LOCALIZATION OF THE ENZYME GERANYLGERANYLPYROPHOSPHATE SYNTHASE IN CAPSICUM FRUITS BY IMMUNOGOLD CYTOCHEMISTRY AFTER CONVENTIONAL CHEMICAL FIXATION OR QUICK-FREEZING FOLLOWED BY FREEZE-SUBSTITUTION - LABELING EVOLUTION DURING FRUIT RIPENING

The enzyme geranylgeranylpyrophosphate synthase (GGPPS), which plays a key role in the synthesis of diterpene compounds, carotenoids and higher terpenoids, has been localized in Capsicum fruit cells by ultrastructural immunogold cytochemistry, after conventional chemical fixation of tissues and quick-freezing followed by freeze-substitution of isolated chloroplasts and chromoplasts. In agreement with previous biochemical studies on cell fractions, the enzyme seems restricted to the plastid compartment. Together with the phenotypic changes of the fruit and the ultrastructural modifications of the plastids during the transition of chloroplasts to chromoplasts, the amount of immunolabelling over plastid sections increases more than a ten-fold factor in the course of fruit ripening. In chemically fixed tissues, the gold labelling of chloroplasts is very faint and erratically localized whereas in further transition stages, and in chromoplasts, most of the gold particles surround the developing plastoglobuli, which are the characteristic carotenoid-bearing structures. Because of the very low and inconstant labelling of chloroplasts in green fruits after chemical fixation, cryofixed and acetone freeze-substituted purified plastids were used as a model system for an accurate localization of the enzyme in these organelles. Quick-freezing in buffered sucrose by slam-freezing on a cold copper block results in optimal preservation of the plastids and improved labelling of GGPPS. The enzyme is not scattered at random throughout the stroma. Gold particles are concentrated in distinct stroma regions, and especially at the sites of initiation of stroma globuli which are the early structural event of carotenoid accumulation. A few gold particles are also present on the margins of thylakoids and, presumably, on the plastid envelope. This paper reports further evidence of the central role of the plastid compartment in the production of C20 isoprenoid intermediates in the plant cell, shows the spatial relationship of the enzyme geranylgeranylpyrophosphate synthase with the plastid substructures and the existence of several GGPPS pools within the plastids. It demonstrates the interest of cryo-methods for an accurate localization of various enzymes in plant cells.