STUDY OF LOCAL ENVIRONMENTAL CONTROL OF ROOT SYSTEM ARCHITECTURE BAO YUN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2013 ACKNOWLEGMENTS I would like to first express my appreciation to my supervisor, JoséR. Dinneny. He offered me the opportunity to study plant biology as a Ph.D student. During my four years of study, he was always encouraging, inspiring and supportive, letting me think on my own project and design the experiments accordingly. I learned how to be a scientist from him. He cared for everyone in the lab and always wanted the best for the lab. Secondly, I would like to thank the Department of Biological Science (DBS) in National University of Singapore (NUS) for providing me the great research environment and the full scholarships for the first two years of my graduation study. I also thank Temasek Life science Laboratory (TLL) for providing great research facilities during the first two years. In addition, I would like to thank Carnegie Institution for Science for providing the excellent research atmosphere and funding during my last two years of my Ph.D study. Thirdly, I would like to thank my former lab member Pooja Aggarwal for her efforts on characterizing the hydropatterning phenomena in rice and current lab member Neil Edwards Robbins II for his efforts on maize experiments. They both contributed to the hydropatterning project. I also want to thank current lab members Muh-ching Yee, Shahram Emami, as well as Bai Yang from Dr. Wang‘s lab for his help to set up the RNA-seq experiment and data analysis. In addition, I am grateful to have had the I opportunity to collaborate with former honors student Xiangling and Simin on the hydrotropism project. Furthermore, I want to thank all the former and current lab members for every live discussion and suggestion on my project. I really had a great time in the lab. You are all my friends and I truly value the friendships. Last but not least, I want to thank all my family for their unconditional love and support at all times. I am very lucky to be your daughter, mom and dad. You always support my decisions and encourage me to continue my life journey. I also want to thank my grandparents for bringing me up and being role models for me when I was little. Additionally, I want to thank my husband, Qin Zhendong, for his understanding, encouragement and support for my study. He is always willing to share my happiness and sadness. I am really lucky to have you all as my family and love you all forever. July, 2013 Bao Yun II TABLE OF CONTENTS ACKNOWLEGEMENTS I TABLE OF CONTENTS III SUMMARY .VII LIST OF TABLES .IX LIST OF FIGURES .X LIST OF ABBREVIATIONS AND SYMBOLS XIII Chapter LITERATURE REVIEW .1 1.1Root system architecture development .2 1.1.1Arabidopsis primary root development 1.1.2Arabidopsis lateral root development .7 1.2 Endogenous hormone regulation of root development 11 1.2.1Abscisic acid 12 1.2.1.1ABA biosynthesis 12 1.2.1.2ABA signaling pathway 13 1.2.1.3ABA regulates root development 15 1.2.2Auxin 17 1.2.2.1Auxin biosynthesis 17 1.2.2.2Auxin signaling pathway and polar transportation .18 1.2.2.3Auxin regulates root development 20 1.2.3Cytokinin 22 1.2.4Gibberellin acid 23 1.2.5Ethylene 25 1.3 Environmental stimuli regulation of the root development .26 1.3.1Water 27 III 1.3.2Gravity .31 1.3.3Nutrients .32 1.4 Objective and significance of this study .34 Chapter MATERIALS AND METHODS 37 2.1Plant materials 38 2.2 Plant growth conditions 39 2.3 Transgenes construction .40 2.4 Transformation of E. coli competent cells .42 2.5 Agrobacteria mediated plant transformation 43 2.6 Luciferase imaging .44 2.7 Microscope analysis .45 2.8 Phenotypic analysis 47 2.9 Protoplasting of roots and isolation of GFP-enriched cell populations by FACS 49 2.9.1Preparing protoplast solutions 49 2.9.2Protoplasting protocol .50 2.9.3FAC sorting 51 2.10 Gene expression 52 2.10.1Q-PCR 52 2.10.2Microarray 55 2.10.2.1RNA isolation and microarray hybridization .55 2.10.2.2Data analysis 55 2.10.3RNA-seq 56 2.10.3.1RNA extraction and library preparation 56 2.10.3.2Data analysis .58 2.11 Genetic analysis 58 2.12 GUS activity analysis 59 IV Chapter RESULTS AND DISCUSSION I 60 3.1 ABSTRACT .61 3.2 INTRODUCTION 62 3.3 RESULTS .64 3.3.1Non-uniformed local environment for roots grown in tissue culture system determines asymmetries in lateral root development .64 3.3.2Lateral root patterning was determined at the founder cell priming stage .74 3.3.3ABA disrupts the hydropatterning process through signaling in the epidermis .79 3.3.4Transcriptome studies showed the importance of the epidermis tissue in response to ABA stimulus in Arabidopsis root 87 3.3.5TAA1 is locally induced by moisture and promotes hydropatterning 100 3.3.6Moisture locally promotes auxin biosynthesis and response 106 3.3.7Auxin transport is critical for hydropatterning determination and ground tissue layers are important for auxin signaling function .112 3.4 DISCUSSION .124 3.4.1Environmental stimuli act as a cue for new organ formation .124 3.4.2Mechanisms underlying lateral root development 126 3.4.3Cell type-specific effect s of different hormones involved in hydropatterning .127 Chapter RESULTS AND DISCUSSION II 129 4.1 ABSTRACT .130 4.2 INTRODUCTION 131 4.3 RESULTS .133 4.3.1ProNCED2:erGFP displays a unique asymmetric expression pattern in the LRC depending on air exposure .133 V 4.3.2Transcriptome profile of LRC revealed an important function of ABA in sensing drought 140 4.4 DISCUSSION .152 4.4.1Root cap is where roots sense environmental stimuli .152 4.4.2Water stress accompanied with other stress in plants .153 Chapter CONCLUSIONS .155 REFERENCES .158 VI SUMMARY Plant development differs fundamentally from that of many animals due to the intimacy with which organogenesis occurs in relation to the external environment. Roots are the primary organ in plants that directly make contact with the underground soil environment. Soil is a heterogeneous environment containing particles and aggregated structures of different sizes, with pockets of air and non-uniform distributions of water and nutrients. Little is known about how roots sense and interpret such micro-scale heterogeneity, partially due to lack of model experimental systems for studying such phenomena. In this study we noticed that standard tissue culture growth condition provides a spatially asymmetric environment for roots and can serve as an effective experimental system to understand the interaction between the root and its local environment. The branched root system of plants serves as a model for understanding pattern formation and is generated through the activity of a transcriptional network with oscillating activity at the root tip, which specifies lateral root pre-branch sites at regular temporal intervals (Moreno-Risueno et al., 2010). Previous work has shown that this process is not affected by different growth conditions. It has been proposed that the external regulation of lateral root development occurs after founder-cell specification. In this study, we reveal a previously uncharacterized dimension with which lateral root patterning occurs, the circumferential axis, and show that VII differences in the environment across this axis create spatial cues that determine the position of lateral roots. Using Arabidopsis as a model system, we show that the ability of roots to distinguish between a wet surface and air environment results in biases in root hair and lateral root initiation. We also observe similar phenomena in maize roots. Using tissue-specific methods to disrupt hormonal signaling, we show that perception of environmental differences likely occurs in the epidermis. The signal perceived in the outer tissue layers regulates the local induction of auxin biosynthesis and transport pathways to promote the development of lateral roots in the inner tissue layers towards the water-exposed surface. In addition to lateral root (LR) patterning, we also characterized the unique expression pattern of a reporter line, ProNCED2:erGFP. We show that the expression is only present in the lateral root cap region on the air side and can be inhibited by exposure to liquid water, indicating that the gene might be involved in sensing local environmental differences. We also investigated the transcriptome profile difference between cells of the entire lateral root cap (marked by another reporter, P83:erGFP) and air-exposed lateral root cap cells (marked by ProNCED2:erGFP). 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Annual review of plant biology 61: 49–64. 177 [...]... section of Arabidopsis root tip 6 Figure2 Lateral root developmental stages 10 Figure3 Diagrams of how to image air side or agar side of the root tip 46 Figure4 Image of root hairs on both sides of a root 67 Figure5 Picture of lateral root growing on agar media 68 Figure6 Roots growing on media containing different concentration of agar 69 Figure7 Lateral root initiation... through their roots Besides absorption of water and inorganic nutrients from soil, plant roots also play important roles in anchoring the plant body to the ground as well as storing food and nutrients Therefore, the development of the root system is important for plants‘ acclimation to environmental changes Root system architecture (RSA) refers to the three-dimensional structure of the root system, including... system, including the primary root, branch roots (lateral roots and adventitious roots) and root hairs (Osmont et al., 2007) Root system architecture development is composed of primary root (PR) and lateral root (LR) development and growth as well as the root hair growth The PR is derived from the embryo and its early developmental process is highly regulated by endogenous signals; environmental stimuli only... XIV YFP Yellow fluorescent protein RFP Red fluorescent protein PR Primary root LRC Lateral root cap LR Lateral root LRP Lateral root primordium RSA Root system architecture PBS Pre-branch site FC Founder cell FACS Fluorescence-activated cell sorting SEM Standard error of the mean XV Chapter 1 LITERATURE REVIEW 1 1.1 Root system architecture development Unlike most animals, plants are immobile They do... of Arabidopsis root tip The root has been color-coded to show different cell types In the meristem region, the quiescent center (QC) is in the center and surrounded by the different initial cells From outer to inner tissue, the radial root structure is composed of lateral root cap, epidermis, cortex, endodermis, and stele 6 1.1.2 Arabidopsis lateral root development The root system architecture is... regulating root growth and shaping the root system architecture Hormones also play important roles in regulating plants responses to the environmental changes Abscisic Acid (ABA), a well-known stress response hormone, can negatively regulate root growth Auxin, which has a major role in coordinating many plant growth and behavioral processes, can promote cell elongation, root growth and lateral root initiation... organogenesis of LRs starts from within the stele of the PR The stele is the innermost tissue of the root, containing the pericyle, xylem and phloem A pair of pericycle cells opposite to the xylem pole can undergo a cyclic auxin dependent pre-initiation event to become primed to become pericyle root founder cells (FCs) and gain stem cell identity to further proliferate The FCs go through several rounds of anticlinal... demonstrated to repress lateral root development ABA-deficient mutants aba2-1 and aba3-1 produce a larger root system than wild type plants(Deak and Malamy, 2005) Exogenously applied ABA in a plant's growth medium can also inhibit the development of lateral roots This process occurs specifically at the LR developmental stage between the emergence of LRP from the PR and the activation of the LR meristem The... (nhr) mutant of Arabidopsis showed both abnormal root cap morphogenesis and reduced sensitivity to ABA (Eapen et al., 2003) Another Arabidopsis phospholipase Dζ2 mutant pldζ2 showed significantly retarded or disturbed root hydrotropic response, and the inhibitory effect of ABA on gravitropism in wild-type roots, was absent in pldζ2 mutant roots In addition, both drought and the presence of exogenous... proposed that environmental regulation might act at later stages of development, such as during the initiation of anticlinal divisions within the FC (LR initiation) or the process of LR outgrowth from the PR LR initiation can be also influenced by tropic responses such as gravitropic curvature and mechanical stimuli such as transient bending of the PR manually (Ditengou et al., 2008) When a root is allowed . STUDY OF LOCAL ENVIRONMENTAL CONTROL OF ROOT SYSTEM ARCHITECTURE BAO YUN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES. primary root, branch roots (lateral roots and adventitious roots) and root hairs (Osmont et al., 2007). Root system architecture development is composed of primary root (PR) and lateral root (LR). development of the root system is important for plants‘ acclimation to environmental changes. Root system architecture (RSA) refers to the three-dimensional structure of the root system, including