SPATIOTEMPORAL CONTROL OF THE SALT STRESS INDUCED TRANSCRIPTIONAL RESPONSE IN ARABIDOPSIS

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), 1943-1945 Liu, J., Ishitani, M., Halfter, U., Kim, C S., & Zhu, J K (2000) The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance Proceedings of the National Academy of Sciences, 97(7), 3730-3734 Liu, Z B., Ulmasov, T., Shi, X., Hagen, G., & Guilfoyle, T J (1994) Soybean GH3 promoter contains multiple auxin-inducible elements The Plant Cell Online, 6(5), 645657 Lové J., Hoke, H A., Lin, C Y., Lau, A., Orlando, D A., Vakoc, C R., & Young, n, R A (2013) Selective inhibition of tumor oncogenes by disruption of superenhancers Cell, 153(2), 320-334 Lozano-Rodriguez, E., Hernandez, L E., Bonay, P., & Carpena-Ruiz, R O (1997) Distribution of cadmium in shoot and root tissues1 Journal of Experimental Botany, 48(1), 123-128 Luan, S., Lan, W., & Chul Lee, S (2009) Potassium nutrition, sodium toxicity, and calcium signaling: connections through the CBL–CIPK network Current opinion in plant biology, 12(3), 339-346 144 Lutts, S., Kinet, J M., & Bouharmont, J (1995) Changes in plant response to NaCl during development of rice (Oryza sativa L.) varieties differing in salinity resistance Journal of Experimental Botany, 46(12), 1843-1852 Ma, S., & Bohnert, H J (2007) Integration of Arabidopsis thaliana stress-related transcript profiles, promoter structures, and cell-specific expression Genome biology, 8(4), R49 Macler, B A., & MacElroy, R D (1989) Productivity and food value of Amaranthus cruentus under non-lethal salt stress Advances in Space Research, 9(8), 135-139 Martınez-Antonio, A., & Collado-Vides, J (2003) Identifying global regulators in transcriptional regulatory networks in bacteria Current opinion in microbiology, 6(5), 482-489 Maruyama, K., Todaka, D., Mizoi, J., Yoshida, T., Kidokoro, S., Matsukura, S., & Yamaguchi-Shinozaki, K (2012) Identification of cis-acting promoter elements in coldand dehydration-induced transcriptional pathways in Arabidopsis, rice, and soybean DNA research, 19(1), 37-49 Mauromicale, G., & Licandro, P (2002) Salinity and temperature effects on germination, emergence and seedling growth of globe artichoke Agronomie-Sciences des Productions Vegetales et de l'Environnement, 22(5), 443-450 Merika, M., & Thanos, D (2001) Enhanceosomes Current opinion in genetics & development, 11(2), 205-208 Michael, T P., Breton, G., Hazen, S P., Priest, H., Mockler, T C., Kay, S A., & Chory, J (2008) A morning-specific phytohormone gene expression program underlying rhythmic plant growth PLoS biology, 6(9), e225 145 Munns, R (2002) Comparative physiology of salt and water stress Plant, Cell & Environment, 25(2), 239-250 Munns, R., & Passioura, J B (1984) Hydraulic resistance of plants III Effects of NaCl in barley and lupin Functional Plant Biology, 11(5), 351-359 Narusaka, Y., Nakashima, K., Shinwari, Z K., Sakuma, Y., Furihata, T., Abe, H., & Yamaguchi‐ Shinozaki, K (2003) Interaction between two cis‐ acting elements, ABRE and DRE, in ABA‐ dependent expression of Arabidopsis rd29A gene in response to dehydration and high‐ salinity stresses The Plant Journal, 34(2), 137-148 Nemhauser, J L., Hong, F., & Chory, J (2006) Different plant hormones regulate similar processes through largely non-overlapping transcriptional responses Cell, 126(3), 467-475 Outlaw, Jr, W H (2003) Integration of cellular and physiological functions of guard cells Critical Reviews in Plant Sciences, 22(6), 503-529 Parida, A K., & Das, A B (2005) Salt tolerance and salinity effects on plants: a review Ecotoxicology and environmental safety, 60(3), 324-349 Passioura, J B., & Munns, R (2000) Rapid environmental changes that affect leaf water status induce transient surges or pauses in leaf expansion rate Functional Plant Biology, 27(10), 941-948 Pathan, M S., Subudhi, P K., Courtois, B., & Nguyen, H T (2004) Molecular dissection of abiotic stress tolerance in sorghum and rice Physiology and biotechnology integration for plant breeding eBook ISBN: 978-0-203-02203-0 Plett, D., Johnson, A., Jacobs, A., & Tester, M (2010) Cell type-specific expression of sodium transporters improves salinity tolerance of rice GM crops, 1(5), 273-275 146 Pruneda-Paz, J L., Breton, G., Para, A., & Kay, S A (2009) A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock Science, 323(5920), 1481-1485 Qasim, M., & Ashraf, M (2006) Time course of ion accumulation and its relationship with the salt tolerance of two genetically diverse lines of canola (Brassica napus L.) Pakistan Journal of Botany, 38(3), 663 Rademacher, W (2000) Growth retardants: effects on gibberellin biosynthesis and other metabolic pathways Annual review of plant biology, 51(1), 501-531 Reece-Hoyes, J S., Diallo, A., Lajoie, B., Kent, A., Shrestha, S., Kadreppa, S., & Walhout, A J (2011) Enhanced yeast one-hybrid assays for high-throughput genecentered regulatory network mapping Nature methods, 8(12), 1059-1064 Rhoades, J D., & Loveday, J (1990) Salinity in irrigated agriculture Agronomy, (30), 1089-1142 Riechmann, J L (2002) Transcriptional regulation: a genomic overview The Arabidopsis book, 1, e0085 Riechmann, J L., Heard, J., Martin, G., Reuber, L., Keddie, J., Adam, L & Yu, G L (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes Science, 290(5499), 2105-2110 Riechmann, L., & Winter, G (2006) Early protein evolution: building domains from ligand-binding polypeptide segments Journal of molecular biology, 363(2), 460-468 Roppolo, D., De Rybel, B., Tendon, V D., Pfister, A., Alassimone, J., Vermeer, J E., & Geldner, N (2011) A novel protein family mediates Casparian strip formation in the endodermis Nature, 473(7347), 380-383 147 Rubio, F., Gassmann, W., & Schroeder, J I (1995) Sodium-driven potassium uptake by the plant potassium transporter HKT1 and mutations conferring salt tolerance Science, 270(5242), 1660-1662 Rubio, F., Gassmann, W., & Schroeder, J I (1995) Sodium-driven potassium uptake by the plant potassium transporter HKT1 and mutations conferring salt tolerance Science, 270(5242), 1660-1662 Saeed, A I., Sharov, V., White, J., Li, J., Liang, W., Bhagabati, N., & Quackenbush, J (2003) TM4: a free, open-source system for microarray data management and analysis Biotechniques, 34 (2), 374 Sakakibara, H (2006) Cytokinins: activity, biosynthesis, and translocation Annu Rev Plant Biol., 57, 431-449 Sato, T., & Theologis, A (1989) Cloning the mRNA encoding 1-aminocyclopropane-1carboxylate synthase, the key enzyme for ethylene biosynthesis in plants Proceedings of the National Academy of Sciences, 86(17), 6621-6625 Savouré A., Hua, X J., Bertauche, N., Van Montagu, M., & Verbruggen, N (1997) , Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana Molecular and General Genetics MGG, 254(1), 104-109 Schoof, H., Lenhard, M., Haecker, A., Mayer, K F., Jü rgens, G., & Laux, T (2000) The Stem Cell Population of Arabidopsis Shoot Meristems Is Maintained by a Regulatory Loop between the CLAVATA and WUSCHEL Genes Cell, 100(6), 635-644 Schultz, E A., & Haughn, G W (1991) LEAFY, a homeotic gene that regulates inflorescence development in Arabidopsis The Plant Cell Online, 3(8), 771-781 148 Schü tzendü bel, A., & Polle, A (2002) Plant responses to abiotic stresses: heavy metal‐ induced oxidative stress and protection by mycorrhization Journal of experimental botany, 53 (372), 1351-1365 Schwechheimer, C (2008) Understanding gibberellic acid signaling—are we there yet? Current opinion in plant biology, 11(1), 9-15 Scott, M P (2000) Development: the natural history of genes Cell, 100(1), 27-40 Shabala, S., & Cuin, T A (2008) Potassium transport and plant salt tolerance.Physiologia Plantarum, 133(4), 651-669 Shabala, S., Cuin, T A., & Pottosin, I (2007) Polyamines prevent NaCl-induced K+ efflux from pea mesophyll by blocking non-selective cation channels FEBS letters, 581(10), 1993-1999 Shahbaz, M., & Ashraf, M (2008) Does exogenous application of 24-epibrassinolide ameliorate salt induced growth inhibition in wheat (Triticum aestivum L.)? Plant Growth Regulation, 55(1), 51-64 Shannon, P., Markiel, A., Ozier, O., Baliga, N S., Wang, J T., Ramage, D., & Ideker, T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks Genome research, 13(11), 2498-2504 Shen, Q., & Ho, T H (1995) Functional dissection of an abscisic acid (ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element The Plant Cell Online, 7(3), 295-307 Shi, H., Ishitani, M., Kim, C., & Zhu, J K (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na +/H+ antiporter Proceedings of the national academy of sciences, 97(12), 6896-6901 149 Shi, H., Kim, Y., Guo, Y., Stevenson, B., & Zhu, J K (2003) The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion The Plant Cell Online, 15(1), 19-32 Shi, H., Xiong, L., Stevenson, B., Lu, T., & Zhu, J K (2002) The Arabidopsis salt overly sensitive mutants uncover a critical role for vitamin B6 in plant salt tolerance The Plant Cell Online, 14(3), 575-588 Shiu, S H., Shih, M C., & Li, W H (2005) Transcription factor families have much higher expansion rates in plants than in animals Plant Physiology, 139(1), 18-26 Siriwardana, N S., & Lamb, R S (2012) The poetry of reproduction: the role of LEAFY in Arabidopsis thaliana flower formation Int J Dev Biol, 56, 207-221 Skirycz, A., Claeys, H., De Bodt, S., Oikawa, A., Shinoda, S., Andriankaja, M., & Inzé D (2011) Pause-and-stop: the effects of osmotic stress on cell proliferation during , early leaf development in Arabidopsis and a role for ethylene signaling in cell cycle arrest The Plant Cell Online, 23(5), 1876-1888 Smyth, G K (2005) Limma: linear models for microarray data In Bioinformatics and computational biology solutions using R and Bioconductor (pp 397-420) Springer New York Soltis, D E., Chanderbali, A S., Kim, S., Buzgo, M., & Soltis, P S (2007) The ABC model and its applicability to basal angiosperms Annals of Botany, 100(2), 155-163 Spychalla, J P., & Desborough, S L (1990) Superoxide dismutase, catalase, and αtocopherol content of stored potato tubers Plant Physiology, 94(3), 1214-1218 150 Stepanova, A N., Robertson-Hoyt, J., Yun, J., Benavente, L M., Xie, D Y., Doležal, K., & Alonso, J M (2008) TAA1-Mediated Auxin Biosynthesis Is Essential for Hormone Crosstalk and Plant Development Cell, 133(1), 177-191 Stewart, G R., & Lee, J A (1974) The role of proline accumulation in halophytes Planta, 120(3), 279-289 Storey, J D., & Tibshirani, R (2003) Statistical significance for genomewide studies Proceedings of the National Academy of Sciences, 100(16), 9440-9445 Storey, J D., Xiao, W., Leek, J T., Tompkins, R G., & Davis, R W (2005) Significance analysis of time course microarray experiments Proceedings of the National Academy of Sciences of the United States of America, 102(36), 12837-12842 Struhl, K (1999) Fundamentally different logic of gene regulation in eukaryotes and prokaryotes Cell, 98(1), 1-4 Sugimoto, M., & Sakamoto, W (1997) Putative phospholipid hydroperoxide glutathione peroxidase gene from Arabidopsis thaliana induced by oxidative stress Genes & genetic systems, 72(5), 311-316 Sun, K., & Hauser, B (2001) Salt stress induces anatomical changes in ovules and embryos, ultimately resulting in seed abortion In The 12th International Meeting on Arabidopsis Research June (pp 23-27) Sun, T P (2008) Gibberellin metabolism, perception and signaling pathways in Arabidopsis The Arabidopsis Book, 6, e0103 Tran, L S P., Urao, T., Qin, F., Maruyama, K., Kakimoto, T., Shinozaki, K., & Yamaguchi-Shinozaki, K (2007) Functional analysis of AHK1/ATHK1 and cytokinin 151 receptor histidine kinases in response to abscisic acid, drought, and salt stress in Arabidopsis Proceedings of the National Academy of Sciences, 104(51), 20623-20628 Trejo, C L., & Davies, W J (1991) Drought-induced closure of Phaseolus vulgaris L stomata precedes leaf water deficit and any increase in xylem ABA concentration Journal of Experimental Botany, 42(12), 1507-1516 Ueguchi-Tanaka, M., Ashikari, M., Nakajima, M., Itoh, H., Katoh, E., Kobayashi, M., & Matsuoka, M (2005) GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin Nature, 437(7059), 693-698 Ulmasov, T., Murfett, J., Hagen, G., & Guilfoyle, T J (1997) Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements The Plant Cell Online, 9(11), 1963-1971 van der Graaff, E., Laux, T., & Rensing, S A (2009) The WUS homeobox-containing (WOX) protein family Genome Biol, 10 (12), 248 Vyroubalová, Š., Václavíková, K., Turečková, V., Novák, O., Šmehilová, M., Hluska, T., & Galuszka, P (2009) Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels Plant Physiology, 151(1), 433-447 Wang, Y., Li, L., Ye, T., Zhao, S., Liu, Z., Feng, Y Q., & Wu, Y (2011) Cytokinin antagonizes ABA suppression to seed germination of Arabidopsis by downregulating ABI5 expression The Plant Journal, 68(2), 249-261 Wang, Y., Liu, C., Li, K., Sun, F., Hu, H., Li, X., & Liu, M (2007) Arabidopsis EIN2 modulates stress response through abscisic acid response pathway Plant molecular biology, 64(6), 633-644 152 Wang, Z Y (2012) Brassinosteroids modulate plant immunity at multiple levels Proceedings of the National Academy of Sciences, 109(1), 7-8 Weigel, D., & Meyerowitz, E M (1994) The ABCs of floral homeotic genes Cell, 78(2), 203-209 Wen, C K., & Chang, C (2002) Arabidopsis RGL1 encodes a negative regulator of gibberellin responses The Plant Cell Online, 14(1), 87-100 Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H., & Schmü lling, T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity The Plant Cell Online, 15(11), 2532-2550 White, P J (1999) The molecular mechanism of sodium influx to root cells Trends in plant science, 4(7), 245-246 Whyte, W A., Orlando, D A., Hnisz, D., Abraham, B J., Lin, C Y., Kagey, M H., & Young, R A (2013) Master transcription factors and mediator establish superenhancers at key cell identity genes Cell, 153(2), 307-319 Willekens, H., Chamnongpol, S., Davey, M., Schraudner, M., Langebartels, C., Van Montagu, M., & Van Camp, W (1997) Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants The EMBO journal, 16(16), 4806-4816 Wu, S J., Ding, L., & Zhu, J K (1996) SOS1, a genetic locus essential for salt tolerance and potassium acquisition The Plant Cell Online, 8(4), 617-627 Xiong, L., Schumaker, K S., & Zhu, J K (2002) Cell signaling during cold, drought, and salt stress The Plant Cell Online, 14(suppl 1), S165-S183 153 Xu, W., Jia, L., Shi, W., Liang, J., Zhou, F., Li, Q., & Zhang, J (2013) Abscisic acid accumulation modulates auxin transport in the root tip to enhance proton secretion for maintaining root growth under moderate water stress New Phytologist, 197(1), 139-150 Yamaguchi-Shinozaki, K., & Shinozaki, K (2005) Organization of cis-acting regulatory elements in osmotic-and cold-stress-responsive promoters Trends in plant science, 10(2), 88-94 Yokoi, S., Quintero, F J., Cubero, B., Ruiz, M T., Bressan, R A., Hasegawa, P M., & Pardo, J M (2002) Differential expression and function of Arabidopsis thaliana NHX Na+/H+ antiporters in the salt stress response The Plant Journal, 30(5), 529-539 Zeevaart, J A D., & Creelman, R A (1988) Metabolism and physiology of abscisic acid Annual review of plant physiology and plant molecular biology, 39(1), 439-473 Zhang, H., Han, W., De Smet, I., Talboys, P., Loya, R., Hassan, A., & Wang, M H (2010) ABA promotes quiescence of the quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary root meristem The Plant Journal, 64(5), 764774 Zhao, X C., & Schaller, G E (2004) Effect of salt and osmotic stress upon expression of the ethylene receptor ETR1 in Arabidopsis thaliana FEBS letters, 562(1), 189-192 Zhong, R., Lee, C., & Ye, Z H (2010) Evolutionary conservation of the transcriptional network regulating secondary cell wall biosynthesis Trends in plant science, 15(11), 625632 Zhu, J K (2000) Genetic analysis of plant salt tolerance using Arabidopsis Plant Physiology, 124(3), 941-948 Zhu, J K (2001) Plant salt tolerance Trends in plant science, 6(2), 66-71 154 Zhu, J K., Liu, J., & Xiong, L (1998) Genetic analysis of salt tolerance in Arabidopsis: evidence for a critical role of potassium nutrition The Plant Cell Online, 10(7), 11811191 Zinzen, R P., Girardot, C., Gagneur, J., Braun, M., & Furlong, E E (2009) Combinatorial binding predicts spatio-temporal cis-regulatory activity Nature, 462(7269), 65-70 Zou, C., Sun, K., Mackaluso, J D., Seddon, A E., Jin, R., Thomashow, M F., & 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