Soybean is a major crop species providing valuable feedstock for food, feed and biofuel. In recent years, considerable progress has been made in developing genomic resources for soybean, including on-going efforts to sequence the genome. These efforts have identified a large number of soybean genes, most with unknown function. Therefore, a major research priority is determining the function of these genes, especially those involved in agronomic performance and seed traits. One means to study gene function is through mutagenesis and the study of the resulting phenotypes. Transposon-tagging has been used successfully in both model and crop plants to support studies of gene function. In this report, we describe efforts to generate a transposon-based mutant collection of soybean. The Ds transposon system was used to create activation-tagging, gene and enhancer trap elements. Currently, the repository houses approximately 900 soybean events, with flanking sequence data derived from 200 of these events. Analysis of the insertions revealed approximately 70% disrupted known genes, with the majority matching sequences derived from either Glycine max or Medicago truncatula sequences. Among the mutants generated, one resulted in male-sterility and was shown to disrupt the strictosidine synthase gene. This example clearly demonstrates that it is possible to disrupt soybean gene function by insertional mutagenesis and to derive useful mutants by this approach in spite of the tetraploid nature of the soybean genome.

The acetyl-CoA/biotin network encompasses genes involved in acetyl-CoA generation and utilization and biotin synthesis, recovery, and transport, as well as biotincontaining enzymes involved in myriad metabolic processes. To expand our understanding of this network and address its control, plants with altered levels of ACL (ATP citrate lyase) and BIOl (encoding adenosylmethionine-8-amino-7-oxononanoate synthase), have been examined. An inducible promoter provides a powerful method to test the effects of alterations in expression of specific genes. The synthetic GVG promoter, reported to be sterol-inducible in Arahidopsis thaliana, was inducible in Glycine max (soybean) when used to control the GUS reporter gene, or theACLA or ACLB genes. It showed the same unique replicable pattern of expression in soybean and Arahidopsis, directing high expression primarily to the vasculature of the leaf and root. ACL is a heteromeric enzyme, responsible for production of cytosolic acetyl-CoA. The ACLA-1 and ACLB-2 genes have been over-expressed individually in Arahidopsis using the GVG promoter. Over-expression of ACL genes led to significantly increased ACLA and ACLB RNA accumulation. Over-expression of one subunit of ACL had no effect on mRNA level of the other subunit or on ACL enzyme activity. Transgenic lines over-expressing high levels of ACLA and ACLB RNA were crossed to yield plants containing both transgenes. ACLA and ACLB RNA levels were greatly increased in ACLA+ACLB plants. For the same plants, there was little or no increase in ACLA or ACLB protein from the wild-type level. ACL enzyme activity and phenotype remained unchanged. In contrast, ACL activity was slightly higher (approximately 1.5-fold increase) in cold-treated ACLA+ACLB plants than in cold-treated wild-type plants. These data suggest that ACL is regulated at the translational and/or post-translational levels, and that this regulation can be modulated environmentally. Global transcript profiling experiments utilizing the Arahidopsis biol mutant deficient in biotin synthesis reveal decreased accumulation ofRNAs encoding processes vi directly related to biotin synthesis and utilization in this mutant. Specifically, subunits of the biotin-containing enzymes acetyl-CoA carboxylase and methylcrotonyl-CoA carboxylase, and L-allo-threonine aldolase/lyase, the catalyst for an early step in biotin synthesis, have decreased transcript accumulation in the biol mutant when compared to wild-type. A number of mRNAs involved in defense responses, stress responses, and production of secondary metabolites show increased accumulation in the mutant plants. At the same time, there are decreases in particular mRNAs for the light reactions of photosynthesis and the TCA cycle. The presence of exogenous biotin caused changes in mRNA profiles in both the biol mutant and wild-type plants. 1 CHAPTER 1. GENERAL INTRODUCTION DISSERTATION ORGANIZATION The alternative paper format has been selected for this dissertation, which consists of five chapters. Where appropriate, formatting gui...