A Dissertation Entitled Stress Induced Silicon Accumulation in the Inducible Accumulator Nicotiana tabacum by Wendy L. Zellner Submitted to the Graduate Faculty as partial fulfillment of the requirements for Doctor of Philosophy degree in Biology ____________________________ Dr. Scott M. Leisner Committee Chair ____________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo August 2012 Copyright © 2012, Wendy Louise Zellner This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Stress Induced Silicon Accumulation in the Inducible Accumulator Nicotiana tabacum by Wendy L. Zellner Submitted to the Graduate Faculty as partial fulfillment of the requirements for Doctor of Philosophy degree in Biology The University of Toledo August 2012 While Silicon (Si) is not a panacea for stress resistance in plants, the element has a number of beneficial effects against both abiotic and biotic stress. Most research to date has been inundated with salt, cold, and fungal resistance induced by Si in high accumulators, with little work performed on viral infections and low accumulators. The low accumulator, N. tabacum infected with Tobacco ringspot virus and supplemented with Si showed a reduction in viral systemic symptoms and a increase in foliar Si levels compared to controls. Si supplementation did not influence the systemic symptoms induced by the unrelated pathogen, Tobacco mosaic virus, nor did infections influence foliar Si levels. Si accumulation in the TRSV-N. tabacum pathosystem was quite variable, so to study stress induced Si accumulation (SISA), a more consistent system was developed. Copper (Cu) toxicity increases foliar Si accumulation in a low accumulator and in N. tabacum. The consistency of this response indicates that it is a good model to study SISA. In addition to Cu, hormones involved in defense iii and environmental perception were tested to determine if they too influenced Si accumulation in leaves. N. tabacum were exposed to exogenous abscisic acid, methyl jasmonate, or salicylic acid and foliar Si content was determined. Plants treated with ABA showed a significant reduction in foliar Si levels, while the other two hormones had no effect. This suggests the ABA has an antagonistic effect on SISA. Along with physiological work, we examined factors involved in the transport of Si into the plant. Aquaporins, belonging to the major intrinsic protein (MIP) family, are involved in water and/or solute transport into and throughout the plant. We identified putative MIPs in N. tabacum, including a novel ntNIP3;1. This protein is likely a Si transporter based on sequence homology to documented transporters. In addition, Si caused a reduction in expression of ntNIP3;1as well as a root-specific tonoplast intrinsic protein (TIP), ntRT-TIP1, which correlates with the expression of other Si transporters. This leads us to believe that in addition to the classical NIP family members involved in Si transport, TIPs may also play a role. Taken together, these data suggest that characterizing plants as low-, intermediate- or high-accumulators can be somewhat misleading, since the ability to accumulate the element is dependent on not just the tissue assayed, but can also be influenced by the environmental conditions in which the plants are grown. In summary, Cu toxicity is a reliable system to begin studying the molecular aspects of SISA in the inducible- accumulator N. tabacum. This process may be under hormonal regulation, since ABA reduces Si levels. With the heavy use of hormone regulators (such as ABA), especially in the floriculture industry, could lead to lowered resistance iv against a number of abiotic and biotic stressors, because of reduced foliar Si levels. v For my husband who encouraged me to pursue my degree, my children who remind me to question the world around me and enjoy all the little discoveries, my family and friends who have supported me throughout my entire schooling and to my teachers and professors who have added to my excitement and love for plant biology. vi Acknowledgments This work could not have been accomplished without the encouragement and guidance from my advisor, Dr. Scott M. Leisner, who gave me the freedom to pursue my interests, while working toward my degree. I also would like to thank the members of my graduate committee Dr. Jonathan Frantz, Dr. John Gray, Dr. James Locke, and Dr. Lirim Shemshedini, who have helped answer questions and shared with me a wide spectrum of skills and knowledge throughout the years. Not a committee member, but equally helpful in my success here at UT is Dr. Charles Krause. I also would like to thank many of my lab members, past and present who have reminded me that there is no better job than a scientist. I especially would like to thank Genevieve Okenka, Dr. Gaurav Raikhy, Dr. Sushant Khandekar, Dr. Jie Li, and Lindy Lutz who have provided great debates and troubleshooting ideas for all the mishaps along the way. In addition, I would like to thank the USDA-ARS technicians Douglas Sturtz, Russell Friedrich, and Alycia Pittenger who were responsible for compiling the ICP-OES data for these studies. Lastly, but not anywhere near the least, I would like to thank Lynn Biltz for everything she has taught me in regards to horticulture and identification of all my unknown species in addition to all that she did keeping plants pest-free. vii Table of Contents Abstract………………………………………………………...……….……......iii Acknowledgments…………………………………………...…………..……....vii Table of Contents…………………………………………...……………..….....viii List of Tables………………………………………….…...……………….….....xi List of Figures ………………………………………….…………...……..…....xiii List of Abbreviations……………………………………………………….......xvii 1 Introduction………………………………………………………….…...1 2 Silicon Delays Tobacco Ringspot Virus Systemic Symptoms In Nicotiana tabacum ………………………..…..…………….....7 2.1 List of Abbreviations……………………………..…………...……....8 2.2 Summary…………………………………………..…..…………..…10 2.3 Materials and Methods………………….………...…………………11 2.4 Results and Discussion………………………………………………14 2.5 Acknowledgments………………………………...…………………19 2.6 References ………………………………………...…………………20 3 Induced Silicon Accumulation in N. tabacum…………...………………24 3.1 Abstract………………………………………………………24 3.2 Introduction……………………………………….…………25 3.3 Materials and Methods………………………………………28 3.4 Results……………………………………………………..…30 3.5 Discussion ………………………………………….…..……43 viii 4 Silicon Regulation of Aquaporin Gene Expression in N. tabacum……...47 4.1 Abstract………………………………………………………47 4.2 Introduction………………………………………….….....…48 4.3 Materials and Methods……………………………….………55 4.4 Results…………………………………………..............……59 4.5 Discussion …………………………………........……………72 5 Additional Si Experiments....…………....………….....…………………75 5.1 Abstract……………………....……………............…………75 5.2 Introduction………………....……………………......………77 5.3 Materials and Methods……………………………….....……80 5.4 Results………………………………………………..............84 5.5 Discussion …………………………………………….......…93 6 Discussion/Future Work……………………………………………....…96 References ………………………………………………...................................106 A Bioinformatic Codes………………………………………………........126 A.1. Introduction..........................................................................126 A.2 AUDPC……………………………………………….........126 A.3 Statistical analysis……………………………………….…128 B Identification and analysis of viruses infecting Pelargonium …………..129 B.1 Abstract……………………………………………….........129 B.2 Introduction………………………………………………...130 B.3Materials and Methods……………………………………...132 B.4 Results………………………………………………...........136 B.5 Discussion……………………………………………….....146 C Contig Sequences………………………………………………............150 ix C.1 Contig Accessions…………………………………….……150 C.2 Contig FASTA Sequences…………………...…….………158 x List of Tables 3.S1: Statistical Parameters of TRSV Symptomatic Area.....................................21 3.S2: Statistical Parameters of TRSV AUDPC......................................................21 3.S3: Statistical Parameters of foliar Si in TRSV infected N. tabacum.................22 3.S4: Statistical Parameters of foliar Si in TMV infected N. tabacum..................22 3.S5: Statistical Parameters of root Si in TRSV infected N. tabacum...................23 3.1: Total elemental analysis of N. tabacum treated with 75 μM Cu……………37 3.2: Total elemental analysis of ABA and SA treated N. tabacum…………..….42 3.3: Total elemental analysis of JA treated N. tabacum…………………..……..43 4.1: MIP ar/R selectivity filter amino acid residues……………………..………52 4.2: Websites used for Computational Analysis……………………………..…..55 4.3: Primers used for Real-Time PCR……………………………..…………….58 4.4: Accession number of Si transporters…………………………..……………60 4.5: Selectivity filter and NPA residues for putative xi N. tabacum MIPs………………………………………………...64 4.6: Accession numbers of A. thaliana and N. tabacum TIPs………..………….67 4.7: Accession number of putative Si transporters……………………..………..70 5.1: Local and systemic TRSV symptoms in A. thaliana ecotypes………..…….84 B.1: Primer sequences for virus detection in OPGC Pelargoniums……............134 B.2 Viruses detected in OPGC Pelargoniums in 2006……………………...….139 B.2: PFBV ecotype study in A. thaliana……………………………..…………146 xii List of Figures 3-1: TRSV systemic symptom spread and detection in N. tabacum……………..16 3-2: Si concentration (mg/kg) within virus-infected plants determined by ICP-OES…………………………………………18 3-1: Symptoms on N. tabacum treated with 50