CDNA CLONING AND EXPRESSION OF A GENE FOR ISOCITRATE LYASE IN PUMPKINCOTYLEDONS

A cDNA clone for isocitrate lyase (ICL) was isolated from a cDNA libra ry prepared from the poly(A)(+) RNA of etiolated pumpkin cotyledons. T he cDNA encoded a polypeptide with 576 amino acids, whose sequence is more than 79% identical to those of ICL from other higher plants and c ontains the C-terminal tripeptide, Ser-Arg-Met, which is a putative ta rgeting signal to microbodies. Immunogold analysis revealed that ICL p rotein is exclusively localized in microbodies in etiolated pumpkin co tyledons. Double labeling experiments with protein A-gold particles of different sizes showed that ICL protein coexists with a leaf-peroxiso mal enzyme, glycolate oxidase, in the microbodies of greening and sene scing pumpkin cotyledons, indicating that transformation between glyox ysomes and leaf peroxisomes occurs directly during greening and senesc ence of the cotyledons. Immunoblot analysis showed that the amount of ICL protein increased markedly during germination and decreased rapidl y when seedlings were exposed to illumination, which induced the micro body transition from glyoxysomes to leaf peroxisomes. In senescing cot yledons, the level of the ICL protein and ICL mRNA increased again wit h the reverse transition of microbodies from leaf peroxisomes to glyox ysomes. Changes in the amount of ICL protein did not correspond to the changes in the level of ICL mRNA during greening and senescence of th e cotyledons, an indication that post-transcriptional regulation plays an important role in the microbody transition.