loss of function of one of three canonical nuclear poly(A) polymerases (PAP) in A. thaliana, PAPS1. By contrast, even complete loss of function of the other two gene-family members only causes subtle phenotypes, indicating functional specialization amongst the different genes. Using promoter-swap and chimaeric-protein constructs, this functional specificity is shown to be largely encoded in the C-terminal non-catalytic domains of the different proteins. Using a mRNA fractionation coupled with microarray, I have defined a set of mRNAs whose poly(A)- tails and stability are changed in the mutant compared to wild type. The microarray analysis suggests that the reduced leaf-growth results from an ectopic upregulation of pathogen- reponse genes. Analysis of plants that are chimaeric for the paps1-1 mutation shows that PAPS1 acts cell autonomously to control petal size and paps1-1 chimerism in the internal layer of the meristem invariably causes the meristem to split into two or more meristems. I propose that different pre-mRNAs are preferentially processed by different canonical PAPs with distinct outcomes in terms of their poly(A) tail lengths. This would open up an additional level of gene expression whereby cells could co-regulate large numbers of transcripts in response to developmental or environmental changes by modulating the balance of canonical PAP isoform activities. I also discuss possible reasons for the identity- dependence of the paps1-1 mutant phenotypes. 2 Acknowledgements My greatest thanks are to my supervisor Michael Lenhard for providing a fantastic work atmosphere, invaluable scientific advices and for his interest in my work. Michael gives me the full freedom to work and create new ideas. I have learnt a lot from the way he analyzes and criticizes the ideas both on this project and on science in general, which he often openly and honestly shares with the lab. His creativity and vision in future research motivate me a lot. I am grateful to my advisors Robert Sablowski, who later becomes my primary supervisor, and John Doonan for constructive suggestions during my supervisory committee meetings. I thank Simon Swiezewski and Fuquan Liu for fantastic discussion and motivation on this project. I thank Cornelia De Moore, James Manley, Nishta Rao, Ghanasyam Rallapalli for collaborating and teaching new techniques on this project. My funding for the PhD is supported by the John Innes Rotation PhD program , BBSRC and the German research foundation DFG. A very big thanks to all past and present members of the Lenhard group and the Baurle group for their help and discussion during my PhD. Special thanks to Holger Breuninger, who always helps me with computing; Adrien Sicard whom I share bench with and for making the lab less lonely during the weekends; Nicola Stacey for her invaluable English support; Peggy Lange, Gerda Trost and Hjördis Czesnick and Christian Kappel who directly involve in some of the work in this project; the new lab members in Potsdam, Germany for making my movement to the new lab in Potsdam, Germany very enjoyable; and two very likable students in the lab at JIC, Lena Stransfeld and Nikolai Adamski. My special thanks to Tung Le and Ngat Tran, my badminton mates Dora Szakonyi, Quynh Le, Thanh Luong, Frank Bauer and many other friends in Norwich and Potsdam with whom I have had so much fun with during my free time here. Lastly, I would like to thank my whole family including my parents, my sister, my cousins and my aunts and my dearest friends Nguyen Thi Thuy Hong, Pham Thi Hong Van, An Dau for their constant communication and encouragements throughout my PhD. 3 Contents Abstract .................................................................................................................................... 2 Acknowledgements .................................................................................................................... 3 Contents.................................................................................................................................... 4 List of Figures ........................................................................................................................... 9 List of Tables and Boxes .......................................................................................................... 12 1 Chapter 1: Introduction ..................................................................................................... 13 1.1 Organ size control in plants: ..................................................................................... 14 1.1.1 The organ growth process: .................................................................................... 14 1.1.2 A definition: What is a size regulator? ................................................................... 16 1.1.3 Properties to be examined for organ size mutants:................................................... 17 1.1.4 The players: Genes involved in organ size control in Arabiodopsis thaliana: ............ 18 1.1.5 General conclusions about the mechanism of organ size control in Arabiodopsis thaliana: .......................................................................................................................... 18 1.1.6 How can different organs reach different size? What is the relationship between organ identity and size regulation ? ............................................................................................. 39 1.2 Pre-mRNA 3’ end formation in plants with comparison to yeast and human: ................ 40 1.2.1 3’ end formation in general: .................................................................................. 40 1.2.2 Roles of 3’ end formation: .................................................................................... 40 1.2.1 Components of the 3’ end formation: ..................................................................... 43 1.3 Poly (A) polymerase: ............................................................................................... 55 1.3.1 General properties of canonical PAPs in yeast and animals: ..................................... 55 1.3.2 Detailed properties of canonical PAPs in yeast and animals: .................................... 57 1.3.3 The genes encoding cPAPs and their mutant phenotypes in yeast and mammalians cPAPs: ...........................................................................................................................59 1.3.4 Plant canonical poly(A) polymerases: .................................................................... 62 1.4 Regulation of canonical polyadenylation: ................................................................... 68 1.4.1 Poly(A)-tail length regulation ............................................................................... 68 1.4.2 Regulation of cleavage and the site of polyadenylation (Alternative polyadenylation) 75 4 1.5 Non-canonical PAPs: ............................................................................................... 79 1.6 Project Aims and Objectives: .................................................................................... 81 1.6.1 Preliminary results: .............................................................................................. 81 1.6.2 Aims and objectives: ............................................................................................ 81 2 .Chapter 2. ds39 is a peculiar mutant with smaller leaves and larger flowers than wild-type. ... 83 2.1 ds39 mutant plants have larger floral organs, but smaller leaves: .................................. 84 2.2 Final cell size is increased in petals, but decreased in leaves of ds39 mutants: ............... 84 2.3 ds39 petals grow at the same rate but for longer period: .............................................. 85 2.4 Cell division is enhanced in ds39: ............................................................................. 90 2.5 Other phenotype of ds39:.......................................................................................... 93 2.6 The opposite phenotypes of ds39 on flower size and leaf size are dependent on the organ identity rather than organ position: ........................................................................................ 93 3 .Chapter 3.The identification of the ds39 mutation as paps1-1 and the phenotypes