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Table of Contents Chapter 1: Introduction MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation Of Desheng Liu Candidate for the Degree: Doctor of Philosophy _____________________________________ (Dr. Christopher A. Makaroff, Director) _____________________________________ (Carole Dabney-Smith, Committee Chair) _____________________________________ (Dr. Michael A. Kennedy, Reader) _____________________________________ (Dr. David L. Tierney, Reader) _____________________________________ (Dr. Eileen K. Bridge, Graduate School Representative) ABSTRACT OVEREXPRESSION OF NTAP:ATCTF7∆B LEADS TO PLEIOTROPIC DEFECTS IN REPRODUCTION AND VEGETATIVE GROWTH IN ARABIDOPSIS by Desheng Liu Eco1/Ctf7 plays a critical role in the establishment of sister chromatid cohesion, which is required for the faithful segregation of replicated chromosomes. Inactivation of Arabidopsis CTF7 (AtCTF7) results in severe reproductive and vegetative growth defects. To further investigate potential roles of AtCTF7 and to identify AtCTF7 interacting proteins, several AtCTF7 constructs were generated and expressed in Arabidopsis plants. 35S:NTAP:AtCTF7∆B (AtCTF7∆299-345) transgenic plants displayed a wide range of phenotypic alterations in reproduction and vegetative growth. Male meiocytes from 35S:NTAP:AtCTF7∆B plants exhibited defective chromosome segregation and ultimately fragmented chromosomes. Mutant ovules developed asynchronously, experienced prolonged meiotic and megagametophytic stages and produced megaspores/embryo sacs that degenerated at various stages. The transgenic plants also exhibited a broad range of vegetative defects, including meristem disruption and apparent epigenetic alterations. Transcripts for epigenetically regulated transposable elements were elevated in transgenic plants. 35S:AtCTF7∆B transgenic plants also exhibited reduced fertility and vegetative defects, with the 35S:AtCTF7∆B defects appearing more severe than those in 35S:NTAP:AtCTF7∆B plants. Additional phenotypes were also observed in 35S:AtCTF7∆B transgenic plants. Therefore, the defects observed in 35S:NTAP:AtCTF7∆B plants are caused by high level expression of AtCTF7∆B and not the presence of the NTAP tag. Finally, Atctf7 plants containing a CTF7pro:AtCTF7∆B construct were obtained and found to grow only slightly better than Atctf7 plants. Therefore, the B motif is required for proper AtCTF7 function. In summary, this study further demonstrates that AtCTF7 plays essential roles in reproduction and in vegetative growth, and that proper levels of AtCTF7 are critical for normal plant growth and development. OVEREXPRESSION OF NTAP:ATCTF7∆B LEADS TO PLEIOTROPIC DEFECTS IN REPRODUCTION AND VEGETATIVE GROWTH IN ARABIDOPSIS A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry by Desheng Liu Miami University Oxford, Ohio December, 2014 Dissertation Director: Dr. Christopher A. Makaroff Table of Contents Chapter 1: Introduction ................................................................................................................... 1 1.1 Mitosis and meiosis............................................................................................................ 1 1.2 Sister chromatid cohesion and chromosome segregation .................................................. 4 1.3 The cohesin complex ......................................................................................................... 4 1.3.1 The cohesin complex in Arabidopsis ...................................................................... 11 1.4 Loading of the cohesion complex on sister chromatids ................................................... 12 1.4.1 Cohesin loading in Arabidopsis ............................................................................. 13 1.5 Cohesion establishment and maintenance ....................................................................... 14 1.5.1 Functions of lysine acetylation .............................................................................. 16 1.6 Cohesin removal .............................................................................................................. 17 1.6.1 Cohesin removal in mitosis .................................................................................... 17 1.6.1.1 Protection and removal of centromeric cohesins ................................................ 19 1.6.2 Cohesin removal in meiosis ................................................................................... 21 1.6.3 Functions of Separase ............................................................................................ 21 1.7 Cohesion establishment, maintenance and release in Arabidopsis .................................. 21 1.7.1 Protection of centromeric cohesion in Arabidopsis ............................................... 22 1.7.2 Separase in Arabidopsis ......................................................................................... 23 1.8 Cohesin complex: functions beyond sister chromatid cohesion ...................................... 24 1.8.1 TranscriptionAL regulation related to the cohesin complex .................................. 24 1.8.2 Cornelia de Lange Syndrome (CdLS).................................................................... 25 1.8.3 TranscriptionAL mis-regulation in Roberts Syndrome (RBS) .............................. 26 1.8.4 DNA double-strand break repair ............................................................................ 27 1.8.5 Human cancers related to mis-function of cohesin ................................................ 28 1.9 References ........................................................................................................................ 30 Chapter 2: 35S:NTAP:AtCTF7∆B leads to various defects in reproduction ................................ 48 2.0 Abstract ............................................................................................................................ 48 2.1 Introduction ...................................................................................................................... 49 2.1.1 Background for Eco1/Ctf7 ..................................................................................... 49 2.1.2 Esco2 mutations in Roberts Syndrome (RBS) ....................................................... 50 2.1.3 The AtCTF7 protein ............................................................................................... 50 2.2 Results .............................................................................................................................. 56 2.2.1 Experimental design and dominant negative defects of 35S:NTAP:AtCTF7∆B plants ............................................................................................................................... 56 2.2.2 35S:NTAP:AtCTF7∆B plants demonstrate defective chromosome segregation during male meiosis ........................................................................................................ 60 2.2.3 35S:NTAP:AtCTF7∆B leads to various defects in ovule development ............... 63 2.2.3.1 Introduction for ovule development in wild type Arabidopsis .......................... 63 2.2.3.2 Ovules display various defects due to variations in AtCTF7 expression............ 67 2.2.3.3 35S:NTAP:AtCTF7∆B leads to various defects in ovule development ............. 67 2.2.4 35S:NTAP:AtCTF7∆B leads to disrupted expression in the genes involved in female gametophyte development and other related processes ...................................... 77 2.3 Discussion ........................................................................................................................ 80 2.4 Materials and methods ..................................................................................................... 83 2.5 References ........................................................................................................................ 90 ii Chapter 3: 35S:NTAP:AtCTF7∆B leads to vegetative growth defects ........................................ 89 3.0 Abstract ............................................................................................................................ 96 3.1 Introduction and background ........................................................................................... 97 3.2 Results .............................................................................................................................. 98 3.2.1 Expression of 35S:NTAP:AtCTF7∆B causes pleiotropic growth defects ............ 98 3.2.2 Epigenetic alterations are present in 35S:NTAP:AtCTF7∆B plants .................... 102 3.2.3 Analysis of 35S:AtCTF7∆B and CTF7:AtCTF∆B plants ................................... 106 3.3 Discussion ...................................................................................................................... 111 3.4 Materials and methods .................................................................................................... 112 3.5 References ....................................................................................................................... 119 Chapter 4: Conclusions and perspectives ................................................................................... 121 4.1 References ...................................................................................................................... 127 iii
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