The major goal of this research proposal is to generate new information on the biogenesis of a plant peroxisome, the glyoxysome, which is pivotally involved in storage lipid metabolism during the critical heterotrophic growth phase of economically - important oilseeds. The main emphasis is on deciphering the mechanisms responsible for targeting and importing membrane phospholipids and enzyme proteins into glyoxysomes in maturing and germinated cotton seeds. Understanding organelle biogenesis (integral to intracellular compartmentation) is an essential part of ultimately comprehending the complexities of cellular metabolism. Formation of glyoxysomes will be analyzed by electron microscopy coupled with immunocytochemistry of enzymes to visualize images in thin sections of developing cotyledons. Phospholipid composition of organelle membranes will be quantitated by Iatrascan TLC/FID, and the subcellular site of phospholipid synthesizing enzymes will be determined from radioactive labeling assays of fractions prepared by density - gradient centrifugation. In vivo (protoplasts) and in vitro (translation products from Poly A+ mRNA) systems will be established to experimentally evaluate critical requirements for import of isocitrate lyase, catalase, and malate synthase into peroxisomes isolated from cotton and other species. Recombinant DNA experiments will be initiated wherein a lambda gt10 library will be sequenced and those with full length open reading frames will be subcloned into vectors for establishing an in vitro transcription/translation system designed to assess molecular modifications on the import of proteins. The timing and appearance of gene transcripts in maturing seeds will be determined with cloned DNA probes to document apparent translation control(s) of enzyme synthesis in maturing seeds.***
