CRYPTOCHROME 1 (CRY1) IS CRITICAL IN MEDIATING DEVELOPMENTAL PROCESS IN RESPONSE TO LIGHT INTENSITY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Tsai-Chi Li, M.S. ***** The Ohio State University 2009 Dissertation Committee: Professor James D. Metzger, Advisor Professor Terrence L. Graham Approved by Professor David J. Barker Professor David Gardner Advisor Horticulture and Crop Science Graduate Program Copyright 2009 Tsai-Chi Li All Rights Reserved ii ABSTRACT Plants can perceive light signals through three major classes of photoreceptors: phytochromes (red/far-red light), cryptochromes (blue and UV-A light), and phototropins (blue and UV-A light). Genome-wide gene expression analysis revealed that CRYPTOCHROME1 (CRY1) is critical in the response of Arabidopsis to high irradiance. Database analysis also showed that expression of CRY1 was highly correlated to the expression of a number of anti-oxidant genes. Therefore, I hypothesized that CRY1 is involved in cellular communication responsible for the integration of irradiance- dependent responses in tobacco. To understand the role of CRY1 in seed germination and early seedling development in response to light intensity, experiments were conducted using transgenic plants overexpressing CRY1. Germination kinetics were determined by measuring the time of radicle emergence in seeds subjected to light intensities of 6, 60, or 600 µmol m-2 s-1. In wild type plants, the final germination percentage was unaffected by different light intensities, but high light intensity caused a significant delay in germination. Plants overexprssing CRY1 showed reduced germination percentage and a delay in the timing of germination under both low and high light intensities. Surprisingly, the lower germination percentages in the CRY1 overexpressors was reversed by high light intensities in contrast to seeds of wild-type. iii Under high light conditions, leaves of CRY1 overexpressing plants contained three-fold more total chlorophyll than wild-type plants, which was probably due to both increased synthesis and a reduction of photooxidative degradation. First, well-developed chloroplasts containing chlorophyll were observed in the cortex of roots of CRY1 overexpressing plants. Second, leaves of CRY1 overexpressing plants contained higher phenolics, flavonoids, and lignin content than wild-type plants. LC-MS results showed that leaves of CRY1 overexpressing plants contained two-fold higher level of chlorogenic acids and ten-fold higher level of rutin than wild-type plants. CRY1 overexpressing plants exhibited reduced ultraviolet-B injury. It may be due to the accumulation of secondary metabolites which have antioxidant activities in protecting cellular structures from oxidative damage. CRY1 overexpression resulted in increased stomatal density. Wild-type plants followed a pattern known as the one cell spacing rule: two stomates are separated by at least one epidermal cell for proper stomatal physiology. In plants overpressing CRY1 there was elevated stomatal density and some of the stomates were clustered in violation of the one cell spacing rule, indicating that stomatal patterning was disrupted in CRY1 overexpression plants. I also observed a severe wilting phenotype in CRY1 overexpression plants when transferred to cold or high light conditions. This may be a result of a reduction in water transport (smaller vessel cells) and the increased transpiration. iv Dedicated to my parents, sister, and brother. v ACKNOWLEDGMENTS I wish to thank my advisor, Dr. James D. Metzger for everything. With his guidance, encouragement, and great efforts, he helped to make research fun for me. I would also like to thank the lab members Adri McKelvey and Dan Lima. With their friendship, I felt so happy to come to the lab everyday. Thanks to Dr. Lian-Mei Graham. With her caring and emotional support, I could pursue this degree. To my committee, Dr. David J. Barker, Dr. David Gardner, and Dr. Terrence L. Graham, I appreciate their help for invaluable suggestions and comments. Thanks to Dr. Terrence L. Graham, Michelle Sinden, and Jiye Cheng in the kind assistance with running HPLC and the LC-MS/MS. Finally, I am most grateful to my parents, sister, brother, and good friends for their love and support. They provided encouragement, advice, and good company to help me get through the difficult times. Without them, I would have given up the degree three years ago. vi VITA 1976……………... Born – Taipei, Taiwan 2000……………… M.S. Biological Science, National Sun Yat-Sen University, Taiwan 2001 …….……….. Research Associate, National Taiwan University, Taiwan 2001-2005………... Graduate Research Associate, Department of Horticulture & Crop Science, The Ohio State University RESEARCH PUBLICATION He, J.X., Fujioka, S., Li, T.C., Kang, S.G., Seto, H., Takatsuto, S., Yoshida, S., and Jang, J.C. (2003). Sterols regulate development and gene expression in Arabidopsis. Plant Physiol 131, 1258-1269. Price, J., Li, T.C., Kang, S.G., Na, J.K., and Jang, J.C. (2003). Mechanisms of glucose signaling during germination of Arabidopsis. Plant Physiol 132, 1424-1438. FIELD OF STUDY Major Field: Horticulture and Crop Science vii TABLE OF CONTENTS Page ABSTRACT.…………………………………………………………………………..... iii DEDICATION………………………………………………….…………...………...…. v ACKNOWLEDGEMENTS……………………………………..………………………. vi VITA…………………………………………………………….…………………….....vii LIST OF TABLES…………………….……………………….…………………….........x LIST OF FIGURES……………………………………………..…………..……………xi CHAPTERS 1. LITERATURE RIVEW………………………………………………….………. 1 1.1 The role of photoreceptor in light perception…...………………………...…. 1 1.2 Phychromes……………………………………………………………………2 1.3 Cryptochromes………………………………………………………………...3 1.4 Phototropins…………………………………………………………………...4 1.5 The photoreceptors in the model plant Arabidopsis thaliana…………………5 1.6 Research objectives………………………………………..…………………10 2. THE ROLE OF CRY1 IN MEDIATING SEED GERMINATION AND EARLY SEEDLING DEVELOPMENT IN RESPONSE TO LIGHT INTENSITY……..12 1.1 Introduction…………………………………………………………………..12 1.2 Materials and methods……………………………………………………….15 1.3 Results………………………………………………………………………..19 1.4 Discussion……………………………………………………………………31 3. CRY1 IS CRITICAL FOR SENSING CHANGES IN IRRADIANCE AND MAY BE INVOLVED IN PHOTOPROTECTION IN TOBACCO………………...…32 3.1 Introduction…………………………………………………………………..32 3.2 Materials and methods……………………………………………………….35 viii 3.3 Results………………………………………………………………………..42 3.4 Discussion……………………………………………………………………68 4. CRY1 MAY BE INVOLVED IN REGULATING SIZE AND SHAPE OF VESSEL ELEMENTS…...………………………………………………………70 4.1 Introduction…………………………………………………………………..70 4.2 Materials and methods……………………………………………………….73 4.3 Results………………………………………………………………………..76 4.4 Discussion……………………………………………………………………90 LIST OF REFERENCES………………………………………………………………...91 APPENDIX GLZ1, A MEMBER OF FAMILY 8 GLYCOSYLTRANSFERASE, MAY PLAY A ROLE IN PROGRAMMED CELL DEATH……..…………………………………….,,97 ix LIST OF TABLES Table Page 2.1. Time of germination of Ns, S168, Nt, S135, and S144 under different light intensities………………………………………………………………………………...27 2.2. The marginal effects of control variables on germination time….………………….28 3.1. Negative ions with corresponding retention time and suggested compound ……….57 3.2. Negative ions with corresponding retention time and suggested compound in root..65 4.1. Stomatal index of wild-type and CRY1 overexpressing plants grown under short and long day conditions………………………………………………………………………83 4.2. Mean length and width of vessels in Ns and S168.………………………...……….85 4.3. The results of ANOVA in mean length of Ns, mean width of Ns, mean length of S168, and mean width of S168…………………………………………………………..86 4.4. The results of paired-t test in mean length of Ns, mean width of Ns, mean length of S168, and mean width of S168.………………………………………………………….87 x LIST OF FIGURES Figure Page 1.1. Domain organization of phytochromes, cryptochromes, and phototropins…………..4 1.2. The data on light responsive expression were compiled by Genevestigator………....7 1.3. The data on stimulus responsive expression were compiled by Genevestigator…..…8 1.4. Co-expressed genes of CRY1 in Arabidopsis thaliana…………...…………………..9 2.1. Molecular characterization of N. sylvestris and N. tabacum cv. Maryland Mammoth transgenic lines. …………………………..………………………………………...…...22 2.2. CRY1 affects seed size …………………………………………………………...…23 2.3. CRY1 affects seed weight……………………………………………………………24 2.4. CRY1 overpression results in a reduced germination .……………………………...25 2.5. The lower germination percentages in the CRY1 overexpressors were reversed by high light intensities …………………………………...………………………………...26 2.6. CRY1 affects the early seedling development …………..…………………………..29 2.7. CRY1 overpression results in an inhibition of early seedling development. ………..30 3.1. CRY1 overexpression reduces photodamage in plants. ……………………………..49 3.2. CRY1 overexpression results in increased chlorophyll content …………………….50 3.3. CRY1 overexpression results in root greening ……………….……………………..51 3.4. Chloroplasts are located in cortex of the maturation zone ………………………….52 3.5. CRY1 overexpression results in increased chlorophyll content in root. …………….53 xi 3.6. CRY1 overexpression results in increased secondary metabolite content. …………54 3.7. HPLC analysis shows increased secondary metabolite content in CRY1 overexpressing plants ……………………………………………………………………55 3.8. Total ion chromatogram of phenolic compounds in wild-type and CRY1 overexpressing plants