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Organellar Signaling Expands Plant Phenotypic Variation and Increases the Potential for Breeding the Epigenome University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Theses, Dissertations, and Student Research in Agronomy and Horticulture Agronomy and Horticulture Department 10-2012 Organellar Signaling Expands Plant Phenotypic Variation and Increases the Potential for Breeding the Epigenome Roberto De la Rosa Santamaria University of Nebraska-Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/agronhortdiss Part of the Agriculture Commons, and the Genetics Commons De la Rosa Santamaria, Roberto, "Organellar Signaling Expands Plant Phenotypic Variation and Increases the Potential for Breeding the Epigenome" (2012). Theses, Dissertations, and Student Research in Agronomy and Horticulture. 57. https://digitalcommons.unl.edu/agronhortdiss/57 This Article is brought to you for free and open access by the Agronomy and Horticulture Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses, Dissertations, and Student Research in Agronomy and Horticulture by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. ORGANELLAR SIGNALING EXPANDS PLANT PHENOTYPIC VARIATION AND INCREASES THE POTENTIAL FOR BREEDING THE EPIGENOME By Roberto De la Rosa Santamaria A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Agronomy (Plant Breeding and Genetics) Under the Supervision of Professor Sally A. Mackenzie Lincoln, Nebraska October, 2012 ORGANELLAR SIGNALING EXPANDS PLANT PHENOTYPIC VARIATION AND INCREASES THE POTENTIAL FOR BREEDING THE EPIGENOME Roberto De la Rosa Santamaria, Ph.D. University of Nebraska, 2012 Adviser: Sally A. Mackenzie MUTS HOMOLOGUE 1 (MSH1) is a nuclear gene unique to plants that functions in mitochondria and plastids, where it confers genome stability. Phenotypic effects of MSH1 down- regulation were studied in sorghum inbreed line Tx430 and Arabidopsis ecotype Columbia-0, with the hypothesis that RNAi suppression of MSH1 triggers retrograde signaling from organelles to the nucleus, alters the epigenome, and derives heritable phenotypic variation suitable for artificial selection. An array of morphological traits and metabolic pathways was detected, including leaf variegation, male sterility and dwarfism, associated with altered gibberellic acid metabolism, higher levels of reactive oxygen species (ROS), and decreased synthesis of ATP. A phenotype that combines dwarf, increased branching, reduced stomatal density and delayed flowering was identified, and designated developmental reprogrammed (MSH1-dr). Reproducible in additional plant species, this phenotypic variation is partially reversed by exogenous GA. In sorghum, the phenotype displays complete penetrance under self-pollination, even after segregation of the transgene, whereas progeny of MSH1-dr transgene null plants x wildtype Tx430 display enhanced growth. Significant differences for agronomic traits and response to selection were observed in the tested F2 to F4 generations, with mean values that surpass the wildtype up to 70% for grain yield/panicle and plant height, and 100% for biomass yield/plant. SSR marker analyses among the parental phenotypes Tx430 and MSH1-dr transgene null, and their derived lines, show no polymorphism, suggesting that the observed changes are non-genetic. In Arabidopsis, this enhanced growth is accompanied by genome methylation changes, whereas genetic hemi-complementation indicates that the novel phenotype results from chloroplast disruption. 4 Dedication I want to dedicate this Thesis to my wife Rosalba, and our kids Roberto Edwin and Shadid Fernanda, because of the love they have shared with me and inspired on me to pursue new goals that alone I never would have reached. I am convinced that beside a great man there is a great woman, and a great family. I also dedicate this to my parents: Liborio Apolinar De la Rosa and Elvira Santamaria; he just gave me my first lessons about agriculture and practical plant breeding, while she just took care of all of the family; both have taught to me that family is the cornerstone for every success in life. For my parents in law Dolores Hernandez and Ismael Montiel (RIP), and sisters in law Esthela and Elizabeth Montiel, who have been always there unconditionally sharing successes and challenges. For my brothers and sister: Pedro, Dionisio, Francisco, Ernesto, Gorgonio (RIP) Reynaldo, Maria del Rocio, and Oscar; for my nieces and nephews. 5 Acknowledgements I want to express my sincere gratitude to my adviser Dr. Sally A. Mackenzie, whose support was determinant for me to reach this goal. Among other things, she taught to me how to write, and encouraged me to see what Mother Nature says through biological phenomena. I thank sincerely the members of my supervisory committee, Drs.: Ismail Dweikat, David Holding, Alan Christensen, and Tom Hoegemeyer for their important suggestions and teaching during my academic program. I am express my gratitude to Dr. John Rajewski, for his support during the development of field experiments, to Osval Montesinos, for his help in the statistical analyses, and to Victoria Lozano, for her logistical help. I thank former and current members of Mackenzie Lab: Maria, Vikas, Jaime, Wilson, Ajay, Saleem, Xue hui, Yashi, Ying Zhi, Fareha, Peibei, Geetanjali, Kamaldeep, Ido, Ray, Omar, and Hardik; in different ways, everyone helped me to be successful in this endeavor. I thank Amy Hilske and Samantha Link for their careful attention of the plant material used in greenhouse experiments, and the administrative staff of the Center for Plant Science Innovation: Barb, Holly, Teresa, Lisa, Laurie, and Tracy. I also thank Colegio de Postgraduados Mexico, the Fulbright program, and the National Science Foundation for the sponsorship I received during these doctoral studies; the latter through the program of my academic adviser. 6 Table of Contents Abstract…………………………………………………………………………... 2 Dedication……………………………………………………………………….. 4 Acknowledgements……………………………………………………………. 5 Table of Contents………………………………………………………………. 6 List of Figures……………………….………………………………………….. 10 List of Tables……………………………………………………………………. 12 Chapter 1…………………………………………………………………………. 14 Literature Review.………………………………………………………… 14 • Introduction………………………………………………………… 14 • Nuclear and organellar plant genome interactions: how they relate to the epigenome to effect plant phenotypy…………….. 1 4 • The nuclear gene MSH1 and plant organelle interactions…… 17 • The nature of MSH1-associated mitochondrial genome recombination……………………………………………………... 19 • MSH1 RNAi down-regulation and plant phenotypic plasticity... 20 • Genetic and epigenetic variation………………………………... 22 • The model plants to be used in this study……………….…….. 23 Arabidopsis………………………………………………………… 23 Sorghum…………………………………………………………… 2 4 • Rationale for epigenetic crop breeding………………………… 26 • References……………………………………………………….. 27 7 Chapter 2…………………………………………………………………………. 34 Chloroplast-mediated plant developmental reprogramming as a result of MUTS HOMOLOG1 suppression……………………………. 34 Abstract…………………………………………………………… 34 Introduction……………………………………………………….. 35 Methods…………………………………………………………… 36 • Plant materials and growth conditions………………… 36 • Microscopy……………………………………………….. 39 • RNA Isolation and Real-Time PCR Analysis ………… 40 • Small RNA analysis…………………..…………………. 40 • Genomic DNA methylation assay……………………… 41 • Metabolite Analysis……………………………………… 41 Results……………………………………………………………. 43 • MSH1 suppression induces similar phenotypic changes in sorghum as in other plant species………… 43 • Phenotypic variants induced in sorghum by MSH1 RNAi suppression are heritable………………………… 43 • Gibberellic acid partially restores the altered phenotype, with significant interaction between GA dosage and the RNAi transgene………………………. 44 • The MSH1-dr phenotype in transgene-null lines displays enhanced growth following crosses with wildtype…………………………………………………… 46 8 • Fertility restoration occurs with wildtype pollen or in CMS MSH1-RNAi lines following segregation of the transgene………………………………………………….. 47 • Different metabolic pathways are altered by MSH1 suppression………………………………………………. 48 • Chloroplast changes are responsible for the MSH1-dr phenotype………………………………………………… 50 Discussion………………………………………………………… 51 References………………………………………………………. 55 Chapter 3………………………………………………………………………… 71 Organelle perturbation alters the epigenome to produce dramatic and heritable changes in plant growth…………………………………. 71 Abstract…………………………………………………………… 71 Introduction……………………………………………………….. 73 Materials and methods………………………………………...... 76 • Plant materials……..…………………………………….. 76 • Field experiments………………………………………… 77 • Phenotypic traits…………………………………………. 78 • Statistical analyses……………………………………… 78 • Genotyping MSH1-dr plants……………………………. 79 • SSR marker screening …………………………………. 80 Results……………………………………………………………. 80 • The MSH1-dr sorghum phenotype is fully penetrant 9 under self-pollination, and derives enhanced growth in crosses to wildtype, with expanded phenotypic variation………………………………………………….. 80 • Derived epi-lines show enhanced growth for agronomic traits with significant environmental interaction effects………………………………………… 81 • Enhanced growth is also observed in derived progenies of msh1 mutant x wildtype Arabidopsis….... 82 • The phenotypic traits are responsive to selection….… 84 • MSH1-dr and derived epi-lines are not polymorphic compared to wildtype……………………….…………… 84 Discussion…………………………………………………………
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