Accumulation and Toxicity of Lanthanum and Neodymium in Horticultural Plants By Arefeh Rezaee A Thesis Presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Applied Science In Engineering Guelph, Ontario, Canada © Arefeh Rezaee, August, 2016 ABSTRACT Accumulation and Toxicity of Lanthanum and Neodymium in Horticultural Plants Arefeh Rezaee Advisors: University of Guelph, 2016 Dr. Emily YW Chiang Dr. Beverley Hale In this study, the dose response of Brassica chinensis L. and Helianthus annuus L. to light rare earth elements (REEs), lanthanum and neodymium, were investigated in vitro. The phytotoxicity of the studied REEs was determined by measuring the aboveground and belowground biomass, and the chlorophyll concentration of the plants. The half maximal inhibitory concentration (IC50) in plant biomass and total chlorophyll concentration were estimated. The available ion species of La and Nd for plant uptake were determined through geochemical modeling. The concentration of these elements in the plants was measured to assay the ability of these plants to hyperaccumulate. Low dosages of La and Nd showed hormetic effects on the shoot and root masses of Helianthus annuus L., while both elements at high concentrations were toxic to the species tested. Tissue content analysis result indicated that the plants were not effective hyperaccumulators. ACKNOWLEDGMENT First, I would like to express my appreciation and gratitude to my advisor, Dr. Emily YW Chiang and my co-advisor Dr. Beverley Hale for their guidance and support of my research, this work would not have been possible without their support. In addition, I would like to mention my enormous appreciation for the guidance and help I received from Dr. Rafael Santos, Dr. Luba Vasiluk, Dr. Loo-Sar Chia, and Mr. Peter Smith. Finally, I would like to thank my family for all their love, constant support, and understanding. My sincere thanks to my mother for her endless love and support, and also continuous encouragement throughout my study. Life would be meaningless without you. iii I dedicate this thesis to my late father iv TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGMENT................................................................................................................ iii LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix INTRODUCTION ....................................................................................................... 1 LITERATURE REVIEW ............................................................................................ 5 2.1. Phytomining Definition and Application ........................................................................ 5 2.2. Internal Factor Affecting Phytomining ........................................................................... 6 2.2.1. Hyperaccumulator Plants ....................................................................................... 6 2.2.2. Metal Uptake Mechanism in Plants ..................................................................... 10 2.2.3. Genetic Mechanism of Metal Hyperaccumulation in Plants ............................... 12 2.3. Hormesis and Metal Effects on Plant Growth............................................................... 13 2.4. Photosynthesis Activity and Metal Accumulation Effects on Chlorophyll .................. 14 2.5. External Factors Affecting Metal Uptake by Plants ..................................................... 15 2.5.1. Metal in Soils ....................................................................................................... 15 2.5.2. Soil pH ................................................................................................................. 16 2.5.3. Fertilizer ............................................................................................................... 17 2.5.4. Chelate ................................................................................................................. 17 2.6. Rare Earth Elements (REEs) ......................................................................................... 18 2.7. An Economically Feasible Model for Phytomining ..................................................... 21 OBJECTIVES ............................................................................................................ 23 v MATERIALs AND METHODs ................................................................................ 24 4.1. Seed Sterilization and Germination .............................................................................. 24 Failed Seed Germination............................................................................................ 25 4.2. Media Preparation and Metal Exposure ........................................................................ 26 4.2.1. Metal Oxide Powder Dissolution and Stock Solution Preparation ...................... 26 4.2.2. Murashige & Skoog (MS) Medium Preparation .................................................. 28 4.3. Growth Condition ......................................................................................................... 29 4.4. Chlorophyll Extraction.................................................................................................. 29 4.5. Biomass Assessment, Plant Tissue Digestion, and Tissue Metal Content ................... 30 4.6. Toxicity Threshold (IC50) ............................................................................................ 30 4.7. Ion Speciation ............................................................................................................... 31 4.8. Gene Expression Analysis ............................................................................................ 31 4.8.1. Total RNA Extraction .......................................................................................... 31 4.8.2. RNA Analysis ...................................................................................................... 33 4.8.3. Primer Design ...................................................................................................... 33 4.9. Statistical Analysis ........................................................................................................ 34 RESULTS .................................................................................................................. 35 Brassica chinensis L. Growth Response to Lanthanum and Estimated IC50 Values ... 35 Brassica chinensis L. Growth Response to Neodymium and Estimated IC50 Values . 38 Helianthus annuus L. Growth Response to Lanthanum and Estimated IC50 Values .. 41 Helianthus annuus L. Growth Response to Neodymium and Estimated IC50 Values . 45 Brassica chinensis L. Chlorophyll Response to Lanthanum and Neodymium, and Estimated IC50 Values .................................................................................................. 48 vi Helianthus annuus L. Chlorophyll Response to Lanthanum and Neodymium, and Estimated IC50 Values .................................................................................................. 49 Rare Earth Element Speciation And Uptake by Plants ................................................. 50 HMA4 Gene Expression ............................................................................................... 54 DISCUSSION ............................................................................................................ 55 6.1. Growth Response and Inhibitory Concentration........................................................... 55 6.2. Chlorophyll Response to Lanthanum and Neodymium ................................................ 57 6.3. Speciation and REE Uptake by The Plants ................................................................... 58 CONCLUSIONS........................................................................................................ 61 RECOMENDATIONS .............................................................................................. 63 REFERENCES .......................................................................................................... 64 APPENDICES ......................................................................................................... 79 10.1. Appendix A: A List of Hyperaccumulator Plants ....................................................... 80 10.2. Appendix B: Fitted Curves ......................................................................................... 85 10.3. Appendix C: MS Medium Components ..................................................................... 92 10.4. Appendix D: Statistical Analysis ................................................................................ 94 vii LIST OF TABLES Table 1- Reported hyperaccumulation concentrations for plants ................................................... 9 Table 2- Five REE hyperaccumulators and REE concentrations (ppm) in their leaves ............... 21 Table 3- Lanthanum stock solution preparation ........................................................................... 26 Table 4- Neodymium stock solution preparation.........................................................................