Molecular Mechanisms Underlying Differentiation of Microbodies.

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A cDNA clone for a leaf-peroxisomal enzyme, glycolate oxidase (E C 1.1.3.1), was isolated by an immunochemical method from a cDNA expression library constructed from poly (A)^+ -RNA of green pumpkin cotyledons. Northern blot analysis showed that the probe could hybridizable with a single 1.5-kb species of mRNA from pumpkin cotyledons and that the amount of the hybridizable mRNA increased dramatically during greening of the cotyledons, Immunoblot analysis showed that the amount of glycolate oxidase was low in darkgrown cotyledons and increased during greening of pumpkin cotyledons. This observation indicates that the induction of a leaf peroxisomal enzyme, glycolate oxidase, during greening of the cotyledons is due to an increase in level of the mRNA.After the function transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and ageinst two gloxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increase strikingly. Double labeling experments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomal are directly transformed to glyoxysomes during, senescence.