The genes encoding the distinct polypeptides of glutamine synthetase (GS) in higher plants present an attractive system to study the molecular mechanisms which regulate nuclear genes involved in two functional processes unique to plants, namely chloroplast development and nitrogen fixation. We propose to examine the genes encoding the distinct forms of GS (chloroplastic and cytosolic), to explore the molecular basis for their differential expression in leaves, roots, and in nitrogen- fixing root nodules of legumes.
The specific aims of this study will address the following questions: 1) Are the mRNAs encoding chloroplast or cytosolic GS polypeptides the products of distinct, uniquely regulated GS genes, or alternatively are they the products of differential transcript initiation or RNA processing? 2) What are the factors which contribute to the specific expression of chloroplast GS mRNA in leaves (i.e. light, tissue- type)? 3) What are the molecular mechanisms involved in the induction of GS mRNA in legume roots infected with nitrogen- fixing Rhizobia? cDNA clones encoding the distinct GS polypeptides will be characterized and oligonucleotide probes specific for each GS mRNA species will be used to examine its expression in vivo in leaves (i.e. light/dark), roots, and during nodule development in legumes. The cognate nuclear GS genes will be isolated, characterized, and used as molecular probes, to determine the molecular events which surround their regulated expression in vivo. In particular, we will examine transcriptional regulation by: light, tissue-type, or Rhizobial infection, in isolated nuclei in vitro. Putative trans-acting transcription initiation factors specific for GS gene transcription will be examined in vitro. Wild type and promoter deletion mutants of GS will be introduced into foreign plants (via Ti vectors) and their expression in the transgenic plants monitored in response to light, tissue-type, or Rhizobial infection to define cis-acting DNA elements required for regulated gene expression. In addition to defining the molecular mechanisms which effect a differential regulation of a family of related genes, in the long term, this information may b applied to the modification of existing genes (i.e. in non-legumes) to effect a successful association with Rhizobium.
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