Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1995 Molecular analyses of group 1 late embryogenic abundant (Lea) genes in soybean (Glycine max (L.) Merr.) Éberson Sanches Calvo Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Genetics Commons, and the Molecular Biology Commons Recommended Citation Calvo, Éberson Sanches, "Molecular analyses of group 1 late embryogenic abundant (Lea) genes in soybean (Glycine max (L.) Merr.) " (1995). Retrospective Theses and Dissertations. 10765. https://lib.dr.iastate.edu/rtd/10765 This Dissertation 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. 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Mi 48106-1346 USA 313.'761-4700 800/521-0600 Molecular analyses of group 1 late embryogenic abundant (Lea) genes In soybean (Glycine max (L) Merr.) by Eberson Sanches Calvo A Thesis Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Zoology and Genetics Major: Genetics Approved: Signature was redacted for privacy. In Chargje of Major Work Signature was redacted for privacy. For the Major Departm^^t Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1995 UMI Number: 9540879 DMI Microform 9540879 Copyright 1995r by DMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17r United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ii DEDICATION This dissertation marl<s the end of a dream and the start of new ones. I dedicate this worl< to the memory of my friend Emilio Carlos Sachi who could not live long enough to see most of his dreams come true. iii TABLE OF CONTENTS GENERAL INTRODUCTION 1 Rationale 1 Literature Review 2 General Aspects of Plant Embryo Development 2 The LEA Proteins 5 Factors Affecting Gene Expression During Plant Seed Development 6 Abscisic Acid 7 Trans-Acting Elements 11 Cis-Acting Elements 13 Conclusions 19 Dissertation Organization 20 Literature Cited 21 CHAPTER 1: CLONING, MAPPING, AND ANALYSES OF EXPRESSION OF THE Em-LIKE GENE FAMILY IN SOYBEAN (Glycine max (L.) Merr.'> 30 Abstract 30 Introduction 31 Materials and Methods 33 Plant Material 33 ABA and Water Deficit Treatments 33 Isolation of cDNA and Genomic Clones 34 DNA Sequencing and DNA Comparisons 35 RFLP Mapping 36 iv Isolation of DNA and RNA, and Hybridization Analyses 36 Reverse Transcription and PGR 38 Results 38 Screening of cDNA Library 38 Genetic Mapping of the Sle Gene Family 39 Sequence Analyses of Genomic Clones 40 Sle mRNA Accumulation in Embryos 42 Sle mRNA Accumulation in Response to ABA and Water Deficit 44 Discussion 45 Acknowledgments 49 References 49 CHAPTER 2: PROMOTER ANALYSES OF A GROUP 1 LEA GENE FROM SOYBEAN (Glycine max (L) Merr.) IN TRANSGENIC TOBACCO 73 Abstract 73 Introduction 74 Experimental Procedures 76 DNA Sequence and DNA Comparisons 76 Construction of Plasmids 76 PCR Mutagenesis 77 Plant Transformations 79 GUS Assays 79 Results 80 Sle Promoters Contain Several Putative Regulatory Elements 80 The Role of the Distal G-Box Core Motifs 81 V The Role of the Proximal ACGT Core Motif 83 The Role of the SphI Box Motif 84 The Role of the CCACAT Repeats 84 Histochemical Staining of Seeds 86 Discussion 86 References 91 CHAPTERS; GENERAL CONCLUSIONS 112 Literature Cited 115 ACKNOWLEDGMENTS 116 1 GENERAL INTRODUCTION Rationale Five years ago the first solid molecular evidence linking the hormone abscisic acid (ABA) as a major positive regulator of late embryogenesis abundant (Lea; Dure et al., 1993) genes in wheat (Marcotte et al., 1989; Guiltinan et al., 1990) and rice (Mundy et al., 1990) was reported. These results were quite intriguing for those interested in soybean seed development mostly because the pattern of endogenous ABA accumulation in soybean embryos (Ackerson, 1984) appeared to be rather distinct from the pattern observed for rice and wheat embryos (Skriever and Mundy, 1990). Furthermore, evidence already existed showing that ABA could also be a positive regulator of transcription of soybean storage protein genes expressed during the maturation phase of seed development (Bray and Beachy, 1985). Since the molecular cloning and detailed analyses of several soybean storage protein genes were already in progress in other laboratories, we decided to clone a Lea gene from soybean as a means to understand the regulation of late embryogenesis during soybean seed development in a manner comparable to storage protein gene regulation. Probably due to its great economic importance, soybean seed development has been subjected to an array of biochemical and physiological studies (Wilcox, 1987). Thus, the study of gene expression in soybean may ultimately establish several links between phenomena at the molecular level and those at the physiological level. Soybean is still far from being a model genetic organism such as maize or Arabidopsis. However, several plant genes are likely to be redundant even in the small genome of Arabidopsis (the Lea genes are a good exemple), and 2 Will not likely be identified and cloned by standard genetic approaches. Soybean seeds, essentially comprised of embryonic material, offer substantial advantage to the application of several molecular biology approaches that rely on the purification of proteins or other molecules, library constructions, etc. The introduction section of this Dissertation will briefly review our current knowledge of seed development in angiosperms with emphasis on the molecular biological aspects of this process. Literature Review General Aspects of Plant Embryo and Seed Development In angiosperms, embryo and seed development comprise the series of events that take place between double fertilization and the germination process. Double fertilization sets the stage for a series of events characteristic to plant embryo and seed development, which includes the limited pattern formation in the embryo without formation of germ line, the accumulation of storage products in a specialized tissue, and the desiccation and acquisition of a quiescent state by the embryo and seed (Goldberg et al., 1989; Goldberg et al., 1994). Despite the noticeable variation that exists in embryo and seed development between different plant taxa (Esau, 1977), these processes can be generally divided into three phases (Gatehouse and Shirsat, 1993; West and Harada, 1993). The first phase, referred to as early embryogenesis, starts with the double fertilization event. Fertilization of the haploid egg cell nucleus by one of the sperm nuclei initiates embryogenesis, whereas the fertilization of the diploid central cell of the embryo sac originates development of triploid endosperm which nourishes the 3 embryo. Embryo, endosperm and ovule-derived integuments form the seed. It is during this first phase that most of the cell divisions and all of the embryo pattern formation occur (de Jong et al., 1993). Early embryogenesis has been extensively described at the morphological level, but very little is known about the molecular nature of the events occurring during this phase, mostly due to the difficulties associated with the small size of the developing embryo and seed. Nevertheless, the development of somatic embryogenesis and in vitro fertilization systems, and the power of Arabidopsis genetics are already making significant contributions to our understanding of this process (de Jong et al., 1993; Goldberg et al., 1994). The second phase of embryo and seed development is commonly referred to as the maturation phase and, at the molecular level, this phase of development is characterized by intensive synthesis of storage products, mainly starch, proteins, and lipids, that will be used by the germinating embryo and the resulting seedling.