The central role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the bio1 mutant of Arabidopsis

Abstract

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...