FUNCTIONAL GENOMICS IN RICE (ORYZA SATIVA L.) USING AC/DS TRANSPOSON TAGGING SYSTEM AND GENE EXPRESSION PROFILING RENGASAMY RAMAMOORTHY THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2008 DEDICATED TO MY FAMILY AND FRIENDS ii ACKNOWLEDGEMENTS I came to Singapore in August 2000 to work in the Rice Functional genomics laboratory at Institute of Molecular Agrobiology (IMA) as an assistant research officer (ARO). Later I started my PhD project in August 2002 at Temasek Life Sciences Laboratory (TLL) and I am very glad today that I could finish my project in appropriate time with the satisfactory outcome of my thesis. This was achieved with collaborative efforts by many people and I would like to acknowledge my sincere thanks to them. I would like to express my profound gratitude to my supervisor Dr. Srinivansan Ramachandran (Ji) for showing his confidence in me, excellent guidance and the constant supports. Ji you are a person with immense intelligence and knowledge and surely I benefited a lot from it. I enjoyed lots of freedom you gave me to design and carry out the research work which made me a very confident and self reliant researcher. It gives me great pleasure to thank and acknowledge my thesis committee members Prof. Prakash Kumar (Dept. of Biological Sciences, National University of Singapore), Dr. Hong Yan and Dr.Yin Zhong Chao (Temasek Life Sciences Laboratory) for their helpful advice and suggestions. I would like to express my sincere thanks to Prof. Venkatesan Sundaresan (Department: Biological Sciences, University of California Davis) who gave me the opportunity to work in his lab at IMA. I take this as an opportunity to thank my very best friend Dr. C. Santhosh Kumar (Group Leader, DuPont, India) who taught me the basics of plant molecular biology and rice transformation techniques, and introduced me to the IMA. I always remember his helping nature and special care to me since we met in India, iii 1995 and I thank his wife Dr. Rajani for being a good friend and her concerns on my research progress. I am deeply indebted to Dr. Jiang ShuYe, without whom this study would have been very difficult, and his stimulating discussions, advices, generous help and the fruitful collaborations which gave us a few co-authored publications. Also I acknowledge and thank Drs. Ildiko Szeverenyi and Tatiana Kolesnik who were Research fellows in our lab in early days of my PhD study with whom I worked together with in several projects of which two were published as co-authors. I am very grateful to all my colleagues who showed lots of care and friendship particularly I would like to express my gratitude and sincerely thank Dr. V. Ramesh Anbazhagan for his critical reading of my thesis, discussions and being a very good friend of mine. I enjoyed working with other colleagues and appreciate their count less help and technical supports of Mr. Nadimuthu Kumar, Prasanna Nori Venkatesh, Ma Zhigang, Lukman Hakim Md., Ms. Kum Yoke Yeong and Lim Geok Boey. I had memorable time in Rice Functional Genomic Lab in both IMA and TLL, thanks to all my former colleagues in particularly thankful to Drs. Doris Bachmann, Leina Mary Joseph, Ritu Bhalla, Mande Kumaran, Mrs. Minne Cai, Hongfen Luan, Mr. Ling Gau Wang, Ms. Peisan Luo and Zhaung Li, Mr. Mayalagu Sevugan, Thanumalayan, Bharathi and Ms. Rajini Sreenivasan. I would like to extent my note of thanks to all the attachment students and summer trainees for their helps in this project especially thankful to Ms. Vinupriya Ganapathy and Mrs. Vanitha Jeevanantham iv My special thanks to my best friends Drs. Mithilesh Mishra, Sivakumar Neelamegam, Srinivas Ramasamy, Sriram Parasuram, and Srinivasan Ramanujam who catalyzed me for venturing into PhD program and encouraged me during my course of studies and beyond. I sincerely thank all my friends Mr. Anup and Kasthurirengan, for the critical reading of thesis. Drs. Kumar, Madhumalar and Bui Thi Ngoc Ha for their attempts to help me in the protein expression, homology modeling and GC analysis, respectively, I would like to extent my thanks to Mr. Vijay Bhaskar, Ravi, Mrs. Angs, Sumathi, and Ms He fang, for their many timely helps during this project. I am sincerely thankful to all the administration staff, store personnel, DNA sequencing laboratory, SEM facilities, Computer section, Media preparation, Electrical, Security and Cleaning service. I am grateful to TLL and Temasek holdings Singapore for their financial support. It may be appropriate to remember and thank here the people who were at SPIC Science Foundation (SSF), India where I started research as my carrier. In particular, I would like the express my gratitude to Drs. K.K.Narayanan, SP.Palaniappan, and George Thomas, who were my group leaders during my five years stay there. Also I would like to extent my sincere thanks to Drs. T.S.Lokeswari, Wheeta Hopper, C.B.Nirmala and Valli Akela who were other group leaders and my well wishers. In the same time, I would like to thank all my SSF friends especially Drs. Loganathan and Saravanakumar. Most importantly, I would also like to thank my family for the support they provided me all my life and in particular, I must acknowledge and thank my wife Mrs. v Angayarkanni and my son Eswarsriram for making my life wonderful and exciting with their love and understanding. Last but not least I would like to thank God for giving me the opportunity, strength and courage to fulfill my dream. vi TABLE OF CONTENTS Title page і Acknowledgements ііi Table of contents νіi Summary xііі List of publications xviii List of figures xіx List of tables xxіi List of abbreviations xxіv CHAPTER 1: Introduction 1.1 Importance of Rice, a model plant for monocots 1.2 Rice Production and Challenges associated with it 1.2.1 Abiotic stresses 1.2.2 Biotic stresses 1.2.3 Challenges in rice production 1.2.4 Strategies to alleviate pressure on rice production 1.3 Genomics 1.4 Bioinformatics 1.5 Plant genome sequencings and annotations 1.6 Transcriptomics 1.7 Proteomics 1.8 Metabolomics 10 vii 1.9 Phenomics 11 1.10 Forward and reverse genetics 12 1.11 Types of mutagenesis to study the gene functions 13 1.12 The insertional mutagenesis: T-DNA vs Transposons 15 1.13 Transposon tagging 18 1.14 Ac/Ds transposon system for functional genomics in rice (Oryza sativa L) 19 1.15 Aim of the project 20 CHAPTER 2: Materials and methods 2.1 2.2 Plant materials 21 2.1.1 Stresses and phytohormone treatments 21 Sexual hybridizations and generation of transposants 22 2.2.1 22 Selection of Ds transposants by GFP and Basta Screens 2.3 Visible phenotype screens 23 2.4 Rescue analysis of semi-dwarf mutant with phytohormones 23 2.5 Cryo-scanning electron microscopy and cell size determinations 24 2.6 Genomic DNA extractions 24 2.7 RNA extractions and cDNA synthesis 26 2.8 Database searches and domain detection of predicted members of GRAM, RIP and WRKY domain gene families 26 2.9 Primer designing, PCR, TAIL-PCR, RT-PCR and Quantitative RT-PCR 27 2.10 Thermal asymmetric interlaced PCR (TAIL-PCR) and FSTs 36 2.11 Reciprocal PCR 36 2.12 Sequencing 36 viii 2.13 Analysis of transposition events in individual panicles 37 2.14 Molecular cloning of OsCYP96B4 gene and its promoter 40 2.15 Rice transformation using Agrobacterium tumefaciens mediated method 40 2.16 Southern blot analysis 44 2.17 Northern blot analysis 45 2.18 Protein purification and Western blot analysis 46 2.19 Histochemical staining for studying Glucuronidase expression 46 2.20 Statistical Analysis 47 2.21 Hetrologous expression of OsCYP96B4 in Schizosaccharomyces pombe 47 2.22 Lipid extraction, gas chromatography and mass spectroscopy analysis 48 CHAPTER 3: A comprehensive transcriptional profiling of three different gene families in rice 3.1 GRAM-Domain Gene Family 3.1.1 Background 49 3.1.2 Genome-wide identification of genes encoding GRAM domaincontaining proteins in rice 51 Expression profile of GRAM-domain genes in different tissues of rice under normal growth conditions 51 3.1.3 3.1.4 3.2 Expression profile of GRAM domain genes upon ABA treatment and under various biotic and abiotic stresses 57 RIP-domain gene family 3.2.1 Background 3.2.2 Genome wide identification of RIP gene family members in rice 3.2.3 Expression of OsRIP genes in different rice tissues at normal growth conditions 66 68 71 ix 3.2.4 3.3 75 WRKY Gene Family of transcription factors 3.3.1 Background 80 3.3.2 Genome wide identification of WRKY gene family members in rice 82 Expression profile of WRKY genes in different tissues at normal growth conditions by RT-PCR 84 Expression profile of WRKY genes under various abiotic and phytohormone treatments by RT-PCR analyses 86 WRKY genes regulated by abiotic stresses, phytohormones and the combinations of abiotic stress and phytohormones 90 3.3.3 3.3.4 3.3.5 3.4 Expression profile of OsRIP genes upon ABA treatment and undervarious biotic and abiotic stresses Discussion 3.4.1 Identification and highly divergent expression patterns of GRAM domain gene family 98 3.4.2 The RIP family members were ancient but not ubiquitous 99 3.4.3 Tissue-specific and stress-induced expression patterns coincide with the,developmental stages sensitive to various environmental factors 100 3.4.4 OsRIP genes may be potentially useful for developing new plant varieties with higher tolerance to various stresses 101 3.4.5 Annotation of WRKY genes in rice genome 101 3.4.6 Possible roles of WRKY genes under normal growth conditions 102 3.4.7 WRKY genes expression in response to abiotic stresses 103 3.4.8 WRKY gene signaling pathways mediated by various hormones 103 CHAPTER 4: Generation of Ds transposon lines and Ac/Ds transposition behavior in rice (Oryza sativa L) genome 4.1 Background 106 x Kurata, N., Miyoshi, K., Nonomura, K., Yamazaki, Y. and Ito, Y. (2005). Rice mutants and genes related to organ development, morphogenesis and physiological traits. Plant Cell Physiol., 46: 48-62 Kuromori, T., Wada, T., Kamiya, A., Yuguchi, M. and Yokouchi, T. et al. A trial of phenome analysis using 4000 Ds-insertional mutants in gene-coding regions of Arabidopsis. Plant J., (2006) 47: 640-651. Lafitte, H.R., Ismail, A. and Bennett, J. (2004). Abiotic stress tolerance in rice for Asia: progress and the future. Proceedings of the 4th International Crop Science Congress, Brisbane, Australia. Lambais, M.R. (2001). In silico differential display of defense-related expressed sequence tags from sugarcane tissues infected with diazotrophic endophytes. Genet. Mol. Biol., 24: 103-111. Li, X.D., Liu, W.Y. and Niu, C.I. (1996) Purification of a new ribosome-inactivating protein from the seeds of Cinnamumum porrectum and characterization of the RNA N-glycosidase activity of the toxic protein. Biol. Chem., 377: 825-831. Lin, J.Y., Liu, K., Chen, C.C. and Tung, T.C. (1971). Effect of crystalline ricin on the biosynthesis of protein, RNA, and DNA in experimental tumor cells. Cancer Res. 31: 921-924. Lippok, B., Birkenbihl, R.P., Rivory, G., Brummer, J., Schmelzer, E., Logemann, E. and Somssich, I.E. (2007). Expression of AtWRKY33 encoding a pathogen-or PAMPresponsive WRKY transcription factor is regulated by a composite DNA motif containing W box elements. Mol. Plant Microbe Interact., 20: 420-429. 199 Liu X., Bai, X., Wang, X. and Chu, C. (2007). OsWRKY71, a rice transcription factor, is involved in rice defense response. J. Plant Physiol., 164: 969-979. Liu, J.H., Luo, M., Cheng, K.J., Mohapatra, S.S. and Hill, R.D. (1999). Identification and characterization of a novel barley gene that is ABA-inducible and expressed specifically in embryo and aleurone. J. Exp. Bot., 50: 727-728. Liu, X.Q., Bai, X.Q., Qian, Q., Wang, X.J., Chen, M.S. and Chu, C.C. (2005). OsWRKY03, a rice transcriptional activator that functions in defense signaling pathway upstream of OsNPR1. Cell Res., 15: 593-603. Liu, Y.G. and Whittier, R.F. (1995). Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics., 25: 674-681. Liu, Y.G., Mitsukawa, N., Ooosumi, T. and Whittier, R.F. (1995). Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J., 8: 457-463 Lorrain, S., Lin, B., Auriac, M.C., Kroj, T., Saindrenan, P., Nicole, M., Balague, C. and Roby, D. (2004). VASCULAR ASSOCIATED DEATH1, a novel gram domaincontaining protein, is a regulator of cell death and defense responses in vascular tissues. Plant Cell, 16: 2217-2232. Lutts, S., Kine,t J.M. and Bouharnont, J. (1995). Changes in plant response to NaCl during development of rice (Oryza sativa L.) varieties differing in salinity resistance. J.Exp. Bot., 46: 1843-1852. 200 Mantri, N.L., Ford, R., Coram, T.E. and Pang, E.C. (2007). Transcriptional profiling of chickpea genes differentially regulated in response to high-salinity, cold and drought. BMC Genomics, 8: 303. Marchive, C., Mzid, R., Deluc, L., Barrieu, F. and Pirrello, J. (2007). Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants. J. Exp. Bot., 58: 1999-2010. Mare, C., Mazzucotelli, E., Crosatti, C., Francia, E., Stanca, A.M. and Cattivelli, L. (2004). Hv-WRKY38: a new transcription factor involved in cold-and droughtresponse in barley. Plant Mol. Biol., 55: 399-416. Margis-Pinheiro, M., Zhou, X.R., Zhu, Q.H., Dennis, E.S., and Upadhyaya, N. M. (2005) Isolation and characterization of a Ds-tagged rice (Oryza sativa L.) GAresponsive dwarf mutant defective in an early step of the gibberellin biosynthesis pathway. Plant Cell Rep., 23: 819-833 Mauch-Mani, B. and Mauch, F. (2005). The role of abscisic acid in plant-pathogen interactions. Curr. Opin. Plant Biol., 8: 409-414. Mendez, E. and Girbes, T. (2005). Molecular characterization and systemic induction of single-chain ribosome-inactivating proteins (RIPs) in sugar beet (Beta vulgaris) leaves. J Exp Bot., 56: 1675-1684. Mishra, M., Karagiannis, J., Sevugan, M., Singh, P., and Balasubramanian, M.K. (2005). The 14-3-3 protein rad24p modulates function of the cdc14p family phosphatase clp1p/flp1p in fission yeast. Curr. Biol., 15: 1376-1383. 201 Montanaro, L., Sperti, S. and Stripe, F. (1973). Inhibition by ricin of protein synthesis in vitro. Ribosomes as the target of the toxin. Biochem. J., 136: 677-683. Mori, M., Nomura, T., Ooka, H., IshizakaM, Yokota, T., Sugimoto, K., Okabe, K., Kajiwara, H., Satoh, K. and Yamamoto, K.(2002). Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid biosynthesis. Plant Physiol., 130: 1152-1161. Muller, M., Dues, G., Balconi, C., Salamini, F. and Thompson, R.D. (1997). Nitrogen and hormonal responsiveness of the 22 kDa alpha-zein and b-32 genes in maize endosperm is displayed in the absence of the transcriptional regulator Opaque-2. Plant J., 12: 281-291. Murashige, T.and Skoog, F. (1962) A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiol. Plant., 15: 473-497 Nakagawa, Y., Machida, C., Machida, Y., and Toriyama, K. (2000). Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants. Plant Cell Physiol. 41, 733-42. Nelson, D.R., Schuler, M.A., Paquette, S.M., Werck-Reichhart, D. and Bak, S. (2004). Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot. Plant Physiol., 135: 756-772. Nguyen, N. V. and Ferrero, A. (2006). Meeting the challenges of global rice production. Paddy Water Environ 4: 1-9. Nielsen, K., Boston, R.S. (2001). Ribosome-inactivating proteins: a plant perspective. Annu Rev Plant Physiol Plant Mol Biol. (2001); 52: 785-816. 202 Oard, J.H., Linscombe, S. D., Braverman, M.P., Jodari, F., Blouin, D.C., Leech, M., Kohli, A., Vain, P., Cooley, J.C. and Christou, P. (1996) Development, field evaluation, and agronomic performance of transgenic herbicide resistant rice. Mol. Breed. 2: 359-368. Obrig, T.G., Irvin, J.D. and Hardesty, B. (1973). The effect of an antiviral peptide on the ribosomal reactions of the peptide elongation enzymes, EF-I and EF-II. Arch. Biochem. Biophys., 155: 278-289. Oh, S.J., Song, S.I., Kim, Y.S., Jang, H.J., Kim, S.Y., Kim, M., Kim, Y.K., Nahm, B.H. and Kim, J.K. (2005). Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth. Plant Physiol., 38: 341-351. Oh, S.K., Yi, S.Y., Yu, S.H., Moon, J.S., Park, J.M. and Choi, D. (2006). CaWRKY2, a chili pepper transcription factor, is rapidly induced by incompatible plant pathogens. Mol. Cells, 22: 58-64. Parinov, S., Sevugan, M., Ye, D., Yang, W.C., Kumaran, M. and Sundaresan, V. (1999). Analysis of flanking sequences from Dissociation insertion lines: a database for reverse genetics in Arabidopsis. Plant Cell, 11: 2263-2270. Park, S.H., Pinson, S.R. and Smith, R.H. (1996). T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices. Plant Mol. Biol., 32: 1135-1148. Park, S.W., Vepachedu, R., Sharma, N. and Vivanco, J.M. (2004). Ribosome-inactivating proteins in plant biology. Planta., 219: 1093-1096. 203 Pawlowski, W.P., Torbert, K.A., Rines, H.W. and Somers, D.A. (1998) Irregular patterns of transgene silencing in allohexaploid oat. Plant Mol. Biol. 38: 597-607. Peumans, W.J., Hao, Q. and Van, Damme, E.J.M., (2001). Ribosome-inactivatingand proteins from plants: more than RNA Nglycosidases? FASEB J., 15: 1493-1506. Phinney, B.O. (1984). Gibberellin A1, dwarfism, and the control of shoot elongation in higher plants. In: Crozier A, Hillman FR(eds) The biosynthesis and metabolism of plant hormones.Cambridge University Press, Cambridge, pp 17-41. Pnueli, L., Hallak-Herr, E., Rozenberg, M., Cohen, M., Goloubinoff, P., Kaplan, A. and Mittler, R. (2002). Molecular and biochemical mechanisms associated with dormancy and drought tolerance in the desert legume Retama raetam. Plant J., 31: 319-330. Pröls, F. and Meyer, P. (1992) The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida. Plant J. 2: 465-475 Qiu, D., Xiao, J., Ding, X., Xiong, M., Cai, M., Cao, Y., Li, X., Xu, C. and Wang, S. (2007). OsWRKY13 mediates rice disease resistance by regulating defenserelated genes in salicylate-and jasmonate-dependent signaling. Mol. Plant Microbe Interact., 20: 492-499. Qiu, Y., Jing, S., Fu, J., Li, L. and Yu, D. (2004). Cloning and analysis of expression profile of 13 WRKY genes in rice. Chin. Sci. Bull., 49: 2159-2168. Raina, S., Mahalingam, R., Chen, F., and Federoff, N. (2002). A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana. Plant Mol. Biol., 50: 93-110. 204 Ramachandran, S. and Sundaresan, V. (2001). Transposons as tools for functional genomics. Plant Physiol Biochem., 39: 243-252. Ramachandran, S., Hans Christensen, E. M., Ishimaru,Y. Dong, C.H., Ming, W. C.,Ann Cleary., A. and Chua N. H. (2000). Profilin plays a role in cell elongation, cell shape maintenance and flowering in Arabidopsis thaliana. Plant Physiology 124: 1637-1647. Rathore, K.S., Chowdhury, V.K. and Hodges, T.K. (1993) Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts. Plant Mol Biol., 21: 871-84 Redei, G.P.(1970). Bibliogr. Genet., 20/2: 1-151. Register, J. C. III, Peterson, D.J., Bell, P.J., Bullock, W.P., Evans, I.J., Frame, B., Greenland, A.J., Higgs, N.S., Jepson, I. and Jiao, S. (1994). Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment. Plant Mol. Biol. 25: 951-961. Reinbothe, S., Reinbothe, C., Lehmann, J., Becker, W., Apel, K. and Parthier, B. (1994). JIP60, a methyl jasmonate-induced ribosome-inactivating protein involved in plant stress reactions. Proc. Natl. Acad. Sci. USA, 91: 7012-7016. Riechmann, J.L., Heard, J., Martin, G., Reuber, L. and Jiang, C.Z.(2000). Arabidopsis transcription factors: genome-wide comparative analysis among Eukaryotes. Science, 290: 2105-2110. Rippmann, J.F., Michalowski, C.B., Nelson, D.E. and Bohnert, H.J. (1997). Induction of a ribosome-inactivating protein upon environmental stress. Plant Mol. Biol., 35: 701-709. 205 Rizhsky, L., Liang, H. and Mittler, R. (2002). The combined effect of drought stress and heat shock on gene expression in tobacco. Plant Physiol., 130: 1143-1151. Rommens, C.M.T., Munyikwa, T.R.I., Overduin, B., Nijkamp, H.J.J., and Hille, J. (1993). Transposition pattern of a modified Ds element in tomato. Plant. Mol. Biol., 21, 1109-1119. Ross, C.A., Liu, Y. and Shen, Q.J. (2007). The WRKY gene family in rice (Oryza sativa). J. Integrative Plant Biol., 49: 827-842. Rowland, O., Ludwig, A.A., Merrick, C.J., Baillieul, F., Tracy, F.E., Durrant, W.E., FritzLaylin, L., Nekrasov, V., Sjolander, K. and Yoshioka, H. (2005). Functional Analysis of Avr9/Cf-9 Rapidly elicited genes identifies a protein kinase, acik1, that is essential for full Cf-9-dependent disease resistance in tomato. Plant Cell, 17: 295-310. Rupasinghe, S.G., Duan, H. and Schuler, M.A. (2007). Molecular definitions of fatty acid hydroxylases in Arabidopsis thaliana. Proteins, 68, 279-293. Rushton, P.J., Torres, J.T., Parniske, M., Wernert, P., Hahlbrock, K. and Somssich, I.E. (1996). Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J., 15: 56905700. Ryu, H.S., Han, M., Lee, S.K., Cho, J.I. and Ryoo, N. (2006). A comprehensive expression analysis of the WRKY gene superfamily in rice plants during defense response. Plant Cell Rep., 25: 836-847. Saitoh, S., Takahashi, K., Nabeshima, K., Yamashita, Y., Nakaseko, Y., Hirata, A. and Yanagida M. (1996) Aberrant Mitosis in Fission Yeast Mutants Defective in Fatty 206 Acid Synthetase and Acetyl CoA Carboxylase The Journal of Cell Biology, 134; 949-961. Sakamoto T, Morinaka Y, Ishiyama K, Kobayashi M, Itoh H, Kayano T, Iwahori S, Matsuoka M, Tanaka H (2003) Genetic manipulation of gibberellin metabolism in transgenic rice. Nat. Biotechnol., 21: 909-913. Sakamoto, T., Miura, K., Itoh, H., Tatsumi, T., Ueguchi-Tanaka, M., Ishiyama, K., Kobayashi, M., Agrawal, G.K., Takeda, S. and Abe, K. (2004). An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiol., 134: 1642-1653 Salanoubat, M., Lemcke, K., Rieger, M., Ansorge, W., Unseld, M., Fartmann, B., Valle, G., Blocker, H., Perez-Alonso, M. and Obermaier, B. (2000). Sequence and analysis of chromosome of the plant Arabidopsis thaliana. Nature, 408: 820822. Schaefer, S.C., Gasic, K., Cammue, B., Broekaert, W., van Damme, E.J., Peumans, W.J.and Korban, S.S. (2005). Enhanced resistance to early blight in transgenic tomato lines expressing heterologous plant defense genes. Planta, 222: 858-866. Schena, M., Shalon, D., Davis, R.W., and Brown, P.O. (1995). Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467-470. Schuler, M.A. and Werck-Reichhart, D. (2003). Functional genomics of P450s. Annu. Rev. Plant Biol., 54: 629-667. 207 Scofield, S.R., Harrison, K., Nurrish, S.J. and Jones J.D.G. (1992). Promoter fusions to the Activator transposase gene cause distinct patterns of Dissociation excision in tobacco cotyledons. Plant Cell 4: 573-582. Sharma, N., Park, S.W., Vepachedu, R., Barbieri, L., Ciani, M., Stirpe, F., Savary, B.J.and Vivanco, J.M. (2004). Isolation and characterization of an RIP (ribosomeinactivating protein)-like protein from tobacco with dual enzymatic activity. Plant Physio., 134: 171-181. Shimamoto, K., Miyazaki, C., Hashimoto, H., Izawa, T., Itoh, K., Terada, R., Inagaki, Y. and Iida, S. (1993). Trans-activation and stable integration of the maize transposable element Ds cotransfected with the Ac transposase gene in transgenic rice plants. Mol. Gen. Genet., 239: 354-60. Shimono, M., Sugano, S., Nakayama, A., Jiang, C.J., Ono, K., Toki, S. and Takatsuji, H. (2007). Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance. Plant Cell, 19: 2064-2076. Shinozaki, K., Yamaguchi-Schinozaki, K. and Seki, M. (2003). Regulatory network of gene expression in the drought and cold stress responses. Curr. Opin. Plant Biol., 6: 410-417. Smith, D., Yanai, Y., Liu, Y.G., Ishiguro, S., Okada, K., Shibata, D., Whittier, R.F. and Fedoroff, N.V. (1996). Characterization and mapping of Ds-GUS-T-DNA lines for targeted insertional mutagenesis. Plant J., 10: 721-732. Song, S.K., Choi, Y., Moon, Y.H., Kim, S.G., Choi, Y.D. and Lee, J.S. (2000). Systemic induction of a Phytolacca insularis antiviral protein gene by mechanical wounding, jasmonic acid, and abscisic acid. Plant Mol. Biol., 43: 439-450. 208 Spielmeyer, W., Ellis, M.H. and Chandler, P.M. (2002). Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene. Proc. Natl. Acad. Sci. USA 99: 9043-9048. Sponsel, V.M., Schmidt, F.M., Porter, S.G., Nakayama, M., Kohlstruk, S. and Estelle, M. (1997). Characterization of new gibberellin-responsive semidwarf mutants of Arabidopsis. Plant Physiol., 115: 1009-1020. Stirpe, F., Barbieri, L., Gorini, P., Valbonesi, P., Bolognesi, A. and Polito, L. (1996). Activities associated with the presence of ribosome-inactivating proteins increase in senescent and stressed leaves. FEBS Lett, 382: 309-312. Sun, T.P., Goodman, H.M. and Ausubel, F.M. (1992). Cloning the Arabidopsis GA1 locus by genomic subtraction. Plant Cell. 4: 119-128. Sundaresan, V., Springer, P., Volpe, T., Haward, S., Jones, J.D., Dean, C., Ma, H. and Martienssen, R. (1995). Patterns of gene action in plant development revealed by enhancer trap transposable element. Genes Dev., 9: 1797-1810. Sundaresan, V., Springer, P., Volpe, T., Haward, S., Jones, J.D., Dean, C., Ma, H., Szabados, L., Kovacs, I., Oberschall, A., Abraham, E., Kerekes, I., Zsigmond, L., Nagy, R., Alvarado, M., Krasovskaja, I., Gal, M., Berente, A., Redei, G., Haim, A., and Koncz, C. (2002). Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome. Plant Journal, 32: 233-242. Szekeres, M., Ne´meth, K., Koncz-Ka´lma´n, Z., Mathur, J., Kauschmann, A., Altmann, T., Re´dei, G.P., Nagy, F., Schell, J. and Koncz, C. (1996), Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell, 85: 171-182. 209 Szeverenyi, I., Ramamoorthy, R., Teo, Z.W., Ma, Z.G. and Ramachandran, S. (2006). Large scale systematic study on stability of Ds element and timing of transposition in rice. Plant Cell Physiol. 47(1): 84-95. The Arabidopsis genome initiative. (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 408: 796-815. Tosti, N., Pasqualini, S., Borgogni, A., Ederli, L., Falistocco, E., Crispi, S. and Paolocci, F. (2006). Gene expression profiles of O3-treated Arabidopsis plants. Plant Cell Environ., 29: 1686-1702. Ueguchi-Tanaka, M., Fujisawa, Y., Kobayashi, M., Ashikari, M., Iwasaki, Y., Kitano, H., and Matsuoka, M. (2000). Rice dwarf mutant d1, which is defective in the alpha subunit of the heterotrimeric G protein, affects gibberellin signal transduction. Proc. Natl. Acad. Sci. USA, 97, 11638-11643. Ulker, B. and Somssich, I.E. (2004). WRKY transcription factors: from DNA binding towards biological function. Curr. Opinion Plant Biol., 7: 491-498. Upadhyaya, N.M., Zhou, X., Zhu, Q.-H., Ramm, K., Wu, L., Eamens, A., Sivakumar, R., Kato, T., Yun, D.-W., Santhoshkumar, Ch., Narayanan, K.K. and Peacock, J.W., Dennis. (2002). An iAc/Ds gene and enchancer trapping system for insertional mutagenesis in rice. Funct. Plant Biol., 29: 547-559. Velculescu, V. E.; Zhang, L.; Vogelstein, B. and Kinzler, K.W. (1995). Serial analysis of geneexpression. Science, 270: 484-487. Vivanco, J.M., Savary, B.J. and Flores, H.E. (1999). Characterization of two novel type I ribosome-inactivating proteins from the storage roots of the Andean crop Mirabilis expansa. Plant Physiol., 119: 1447-1456. 210 Walbot, V. (1992). Strategies for mutagenesis and gene cloning using transposon tagging and T-DNA insertional mutagenesis. Annu. Rev. Plant Phys. Plant Mol. Biol., 43: 49-82. Walsh, T.A., Morgan, A.E. and Hey, T.D. (1991). Characterization and molecular cloning of a proenzyme form of a ribosome-inactivating protein from maize: novel mechanism of proenzyme activation by proteolytic removal of a 2.8kilodalton internal peptide segment. J. Biol. Chem., 266: 23422-23427. Wang, S.H., Chen, F. and Zhou, K.D. (2000). In vitro pollen germination of rice. Acta. Agronomica Sinica, 26: 609-612 (in Chinese). Wang, Y., Xue, Y. and Li, J. (2005). Towards molecular breeding and improvement of rice in China. Trends Plant Sci., 10: 610-614. Wang, Z.P., Yang, P.Z., Fan, B.F. and Chen, Z.X. (1998). An oligo selection procedure for identification of sequence-specific DNA-binding activities associated with the plant defence response. Plant J., 16: 515-522. Weigel, D., Ahn, J.H., Blazquez, J., Borevitz, J.O., Christensen, S.K., Frankhauser, C., Ferrandiz, C., Kardailsky, I., Neff, M.M. and Nguyen, J.T., et al. (2000). Activation tagging in Arabidopsis. Plant Physiol., 122: 1003-1013. Wellesen, K., Durst, F., Pinot, F., Benveniste, I., Nettesheim, K., Wisman, E., SteinerLange, S., Saedler, H. and Yephremov, A. (2001). Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid omega-hydroxylation in development. Proc. Natl. Acad. Sci. USA 98: 9694-9699. Wilkins, M. R., Williams, K. L., Appel, R.D. and Hochstrasser, D.F. (1997). Proteome Research: New Frontiers in Functional Genomics. Springer-Verlag. 211 Wilson, K., Long, D., Swinburne, J. and Coupland, G. (1996). A Dissociation insertion causes a semidominant mutation that increases expression of TINY, an Arabidopsis gene related to APETALA2. Plant Cell, 8: 659-671. Wishart, D. S., Tzur, D., Knox, C., Eisner, R., Guo, A. C., Young, N., et al. (2007). HMDB: the human metabolome database. Nucleic Acids Research, 35(Database issue), D521-D526. Wong, R.N.S., Mak, N.K., Choi, W.T. and Law, P.T.W. (1995). Increased accumulation of trichosanthin in Trichosanthes kirilowii induced by microorganisms. J. Exp. Bot., 46: 355-358. Wu, K.L., Guo, Z.J., Wang, H.H. and Li, J. (2005). The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Res., 12: 9-26. Xie, Z., Zhang, Z.L., Hanzlik, S., Cook, E. and Shen, Q.J. (2007). Salicylic acid inhibits gibberellin-induced alpha-amylase expression and seed germination via a pathway involving an abscisic-acid-inducible WRKY gene. Plant Mol. Biol., 64: 293-303. Xie, Z., Zhang, Z.L., Zou, X., Huang, J., Ruas, P., Thompson, D. and Shen, Q.J. (2005). Annotations and Functional Analyses of the Rice WRKY gene Superfamily Reveal Positive and Negative Regulators of Abscisic Acid Signaling in Aleurone Cells. Plant Physiol., 137: 176-189. Xie, Z., Zhang, Z.L., Zou, X., Yang, G., Komatsu, S. and Shen, Q.J. (2006). Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleurone cells. Plant J., 46: 231-242. 212 Xiong, Y., Liu, T., Tian, C., Sun, S., Li, J. and Chen, M. (2005). Transcription factors in rice: a genome-wide comparative analysis between monocots and eudicots. Plant Mol. Biol., 59: 191-203. Xu, J., Wang, H. and Fan, J. (2007). Expression of a ribosome-inactivating protein gene in bitter melon is induced by Sphaerotheca fuliginea and abiotic stimuli. Biotechnol Lett, 29: 1605-1610. Xu, X., Chen, C., Fan, B. and Chen, Z. (2006). Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors. Plant Cell, 18: 1310-1326. Xu, Y.L., Li, L., Wu, K.Q., Peeters, A.J.M., Gage, D.A. and Zeevaart, J.A.D. (1995). The GA5 locus of Arabidopsis thaliana encodes a multifuctional gibberellin 20oxidase-molecular cloning and functional expression. Proc. Natl. Acad. Sci. USA, 92: 6640-6644. Yamaguchi S. (2008). Gibberellin Metabolism and its Regulation. Annu. Rev. Plant Biol., 59: 225-251. Yoda, H., Ogawa, M., Yamaguchi, Y., Koizumi, N., Kusano, T. and Sano, H. (2002). Identification of early-responsive genes associated with the hypersensitive response to tobacco mosaic virus and characterization of a WRKY-type transcription factor in tobacco plants. Mol. Genet. Genomics, 267: 154-161. Yu, J., Hu, S., Wang, J., Wong, G.K., Li, S., Liu, B., Deng, Y., Dai, L., Zhou, Y. and Zhang, X. (2002). A draft sequence of the rice genome (Oryza sativa L. ssp. Indica). Science, 296: 79-92. 213 Yu, J., Wang, J., Lin, W., Li, S., Li, H., Zhou, J., Ni, P., Dong, W., Hu, S. and Zeng, C. (2005). The genomes of Oryza sativa: a history of duplications. PLoS Biol. (e38): 0266-0281. Yuan, H., Ming, X., Wang, L., Hu, P., An, C. and Chen, Z. (2002). Expression of a gene encoding trichosanthin in transgenic rice plants enhances resistance to fungus blast disease. Plant Cell Rep., 20: 992-998. Zhang, S., Warkentin, D., Sun,B., Zhong, H. and Sticklen, M. (1996). Variation in the inheritance of expression among subclones for unselected (uidA) and selected (bar) transgenes in maize (Zea mays L.). Theor. Appl. Genet., 92: 752-761. Zhang, Y. and Wang, L. (2005). The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evol. Biol., 5: 1. Zhang, Y., Zhu, Y., Peng, Y., Yan, D., Li, Q., Wang, J., Wang, L. and He, Z. (2008). Gibberellin homeostasis and plant height control by EUI and a role for gibberellin in root gravity responses in rice. Cell Research, 18: 412-421. Zhang, Z.L., Xie, Z., Zou, X., Casaretto, J., Ho, T.H. and Shen, Q.J. (2004). A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells. Plant Physiol., 134: 1500-1513. Zheng, Z., Mosher, S.L., Fan, B., Klessig, D.F. and Chen, Z. (2007). Functional analysis of Arabidopsis WRKY25 transcription factor in plant defense against Pseudomonas syringae. BMC Plant Biol., 7: 2. Zou, X., Seemann, J.R., Neuman, D. and Shen, Q.J. (2004). A WRKY gene from creosote bush encodes an activator of the abscisic acid signaling pathway. J. Biol. Chem., 279: 55770-55779. 214 [...]...4.2 4.3 Generation of Ac/ Ds parental lines Large-scale generation of unlinked transposants 109 113 4.4 Ds Starter lines maintained their activity through several generations 116 4.5 Stable BAR gene expression in F3, F4 and F5 generations of Ds transposon lines 119 Ds element can be remobilized in the F5 generation of stable transposants 121 4.7 Developmental timing of Ds transposition 125... Sundaresan and S Ramachandran Establishing an efficient Ac/ Ds tagging system in rice: large-scale analysis of Ds flanking sequences The Plant Journal (2004) 37, 301-314 xviii LIST OF FIGURES Figure 2.1 Constructs used in this study 41 Figure 2.2 Construct used for dsRNAi analyses 42 Figure 3.1A Structural organizations of three different GRAM domain-containing proteins in rice 53 Figure 3.1B Similar expression. .. of gene expressions and regulations and thus to narrow down specific genes that can combat multiple stresses 1.3 Genomics Genomics is a discipline focusing on the study of poly deoxyribonucleic acid (DNA) molecules within a single cell of an organism Genomics includes intensive 4 efforts to determine the entire set of DNA sequences and fine genetic mapping of genomes, as well as the analysis of the information... both factors, suggesting an interaction between abiotic stress and hormone signaling Based on our expression analysis, we suggest that each member from GRAM domain, RIP and WRKY gene families may play a specialized roles in a specific tissue or stress condition and may function as a regulator of environmental and hormonal signaling A two-element Ac/ Ds gene trap system was successfully established in rice. .. climatic factors, which damages many crop species and every year about two thirds of the world’s land is facing the low temperatures including freezing Cold reduces the cultivated land area as well as the growing seasons of crops including rice Cold increases the solute concentration and decreases the availability of liquid water for plant growth Low temperature affects rice in both seedling and heading... transposons silencing during the propagation of parental lines at least up to T5 generation Moreover, the stably transposed Ds element was active even at F5 generation, since Ac could remobilize Ds element as indicated by the footprint analysis of several revertants The BAR gene expression was monitored from F3 to F6 generations in more than thousand lines Strikingly, substantial transgene silencing was not... Venkatesh, Minne Cai and Srinivasan Ramachandran Genomewide survey of the RIP domain family in Oryza sativa and their expression profiles under various abiotic and biotic stresses Plant Mol Biol 67: 603-614 (2008) 2 Rengasamy Ramamoorthy, Shu-Ye Jiang, Nadimuthu Kumar, Prasanna Nori Venkatesh and Srinivasan Ramachandran A Comprehensive Transcriptional Profiling of the WRKY Gene Family in Rice under... mutagenesis techniques were used for functional genomics in rice We examined the expression profile of rice genes by transcriptomic approach to identify novel genes which were responsive to multiple stresses like abiotic and/ or biotic stresses In another approach, the maize Ac/ Ds transposable element system was utilized to generate a large pool of Ds insertion lines This thesis consists of different... observed in all the generations tested We analyzed the timing of transposition during rice development and provide evidence that Ds transposes xv late after tiller formation The secondary transposition events were ruled out by analyzing possible footprints with reciprocal PCRs Our study validates the Ac/ Ds system as a tool for large-scale mutagenesis in rice We propose that harvesting rice seeds from individual... suggesting a defect in cell elongation Flanking sequence tag analysis showed the Ds insertion into OsCYP96B4 gene This gene was devoid of intron and encoded a 60.5 kDa protein Segregation analysis revealed that the phenotype co-segregated with the Ds and revertants confirmed that the dwarf phenotype was due to the insertion of Ds element into OsCYP96B4 gene The exogenous treatment of brassinosteroid and . FUNCTIONAL GENOMICS IN RICE (ORYZA SATIVA L.) USING AC/ DS TRANSPOSON TAGGING SYSTEM AND GENE EXPRESSION PROFILING RENGASAMY RAMAMOORTHY . gene signaling pathways mediated by various hormones 103 CHAPTER 4: Generation of Ds transposon lines and Ac/ Ds transposition behavior in rice (Oryza sativa L) genome 4.1 Background 106. approaches, it is imperative to identify and functionally characterize rice genes through functional genomics. In the present investigation transcriptomics and transposon insertional mutagenesis