A molecular and biochemical analysis of inositol metabolism in cotyledons of Phaseolus vulgaris and Arabidopsis thaliana.

摘要

Inositol is a cyclic molecule with six hydroxyl groups and is found in eukaryotes and prokaryotes. Derivatives of inositol are involved in signal transduction involving hormones, growth factors and neurotransmitters, and in membrane formation through their role as precursors of phospholipid biosynthesis. Additionally, in plants, inositol is involved in seed germination, sugar transport, stress responses, and cell wall biogenesis.; The rate limiting step for inositol synthesis is the conversion of glucose 6-phosphate to myo-inositol 1-phosphate by the enzyme, myo-inositol 1-phosphate synthase (MI-1-P synthase). Free inositol is generated by the removal of phosphate by the enzyme, myo-inositol 1-monophosphatase.; My dissertation research addresses the temporal and spatial regulation of MI-1-P synthase in the development of Phaseolus vulgaris, a member of the kidney bean family, and Arabidopsis thaliana, the common thale cress. Molecular and biochemical analyses were employed to study expression of MI-1-P synthase in embryonic leaves, called cotyledons, in both plant systems. Cotyledons contain storage microbodies that are essential for seed germination and the development of the early seedling.; A genomic clone encoding MI-1-P synthase of Phaseolus vulgaris was isolated to study, at the molecular level, the expression of different isoforms and to identify transit peptide sequences and conserved motifs. Southern blot analysis demonstrated the presence of two genes encoding the synthase. A developmental profile of MI-1-P synthase expression in cotyledons was generated in order to study the temporal expression of the different isoforms. Immunolocalization studies utilizing confocal and light microscopy generated data that demonstrated the association of the synthase to structures that resemble organelles. Organelle isolation and biochemical analysis of chloroplasts, mitochondria, microsomes and plasma membrane from cotyledons demonstrated MI-1-P synthase activity in the organelles.