Functional Analysis of Receptor-like Kinases in Pollen-Pistil Interactions in Arabidopsis thaliana PDF
Preview Functional Analysis of Receptor-like Kinases in Pollen-Pistil Interactions in Arabidopsis thaliana
University of Massachusetts Amherst ScholarWorks@UMass Amherst Masters Theses 1911 - February 2014 2009 Functional Analysis of Receptor-like Kinases in Pollen-Pistil Interactions in Arabidopsis thaliana Mini Aggarwal University of Massachusetts Amherst Follow this and additional works at:https://scholarworks.umass.edu/theses Part of theBiochemistry Commons,Molecular Biology Commons, and thePlant Biology Commons Aggarwal, Mini, "Functional Analysis of Receptor-like Kinases in Pollen-Pistil Interactions in Arabidopsis thaliana" (2009).Masters Theses 1911 - February 2014. 347. Retrieved fromhttps://scholarworks.umass.edu/theses/347 This thesis is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Masters Theses 1911 - February 2014 by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. FUNCTIONAL ANALYSIS OF RECEPTOR-LIKE KINASES IN POLLEN- PISTIL INTERACTIONS IN ARABIDOPSIS THALIANA A Thesis Presented by MINI AGGARWAL Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE September 2009 Plant Biology © Copyright by Mini Aggarwal 2009 All Rights Reserved FUNCTIONAL ANALYSIS OF RECEPTOR-LIKE KINASES IN POLLEN- PISTIL INTERACTIONS IN ARABIDOPSIS THALIANA A Thesis Presented by MINI AGARWAL Approved as to style and content by: __________________________________________ Alice Y. Cheung, Chair __________________________________________ Om Parkash, Member __________________________________________ Ana Caicedo, Member ________________________________________ Elsbeth Walker, Department Head Plant Biology ACKNOWLEDGEMENTS It gives me great pleasure to acknowledge my supervisor, Dr. Alice Cheung for her constant support, expert guidance and vision all along the course of my research work and in preparation of this thesis. I am grateful to Dr. HenMing Wu for his invaluable guidance and timely inputs on my research work in the lab. I owe special thanks to my lab-mate, Dr. Qiaohong Duan for her help in designing and executing experiments. I would like to express my gratitude to all my other lab mates especially Daniel, Yongsheng, Chao, WenGuang and Yanjiao for their help and encouragement in the progress of my research work. Also, I duly appreciate assistance by Andre and Victor in furthering my research work. I am really grateful to Dr. Elsbeth Walker (Director) and Tobias Baskin (Graduate Program Director), Plant Biology Graduate Program, who made sure that administrative hiccups never disrupted academic progress. I sincerely thank Susan Capistran for meticulously taking care of administrative details at every step of my education in University of Massachusetts, Amherst. I would like to express my gratitude to my thesis committee, Dr. Om Parkash and Dr. Ana Caicedo for their cooperation and for providing me their invaluable insights on my research. I would like to thank Plant Biology Graduate Program for awarding me with the Davis and Delisle Research Grant for the year 2008 and 2009. iv I am thankful to my batch-mate and dear friend, Burcu who has been a constant motivator and support system during the course of my studies in UMass. I would like to thank all my friends- Vandita, Sharad, Neetu, Mayank, Anirudha, Ajanta, Supratim, Shilpi, Mona and Navdeep who proved to be not only able playmates but also formed a support group during tough and trying times. I am deeply grateful to my dearest family- my Parents and my Siblings- Smita and Aditya who have always stood by me and showed their unfailing belief in my abilities. I am most indebted to my husband- Naveen whose unconditional love, sacrifice and cooperation made a world of difference during my research and stay in Amherst. Last but not least, I would really like to thank my dear Parents-in-law and Sumeet-Sandhya for their patience, encouragement and support throughout my research work in Amherst. v ABSTRACT FUNCTIONAL ANALYSIS OF RECEPTOR-LIKE KINASES IN POLLEN- PISTIL INTERACTIONS IN ARABIDOPSIS THALIANA September 2009 MINI AGGARWAL, B.S., PANJAB UNIVERSITY INDIA M.S., PANJAB UNIVERSITY INDIA M.S., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Alice Y. Cheung Rac/Rop GTPases are molecular switches in plants that control the growth of polarized cells such as pollen tubes and root hairs, differentiation, development, actin dynamics, production of reactive oxygen species (ROS) and disease resistance. These small GTPases are activated by guanine nucleotide exchange factors (GEFs) that replace GDP for GTP and are referred to as RopGEFs in plants. To identify upstream components of the RopGEF regulated signaling pathways, GEF1 from Arabidopsis thaliana was used as a bait to screen a seedling cDNA library in a yeast two-hybrid system. This yielded members of a small family of the Catharanthus roseus Receptor- like kinase (CrRLK), referred to as the Feronia-like Receptor-like kinase (FlRLK) family as the potential GEF interactors. A synergid cell-expressed member of this family, FERONIA/SIRENE (RLK-10), regulates pollen tube reception by the female gametophyte and along with the other two plasma-membrane bound receptors of this family, THESEUS (RLK-3) and HERCULES (RLK-4) promote cell elongation in Arabidopsis. I have chosen two of these FlRLKs for my studies: RLK-5, the most abundant pollen expressed member of the family and RLK10. My studies with RLK-5 suggest that vi it plays a significant role in pollination or fertilization since homozygous rlk-5 was never recovered from selfed heterozygous rlk-5 mutant. In addition to this, the reciprocal crosses with RLK-5/rlk-5 and wild type resulted in severe male transmission defect indicating that the rlk-5 mutation induces male sterility. This observation was found consistent with the pollen-specific expression pattern of RLK-5 suggested by microarray data and confirmed by histochemical GUS staining analysis of the RLK5p-GUS transgenic Arabidopsis. The RLK-5/rlk-5 pollen displayed no pollen-viability defects and the pollen tube growth in-vivo appears normal. Despite the unaffected pollen viability and apparent normal in-vivo pollen tube growth, the RLK-5/rlk-5 plants formed low seed set suggesting compromised fertilization. Additional analysis will be required to determine the basis of male deficiency and reduced seed sets in RLK5/rlk5 mutants. Loss of function mutations of RLK-10 (FERONIA/SIRENE) gene show failure in pollen tube growth arrest upon penetration of the female gametophyte, supernumerary pollen tube penetration of the ovule and reduced female fertility. Our studies with RLK- 10 suggest that it is important for mediating a proper oxido-reductive condition within the ovule necessary for pollen tube rupture and fertilization of the female gametophyte. The multiple pollen tube entry phenotype of the Arabidopsis knockout mutant, rlk-10 correlated with decreased ROS level and de-esterified pectin in the filiform apparatus lining the synergid cell of its female gametophyte. These results establish an important signaling link between RLK-10 and ROS and pectin in a GEF-Rac/Rop regulated pathway in pollen tube-ovule interaction. vii TABLE OF CONTENTS Page ACKNOWLEDGMENTS..................................................................................................iv ABSTRACT.......................................................................................................................vi LIST OF TABLES..............................................................................................................x LIST OF FIGURES...........................................................................................................xi CHAPTER 1. INTRODUCTION...................................................................................................1 1.1 G Proteins: Rac/Rop GTPases...........................................................................1 1.2 Plant Receptor-like Kinases (RLKs)..................................................................5 1.3 ROS/H O ..........................................................................................................9 2 2 2. MATERIALS AND METHODS...........................................................................12 2.1 Plant material...................................................................................................12 2.2 DNA extraction and PCR analysis...................................................................13 2.3 GUS analysis....................................................................................................13 2.3.1 Flowers..............................................................................................13 2.3.2 Embryos............................................................................................14 2.4 Stages of flowers..............................................................................................14 2.5 Hand-pollination or Crosses............................................................................15 2.5.1 Reciprocal crosses.............................................................................15 2.5.2 Limited pollination............................................................................15 2.5.3 Self-cross...........................................................................................16 2.6 In-vivo pollen tube growth analysis.................................................................16 2.7 Seed-set analysis..............................................................................................16 2.8 H O /Ascorbic Acid/OH. radical bursting.......................................................17 2 2 2.9 Pectin staining of ovules..................................................................................17 2.9.1 Paraffin sectioning............................................................................18 3. CHARACTERIZATION AND MUTANT ANALYSIS OF POLLEN- EXPRESSED RECEPTOR-LIKE KINASES.......................................................20 3.1 Introduction......................................................................................................20 3.2 rlk-5 mutation has male transmission defect...................................................21 3.3 RLK5pGUS is localized in pollen....................................................................26 3.4 RLK-5/rlk-5 anthers yield viable pollen...........................................................26 viii 3.5 rlk-5 mutation does not affect pollen tube penetrance.....................................27 3.6 RLK-5/rlk-5 plants form lower seed set...........................................................29 3.7 Discussion........................................................................................................31 4. SYNERGID-EXPRESSED RECEPTOR-LIKE KINASE, FERONIA/SIRENE/RLK-10 MEDIATES ROS- AND PECTIN-GATED POLLEN TUBE-OVULE INTERACTIONS........................................................35 4.1 Introduction......................................................................................................35 4.2 Stages of flowers..............................................................................................38 4.3 RLK-10 mediates ROS-gated pollen tube reception........................................39 4.4 H O /OH. radical cause the pollen tube bursting.............................................42 2 2 4.5 Pectin accumulates asymmetrically in the synergid cell area..........................46 4.6 Discussion........................................................................................................50 APPENDICES A. DISTINCT EXPRESSION PATTERNS OF TYPE II ARACS IN ARABIDOPSIS.....................................................................................................53 B. DOUBLE MUTANT CROSS STATUS FOR RECEPTOR-LIKE KINASES.....58 BIBLIOGRAPHY..............................................................................................................59 ix
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