Tissue specific gene expression is central to the development of complex eukaryotes. This project addresses the long term goal of understanding the molecular mechanisms regulating the tissue-specific expression of the zein family of maize seed storage protein genes. Molecular and genetic analyses have shown that the developmental and tissue-specific expression of zeins is dependent on the trans-acting regulatory locus, opaque-2 (o2). Our research has demonstrated that the product of this locus is a regulatory protein belonging to the basic leucine zipper (bZIP) class of eukaryotic transcription factors. We have shown that O2 affects the expression of only certain members of this gene family through recognition of a specific binding site (the O2 box) in promoters of 22-kD zein genes. From characterizations of mutant o2 alleles, analyses of DN4 binding properties, and assays of transcriptional activation in yeast and maize cells, it appears that the capacity of O2 to bind to specific zein promoters in vivo is influenced by interactions with other endosperm proteins. As part of our efforts to understand how these proteins may affect the activity of O2, we have cloned the cDNAs encoding two proteins that, like O2, bind the O2 box in a sequence specific manner. One of these encodes a bZIP protein that forms DNA-binding heterodimers with O2. We intend to characterize these proteins as to their possible interaction with O2 in regulating zein gene expression. In addition, the question will be addressed as to what regulates the expression and activity of O2 in maize endosperm. We have determined that O2 is multiply phosphorylated in vivo, with phosphorylation affecting DNA binding. We will determine if this phosphorylation is influenced by the carbon-nitrogen balance or by availability of nitrogen or sulfur. This study may establish an important link between nutrient availability and storage protein gene activation. These studies will also include an analysis of O2 promoter activity in transgenic plants to identify regulatory elements important to the developmental expression of O2. Finally, new mutant alleles of O2 will be generated by transposon mediated mutagenesis to identify domains in this regulatory protein. The knowledge gained from these studies will not only contribute to our basic understanding of the mechanisms underlying seed storage protein gene expression, but should also provide insight into the general mechanism by which eukaryotes control the process of tissue specific gene expression.