SPATIOTEMPORAL CONTROL OF THE SALT STRESS INDUCED TRANSCRIPTIONAL RESPONSE IN ARABIDOPSIS RUI WU NATIONAL UNIVERSITY OF SINGAPORE 2013 SPATIOTEMPORAL CONTROL OF THE SALT STRESS INDUCED TRANSCRIPTIONAL RESPONSE IN ARABIDOPSIS RUI WU A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Rui Wu August 20th, 2013 ACKNOWLEGEMENT The first big “thank you” I want to send to my supervisor, Dr Jose R Dinneny Thanks for providing the good research environment and challenging ideas during my PhD study; positive attitude and kind encouragement when I encountered depression and frustration from the projects and life; as well as his kind and open support for my decisions on both work and life, just like a friend I became more independent as a scientific thinker and problem solver, and closer to a real researcher Thanks to the companionship of my kind and supportive lab mates, I did not feel alone abroad doing this challenging thing Whenever I needed help, they gave me hands without hesitation Thanks to Lina for her generous help and guidance when I was first in the lab and her companion for the entire years Thanks to Jeffrey, Chonghan, Xie Fei, Pooja, Geng Yu, Shahram, MC, Neil, Bao Yun, Ruben and Jose for the valuable discussions and advice for my projects Thank Penny, Han-qi and Cliff for their work assisting my study Thanks to all intern students and undergraduate students doing the final year projects for the work they have done I would like to thank the Department of Biological Sciences in NUS for providing the precious opportunity for me to pursue my PhD degree; it is really a great university that gives access to advanced research, excellent people and valuable resources And I would also like to thank Temasek Life Sciences Laboratory and Carnegie Institution of Plant Sciences for providing the facilities and platform for me to my study and communicate with excellent people Thank all my friends in Singapore and the US for making my life abroad like at home Thanks to our collaborators, Dr Jose Pruneda-Paz and Dr Steve Kay, for their efforts on transcription factor screening And thanks to Dr Hao Yu lab (Temasek Lifesciences I Laboratory) for providing the vectors for GUS reporter and seeds of RGA::GFP:GRA Thanks to Dr Joseph Horecka in Prof Ronald Davis’ lab (School of Medicine Department of Biochemistry, Stanford University) for his generous advice and reagents for yeast transformation and colony PCR At last, I want to give the biggest “Thank you” to my family Thank you for giving me continuous love and support This will be the most precious gift in my life July 27, 2013 Rui Wu II TABLE OF CONTENTS ACKNOWLEGEMENT I TABLE OF CONTENTS III SUMMARY VII LIST OF TABLES IX LIST OF FIGURES X CHAPTER LITERATURE REVIEW 1.1 HIGH SALINITY STRESS IN PLANTS 1.1.1 High salinity affects different developmental events of plants 1.1.2 Evolutionary variations of plant adaption to high salinity stress 1.1.2.1 Halophytes 1.1.2.2 Glycophytes 1.1.3 Secondary physiological responses involved in high salinity stress 1.1.3.1 Hyper-osmotic stress 1.1.3.2 Dehydration (drought stress) 1.1.3.3 Ion disequilibrium 1.1.3.4 Oxidative stress 1.1.4 Hormone involvement in salt response 10 1.1.4.1 Abscisic acid (ABA) 10 1.1.4.2 Ethylene 12 1.1.4.3 Gibberellic acid (GA3) 14 1.1.4.4 Brassinosteroids 15 III 1.1.4.5 Cytokinin 16 1.1.4.6 Auxin 17 1.1.5 Studies of high salinity stress in Arabidopsis 18 1.1.5.1 Arabidopsis is a model plant in salt stress studies 18 1.1.5.2 Root—a multicellular organ directly responsive to salt stress 20 1.2 TRANSCRIPTIONAL REGULATION AND TRANSCRIPTIONAL NETWORK 24 1.2.1 Transcriptional regulation is an indispensable process involved in developmental process and environmental stimuli response 24 1.2.2 Mechanisms of transcriptional regulation 26 1.2.3 Approaches to generate a transcriptional network 28 1.3 OBJECTIVES AND SIGNIFICANCE OF THIS STUDY 30 CHAPTER MATERIALS AND METHODS 32 2.1 PLANT MATERIALS 33 2.2 PLANT GROWTH CONDITIONS AND STRESS TREATMENT 33 2.3 GENERATION OF TRANSGENIC LINES 34 2.3.1 Sequences design of synthetic promoters 34 2.3.2 Constructs 36 2.3.3 Agrobacterium mediated plant transformation 38 2.4 YEAST ONE HYBRID SCREEN 38 2.4.1 Constructs generation 39 2.4.2 Yeast transformation 39 2.4.3 Yeast one hybrid screening 40 2.5 BIOINFORMATICS DATA ANALYSIS 41 2.6 LIVE IMAGING 44 2.7 CONFOCAL MICROSCOPIC ANALYSIS 44 IV 2.8 GUS STAINING 45 2.9 LUC ANALYSIS 45 2.10 GENE EXPRESSION 46 2.11 GENETIC ANALYSIS 51 CHAPTER RESULTS AND DISCUSSIONS I 52 3.1 ABSTRACT 53 3.2 INTRODUCTION 54 3.3 RESULTS 56 3.3.1 A global spatiotemporal transcriptional map of the salt stress response in Arabidopsis root 56 3.3.2 Different strategies were used to adapt salt stress at early and late stages 65 3.3.3 A cluster-comparison method identifies targets mediating hormone signaling in salt stress response 67 3.3.4 Spatiotemporal understanding of hormone biosynthesis and signaling pathway ABA as an example 69 3.3.5 ABA signaling mediated transcriptional response to salt stress showed tissue specificities 74 3.3.6 Dynamic involvement of GA signaling during salt stress response 77 3.4 DISCUSSIONS 79 CHAPTER RESULTS AND DISCUSSIONS II 82 4.1 ABSTRACT 83 4.2 INTRODUCTION 84 4.3 RESULTS 86 V 4.3.1 Schematic description of the pipeline for setting up the transcriptional network 86 4.3.2 Identification of the salt responsive cis-regulatory elements based on the spatiotemporal transcriptional map of Arabidopsis roots 90 4.3.3 Synthetic promoters harboring CREs confer the ability to drive specific expression patterns under normal or stress conditions 94 4.3.4 Synthetic promoters containing CREs confer the ability to respond to salt stress in a dynamic manner 109 4.3.5 Synthetic promoter strategy for screening using the TF library 117 4.4 DISCUSSION 128 4.4.1 Synthetic promoters drive tissue-specific and salt responsive patterns 128 4.4.2 ABRE’s expression pattern indicates the location of ABA signaling in root development and environmental response 129 4.4.3 Combinatorial properties of regulatory elements necessary for environmental stress response 130 CHAPTER CONCLUSIONS 131 REFERENCES 133 APPENDIX 156 CURRICULUM VITAE 157 VI SUMMARY For all living things, the ability to respond to environmental stress is an essential property Various environmental stimuli can be processed by organisms, resulting in different kinds of responses, such as morphological and physiological changes as well as actual behaviors With these responses, organisms can acclimate effectively for survival Different from animals that can escape from a poor environment, the only strategy plants can use is to acclimate However, an organism is just like a black box, because how the input environmental signals are processed is not clear, but what is known is that intricate signal transduction and transcription networks must be involved My study is focused on how different signaling pathways are integrated spatiotemporally under high salinity stress and how transcriptional regulation occurs Firstly, I did an analysis on a previously generated spatiotemporal transcriptional map of salt stress in Arabidopsis roots, covering core cell types and time points for salt treatment Compared with the previous study showing tissue-specific responses at hour to high salinity, this map provided higher temporal resolution, giving a more dynamic view of how different cell types respond to salt stress at different time periods of salt treatment Based on this spatiotemporal map, the transcriptional changes of key components in hormone biosynthesis and signaling were identified, suggesting that these hormones function in specific cell types and at particular stages during acclimation to high salinity A bioinformatics method was also developed to systematically de-convolve the hormone crosstalk network with salt stress, identifying some salt stress response submodules controlled by hormone signaling A good portion of these modules were validated using high throughput q-RT PCR The dynamic transcriptional regulation and homeostasis mediated by hormone signaling is well correlated to the dynamic root growth illustrated by my colleague VII lineages during Drosophila embryogenesis Proceedings of the National Academy of Sciences, 91(12), 5662-5666 Lin, C C., & Kao, C H (1995) NaCl stress in rice seedlings: starch mobilization and the influence of gibberellic acid on seedling growth Botanical Bulletin of Academia Sinica, 36, 169-173 Liu, J., & Zhu, J K (1998) A calcium sensor homolog required for plant salt tolerance Science, 280(5371), 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& Shiu, S H (2011) Cis-regulatory code of stress-responsive transcription in Arabidopsis thaliana Proceedings of the National Academy of Sciences, 108(36), 14992-14997 155 Appendix Appendix Figure Summary of the patterns of tissue-specific cis-regulatory elements Bar charts show the expression levels of the two genes that I used to generate the synthetic promoters, and it was generated based on the transcriptional root map (Brady et al., 2007) 156 Curriculum Vitae Personal information Name: Wu, Rui Nationality: Chinese Gender: Female Address: 141 Del Medio Ave, Mountain view, CA 94040 Email: wur587@gmail.com or ruiwu@stanford.edu Education and Research experience Education     Nov., 2011- present: Pre-doctoral Fellow, Carnegie Institution of Plant Science, Stanford University, the USA; PhD candidate, department of biological sciences, National University of Singapore, Singapore Sep., 2009- Oct., 2011: Junior Research Fellow, Temasek Lifesciences Laboratory, Singapore; PhD candidate, department of biological sciences, National University of Singapore, Singapore Sep., 2006- Jul., 2009: Master of sciences (Major – Genetics), Key Laboratory of Molecular Epigenetics of MOE, Northeast Normal University, China Sep., 2002- Jul., 2006: Bachelor of Sciences (Major – Biological Sciences), GPA (3.81), Northeast Normal University, China Research experience   Sep., 2009- present Dr Jose Dinneny lab Temasek Lifesciences Laboratory & Dept of Plant Biology, Carnegie Institution for Science My current work focuses on the functional study of the mechanisms involved in the spatiotemporal regulation of gene expression in salt stress response Specifically, (1) I did a bioinformatics analysis of the spatiotemporal microarray data set of Arabidopsis roots under salt treatment, and revealed how different clusters of genes and biological processes were dynamically regulated in different cell types of roots (Geng and Wu et al., 2013); (2) I systematically showed how different hormone signals are integrated in the spatiotemporal transcriptional regulation and what targets they use to mediate the salt stress response I showed that ABA biosynthesis and signaling functions early in salt stress response in endodermis using high throughput q-RT PCR technology, as well as the crosstalk between different hormones (Geng and Wu et al., 2013); (3) I am setting up a synthetic pipeline allowing to efficiently study the biological functions of cis-regulatory elements (CREs) leading to tissue-specific and dynamic salt responsive patterns This work will help to provide the essential foundations for promoter engineering on practical purpose (Wu et al in preparation); (4) Based on the synthetic promoter strategy, I have obtained transcription factors from yeast one hybrid screening, and set up the CREs centered transcriptional network in spatiotemporal salt response (Wu et al., in preparation) Sep., 2005-Sep., 2009 Dr Bao Liu lab Key Laboratory of Molecular Epigenetics of MOE, Northeast Normal University I studied the genetic and epigenetic consequences on the plant genomes induced by different kinds of stresses, such as alien-DNA integration, hybridization, grafting, and tissue culture (Details in the following publications) 157 Publications         Rui Wu, Shahram Emami, Jose Pruneda-Paz et al Functional exploration of the mechanism for transcriptional regulation in salt stress response (Manuscript in preparation) Yu Geng*, Rui Wu*, Choon Wei Wee* et al A spatio-temporal understanding of growth regulation during the salt stress response in Arabidopsis The Plant Cell, 2013, 25: 1-23 Xiaoran Wang*, Rui Wu*, Xiuyun Lin et al Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids BMC Plant Biology, 2013, 13:77 Rui Wu*, Xiaoran Wang*, Yan Lin et al Inter-species grafting caused extensive and heritable alterations of DNA methylation in Solanaceae plants PLoS ONE, 2013, 8(4): e61995 Ying Wu*, Rui Wu*, Bangjiao Zhang et al Epigenetic instability in genetically stable micropropagated plants of Gardenia jasminoides Ellis Plant Growth Regul, 2012, 66: 137-143 Rui Wu, Wanli Guo, Xiaoran Wang et al Unintended consequence of plant transformation: transgene integration has caused transpositional activation of an endogenous retrotransposon Tos17 in rice ssp Japonica cv Matsumae Plant Cell Rep, 2009, 28:1043–1051 Mu Li*, Wanli Guo*, Lanjuan Hu, Xiaoming Liu, Yufei Zhang, Rui Wu et al Genetic variation in natural populations of Hordeum brevisubulatum native to the Songnen Prairie in northeastern China: comparison of four nuclear DNA markers CAN J PLANT SCI, 2007, 87: 773–780 Wanli Guo*, Rui Wu*, Yufei Zhang et al Tissue culture - induced locus specific alteration in DNA methylation and its correlation with genetic variation in Codonopsis lanceolata Benth et Hook F Plant Cell Rep, 2007, 26:1297–1307 *Authors contributed equally Presentations     Rui Wu et al Functional exploration of CRE centered transcriptional network in Arabidopsis roots ASPB Plant Biology 2013 Poster presentation Rui Wu et al Generation of CRE centered transcriptional network in the Arabidopsis root WS-ASPB Annual Meeting 2013 Poster presentation Rui Wu et al A newly developed spatiotemporal map enables a systematic study of hormone crosstalk during the salt stress response in Arabidopsis roots Cold Spring Harbor 77th Symposium: Plants 2012 Poster presentation Rui Wu and Bao Liu Unintended Genetic and Epigenetic Consequences of Plant Transformation: Transgene Integration in Rice Has Caused Mobilization of an Endogenous Retrotransposon Tos17 Proceedings of International Symposium on Chloroplast Genomics and Genetic Engineering Dec., 2007 Oral presentation 158 .. .SPATIOTEMPORAL CONTROL OF THE SALT STRESS INDUCED TRANSCRIPTIONAL RESPONSE IN ARABIDOPSIS RUI WU A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL... of cis-regulatory elements which are good starting points for the generation of a CRE centered transcriptional network involved in the salt stress response in the Arabidopsis roots The pipeline... for the change of GAs According to previous studies, it was known that GA participates in the stress response, including salt stress On the one hand, the biosynthesis of GA can be affected by salt