Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 Molecular mechanisms for regulating seed amino acid levels in maize (Zea mays) Marie Adams Hasenstein Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agricultural Science Commons, Agronomy and Crop Sciences Commons, and the Genetics and Genomics Commons Recommended Citation Hasenstein, Marie Adams, "Molecular mechanisms for regulating seed amino acid levels in maize (Zea mays)" (2007). Retrospective Theses and Dissertations. 14677. https://lib.dr.iastate.edu/rtd/14677 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Molecular mechanisms for regulating seed amino acid levels in maize (Zea mays) by Marie Adams Hasenstein A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Genetics Program of Study Committee: Paul Scott, Co-Major Professor Erik Vollbrecht, Co-Major Professor Phil Becraft Iowa State University Ames, Iowa 2007 Copyright © Marie Adams Hasenstein, 2007. All rights reserved. UMI Number: 1447529 UMI Microform 1447529 Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ii TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................................. iii ABSTRACT ............................................................................................................................ iv CHAPTER 1: GENERAL INTRODUCTION ..................................................................... 1 OBJECTIVE ............................................................................................................................. 1 DISSERTATION ORGANIZATION .............................................................................................. 2 LITERATURE REVIEW ............................................................................................................. 3 Importance of Maize for Food/Feed ................................................................................. 3 Lysine ................................................................................................................................ 4 Methionine ...................................................................................................................... 18 REFERENCES ........................................................................................................................ 26 CHAPTER 2: MOLECULAR CHARACTERIZATION OF ZMBZIP1, A DOWNSTREAM TARGET OF THE OPAQUE-2 TRANSCRIPTION FACTOR ....... 35 ABSTRACT ........................................................................................................................... 35 MATERIALS AND METHODS ................................................................................................. 36 ZmbZIP1 Expression Levels ........................................................................................... 36 Physical Mapping of the ZmbZIP1 Gene ........................................................................ 39 Creation of a ZmbZIP1 TILLING Mutant to Attempt to Alter ZmbZIP1 Protein Expression ....................................................................................................................... 41 DNA Isolation ................................................................................................................. 41 RESULTS AND DISCUSSION ................................................................................................... 46 ZmbZIP1 Has Varying Transcript Levels in Different Tissues ....................................... 46 Physical Mapping of the ZmbZIP1 Locus ....................................................................... 52 SNaPshot Genotyping ..................................................................................................... 57 CONCLUSION ........................................................................................................................ 59 REFERENCES ........................................................................................................................ 60 CHAPTER 3: MOLECULAR MECHANISM OF METHIONINE DIFFERENTIATION IN THE BS11 HIGH AND LOW MET MAIZE LINES ............ 62 ABSTRACT ........................................................................................................................... 62 MATERIALS AND METHODS ................................................................................................. 62 Differential Expression of Cys2 and Dzs10 via Quantitative Real-Time RT-PCR ......... 64 Cys2 and Dzs10 Sequencing and Allele Comparison ..................................................... 65 Amino Acid Assay to Determine Kernel Methionine Levels ........................................... 66 RESULTS AND DISCUSSION ................................................................................................... 67 The BS11HM Population Contains Significantly More Methionine than BS11LM ....... 67 Correlation Between Population Methionine Levels and Cys2 or Dzs10 mRNA Levels 69 Correlation Between Kernel Methionine and Cys2 or Dzs10 Haplotypes ..................... 73 CONCLUSION ........................................................................................................................ 76 REFERENCES ........................................................................................................................ 78 GENERAL CONCLUSIONS .............................................................................................. 79 iii ACKNOWLEDGEMENTS I would like to express my gratitude to a number of people who have been indispensible to me during my graduate career. First, to Dr. Paul Scott, my major professor, for his supervision, encouragement and enthusiasm for the work that I was doing, as well as for our many discussions on statistical analysis. I would also like to thank my committee members Drs. Erik Vollbrecht and Phil Becraft for their guidance, concern about my research, and availability throughout my time at Iowa State. Special thanks are extended to Anastasia Bodnar and Dr. Adrienne Moran-Lauter for their help with HPLC and amino acid analysis, both of which were instrumental for the completion of my project. On a more personal level, I would like to thank my husband, Jason Hasenstein, for his constant support through both my best and worst days, and to Bart and Michelle Adams, Lisa Haney, Jamie O’Rourke, Oliver Couture, and Ryan Benson for their friendship and calming and positive influences on me as I was completing my research. iv ABSTRACT The majority of worldwide maize production is used for food or feed; as such it is imperative that its amino acid balance mirror the needs of humans and animals. However, lysine and methionine, two essential amino acids necessary for proper growth and development, are lacking in the maize endosperm. In this thesis, molecular mechanisms to increase both lysine and methionine deposition in the kernel are explored. ZmbZIP1, a maize transcription factor, was identified through differential expression in a microarray experiment as a potential candidate to increase the lysine content of the endosperm while lessening the pleiotrophic effects of prior candidates, such as Opaque-2; however, RT-PCR shows this level of differential expression may be genotype specific. ZmbZIP1 was physically mapped to maize chromosome 9, contig 380, and a rice orthologue, RISBZ5 was found through synteny. Expression data shows that ZmbZIP1 mRNA is present in all tested maize tissues to some extent. An attempt to make a ZmbZIP1 knock- down/knock-out mutant did not result in any phenotypic changes in the maize plant as a whole. Further no changes were seen in kernel alpha-zein deposition, which is the main determinant of endosperm lysine content. These results suggest either a functionally redundant protein to ZmbZIP1 exists, the knock-out alleles retained sufficient activity to be functional, or phenotypes produced by disruption of ZmbZIP1 are not detectable by the methods we used. In all cases, these results suggest more work must be done to determine the efficacy of ZmbZIP1 in regulating endosperm lysine levels. A molecular mechanism for increasing methionine deposition into maize endosperm was investigated with regards to two candidate genes: the 10kDa delta-zein (Dzs10), a v methionine-rich storage protein in endosperm, and cysteine synthase (Cys2), a key regulator in cysteine/methionine biosynthesis. mRNA expression levels and haplotypes were determined for each population, and were correlated to the average methionine content. At the mRNA level, Dzs10 was found to be differentially expressed in maize endosperm, while Cys2 is likely not differentially expressed. Further, a correlation of 0.4535 was found between Dzs10 mRNA and kernel methionine levels, suggesting part of the increase in kernel methionine deposition may be due