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«Title of Dissertation: LEUNIG, LEUNIG HOMOLOG, AND SEUSS ARE TRANSCRIPTIONAL CO-REPRESSORS THAT REGULATE FLOWER DEVELOPMENT, MUCILAGE SECRETION, AND ...»

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ABSTRACT

Title of Dissertation: LEUNIG, LEUNIG HOMOLOG, AND SEUSS

ARE TRANSCRIPTIONAL CO-REPRESSORS

THAT REGULATE FLOWER

DEVELOPMENT, MUCILAGE SECRETION,

AND PATHOGEN RESISTANCE

Minh Bui, Doctor of Philosophy, 2009 Directed By: Associate Professor William J. Higgins, Department of Biology Transcriptional repression is an important regulatory mechanism for development.

My thesis focuses on dissecting the function of Groucho (Gro)/Transducin-Like Enhancer of split (TLE) family of transcriptional repressors in plant development. My work characterizes two Arabidopsis thaliana genes, LEUNIG (LUG), first discovered to repress transcription of the floral homeotic gene AGAMOUS (AG), and LEUNIG_HOMOLOG (LUH), a gene with the highest sequence similarity to LUG. To investigate the functional redundancy between LUG and LUH, I constructed and analyzed lug; luh double mutants, and concluded that both LUG and LUH repress AG expression in the flower, with LUG playing a more prominent role than LUH. The double mutant also revealed a previously unknown function of LUG and LUH in embryogenesis because lug-3; luh-1 double mutants are embryo lethal, while the single mutants develop normal embryos. During the course of this study, I developed a new genotyping method called Simple Allele- discriminating PCR (SAP), which is cost-effective, quick, and easy to perform. This method has greatly facilitated my research as well as others in the lab.

A second part of my thesis addresses the role of LUG and LUH in other developmental processes besides flower development. My data indicate that these two genes, like their counter parts in fungi and animals, act as “global co-repressors” in various developmental and physiological processes. My thesis work revealed that both co-repressors, together with its interacting protein SEUSS (SEU), repress the Salicylic Acid (SA) pathogen defense pathway. Although lug-3, luh-1, and seu-1 mutants induced PR1 expression at higher levels than wild-type, only lug-3 and seu-1 mutants were pathogen resistant. Furthermore, LUH functions as a positive regulator in seed mucilage secretion, a process important for proper seed germination, hydration, and dispersal. I propose a possible connection between the defect in mucilage secretion and pathogen defense in luh-1 mutant plants and seeds, which places the foundation for further investigation and may uncover mucilage secretion as a major defense mechanism. My thesis has provided important insights into how transcriptional co-repressors regulate diverse developmental and physiological pathways in higher plants.

LEUNIG, LEUNIG HOMOLOG, AND SEUSS ARE TRANSCRIPTIONAL COREPRESSORS THAT REGULATE FLOWER DEVELOPMENT, MUCILAGE

SECRETION, AND PATHOGEN RESISTANCE

–  –  –

Advisory Committee:

Associate Professor William J. Higgins, Chair Associate Professor Zhongchi Liu Associate Professor Caren Chang Associate Professor Irwin Forseth Associate Professor Eric Haag Assistant Professor June Kwak © Copyright by Minh Bui 2009 Dedication This dissertation is dedicated to my wonderful and loving grandmother, Gai T.

Nguyen (1916-April 28th, 2007). May she rest in peace. I miss you, I love you, will never forget you, and I hope you are proud of me.

–  –  –

My success would not be possible without the help and support from so many people throughout my academic career. I would like to thank my parents Quang Bui and Hoa Phan, sister Kathy Bui, and cousin Khanh Dinh, all who have provided a great deal of love, emotional, and financial support throughout the years.

Secondly, my co-advisors Drs. Zhongchi Liu and William Higgins who have invested a lot of their time and effort in helping me grow as a researcher, critical thinker, and teacher/mentor.

Other affiliations that played a key role in my success include members in the Biology Department such as Drs. Marco Colombini and Justicia Opoku, who provided valuable guidance and nurtured my love for teaching, and Lois Reid and Karen Speorl for their help and support throughout the years. Also, life partner Nathan Morris and dear friends including Dr. Leah Siskind, Amy Wayne, Boy and Khang Sharp, because without you guys, I would not have made it this far. Last but not least, past and present lab members including Channa Amarasekera, Boyana Grigorova, Fei Huang, Ranjani Ganesan, Paja Sijacic, Chloe Mara, and Courtney Hollender for your friendship, support, and thanks for making lab fun!

–  –  –

Dedication

Acknowledgements

Table of Contents

List of Tables

List of Figures

Chapter 1: Introduction and Literature Review

1.1 Introduction

1.2 Flower Development

1.2.1 ABCE Model for Floral Organ Identity & Specification

1.2.2 Flower Development & Co-Repressors

1.3 Seed Coat Development and Mucilage Production & Secretion

1.3.1 Seed Development

1.3.2 Developmental Stages of the Seed Coat





1.3.3. Mucilage Composition and Synthesis

1.3.4 Genetic Control of Epidermal Seed Development

1.4 Bacterial Pathogen Resistance

1.4.1 Plant Innate Immunity

1.4.2 Plant Resistant (R)-Genes

1.4.3 The Salicylic Acid (SA) Pathway in Plant Defense

1.4.4 PATHOGENESIS RELATED (PR) 1-5 Genes

1.4.5 Hormonal Cross-talk Among Pathogen Resistance Pathways

1.5 Transcriptional Co-repressors, Structure, & Function

1.5.1 Groucho and TLE Co-repressors

1.5.2 TUP1 Functions as a Global Co-Repressor in Yeast

1.5.3 Plant Co-Repressors

1.5.4 Co-Repressor Mechanisms

1.6 Significance

Chapter 2: LEUNIG_HOMOLOG and LEUNIG Are Partially Redundant During Arabidopsis Embryo and Floral Development

2.1

Abstract

2.2 Introduction

2.3 Materials and Methods

2.3.1 Plant Growth and Mutant Identification

2.3.2 Double Mutant Construction and Genotyping

2.3.3 Microscopy and Photography

2.3.4 Molecular Analyses of LUH

2.3.5 Yeast-two-hybrid Assay and Repression Assay

2.4 Results

2.4.1 luh-1 Mutants Exhibit Vegetative and Development Defects

2.4.2 luh-1 Mutation Enhances lug Mutant Flower Phenotype

iv 2.4.3 lug; luh Double Mutants Are Embryo Lethal

2.4.4 LUG and LUH Have Divergent Functions and Expression Patterns......... 64 2.4.5 LUH Interacts Directly with SEU But Not with LUG

2.5 Discussion

2.5.1 LUG and LUH Exhibit Partially Redundant But Not Identical Functions 70 2.5.2 LUH and SEU Act in the Same Pathway to Regulate Flower Development

2.5.3 LUG and LUH Have Divergent Regulation During Environmental Stress71 2.5.4 Proposed Model for Transcriptional Repression of AGAMOUS.............. 72

2.6 Conclusion

2.7 Acknowledgements

Chapter 3: LUH Regulates Mucilage Secretion and Both LUG and LUH Regulate Pathogen Resistance in Arabidopsis

3.1 Abstract

3.2 Introduction

3.3 Materials and Methods

3.3.1 Plant Growth and DNA Extraction

3.3.2 Resin Embedding

3.3.3 Mucilage Staining

3.3.4 Scanning Electron Microscopy

3.3.5 RNA/Northern Gene Expression Analysis

3.3.6 Pathogen Inoculation

3.4 Results

3.4.1 luh-1 and lug-3 Exhibit Seed Mucilage Defects Upon Imbibition............ 88 3.4.2 Genetic Interaction Between LUH and SEU

3.4.3 luh-1 Mutant Seeds Fail to Secrete Mucilage

3.4.4 luh-1 Mutant Plants Are Susceptible While lug-3 and seu-1 Mutants Are Resistant to Bacterial Pathogen

3.4.5 lug-3, luh-1, and seu-1 Exhibit a Constitutively Active, Hypersensitive Response to Pathogen Infections

3.4.6 Most mum Mutants Are More Susceptible to Bacterial Pathogens........... 96

3.5 Discussion

3.6 Acknowledgements

Chapter 4: Conclusions

4.1 Summary

4.2 Future Direction

4.2.1 Linking Mucilage Synthesis in Seed to Pathogen Defense

4.2.2 Pathogen Defense

Appendix I: Simple allele-discriminating PCR for cost-effective and rapid genotyping and mapping (SAP)

A1.1 Abstract

A1.1.1 Background

A1.1.2 Results

v A1.1.3 Conclusions

A1.2 Introduction

A1.3 Materials and Methods

A1.3.1 Plant Growth and DNA Extraction

A1.3.2 Primers and PCR

A1.3.3 High-throughput Application

A1.4 Results

A1.4.1 SAP Primer Design for Genotyping three mutant alleles in Arabidopsis thaliana

A1.4.2 Feasibility in High-throughput Applications

A1.5 Discussion

A1.6 Conclusion

A1.7 List of Abbreviations

Appendix II: Ethane Methyl Sulfonate (EMS) Induced Mutagenesis Screen for LUH Suppressors

Bibliography

–  –  –

Table 1.1: The four classes of floral homeotic genes……………………………………….

3 Table 1.2: Genes that regulate seed coat and/or mucilage development, their molecular nature, and mutant phenotypes……………………………………………………………… 22 Table 3.1: Primers used in RT-PCR during pathogen defense and mucilage studies……… 86 Table A1.1: The strength of destabilization for all combinations of nucleotide pairing …. 120 Table A1.2: An alternative comprehensive table for the design of SAP primers…………. 120 Table A1.3: Primer sequences for three different alleles………………………………….. 123 Table A2.1: Photograph of luh-1 seed (left) and a potential luh-1 suppressor mutant seed. 130

viiList of Figures

Fig. 1.1: The four floral whorls and the tetrameric complexes that determine floral organ identity…………………………………………….………………………………………...6 Fig. 1.2: Model depicting the transcriptional machinery that regulates AG expression…… 8 Fig. 1.3: The cellular structure of the seed coat……………………………………………. 11 Fig. 1.4: Different developmental stages of the outer integument…………………………. 13 Fig. 1.5: Rhamnogalacturonan I (RGI) is the primary pectin component of mucilage that consists of galactose and arabinose branches……………………………………………….15 Fig. 1.6: An illustration of the plant cell membrane structures including the primary and secondary cell walls, and the middle lamella that “glues” the cells together……………….16 Fig. 1.7: Three distinct pathways for the synthesis of mucilage in Arabidopsis seed….….. 21 Fig. 1.8: Mutants affecting different stages of seed coat development……………………. 23 Fig. 1.9: Flowchart depicting the multi-layered defense mechanisms found in plants……. 26 Fig. 1.10: The different types of plant Resistance (R) proteins……………………………. 27 Fig. 1.11: The three primary pathogen defense pathways in plants………………………...31 Fig. 1.12: Depiction of the similar and different domain structures of the Gro/Tup1 and SEU classes of transcriptional repressors………………………………………………….. 36 Fig. 1.13: Groucho “repressosome” complex……………………………………………… 37 Fig. 2.1: LUH protein structure and gene sequence………………………………………...57 Fig. 2.2: luh-1 develops normal flowers but exhibits defects in vegetative growth……….. 59 Fig. 2.3: luh-1 enhances lug-16 and lug-3 during flower development…………………… 62 Fig. 2.4: luh-1; lug-16 double mutants are embryo lethal…………………………………. 64 Fig. 2.5: 35S::LUH failed to rescue lug-16 mutants……………………………………….. 65 Fig. 2.6: LUH expression in comparison to LUG and SEU…………………………….….. 68 Fig. 2.7: LUH interacts with SEU but not LUG in yeast…………………………………... 70 Fig. 2.8: A model on the repression of AG by LUG, LUH, and SEU during flower development………………………………………………………………………………... 74 Fig. 3.1: Mucilage secretion after imbibement across different genotypes…………….….. 90 Fig. 3.2: Plastic section of seed epidermal cells, comparing wild-type (Col-er) to luh-1…. 92 Fig. 3.3: RT-PCR gene expression analysis of cDNA from siliques/seeds………………... 93 Fig. 3.4: lug-3, seu-1, and 35S::LUH; luh-1 plants are resistant, while luh-1 is susceptible to bacterial pathogen……………………………………………………………………….. 94 Fig. 3.5: RT-PCR analysis using molecular markers in the SA and JA/ET pathogen defense pathways…………………………………………………………………………... 96 Fig. 3.6: Bacterial titer of infected mum mutants………………………………………….. 97 Fig. 3.7: Microarray data indicating the expression fold changes of the βGALACTOSIDASES (BGAL) genes………………………………………………………... 98 Fig. 3.8: RT-PCR analysis using putative SA pathway genes and genes involved in mucilage synthesis and secretion…………………………………………………………... 99 Fig. 4.1: RT-PCR analysis semi-quantitatively measuring FAS1 and FAS2 expression in lug-3 and luh-1 mutant leaves compared to wild-type, Ler and Col-er, respectively……… 108 Fig. 4.2: Different genotypes grown on MS media supplemented with 270 mM mannitol for 21 days to induce osmotic stress, resulting in increased ROS levels and leaf chlorosis.. 110 Fig. A1.1: Illustration of the SAP principle………………………………………………... 121 Fig. A1.2: SAP-based genotyping of three different mutant alleles……………………….. 123 Fig. A1.3: Adaptation of SAP for high throughput applications…………………………... 126 viii Fig A2.1: Identifying genes that repress mucilage secretion and are repressed by LUH via EMS mutagenesis in luh-1 seeds……………………………………………………………130

–  –  –



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