Imaging oscillations in Gonyzaulax: a chloroplast rhythm of nitrate reductase visualized by immunocytochemistry

RESUMO

Gonyaulax polyedra is a unicellular marine photosynthetic dinoflagellate known to display numerous circadian rhythms, including bioluminescence, motility, cell division and several chloroplast-related rhythms. Due to this, Gonyaulax has become a widely used model organism for studying the cellular biological clock. In this work we describe another rhythm for Gonyaulax cells also associated with the cell's chloroplasts, a rhythm in localization of the enzyme nitrate reductase (NR). A polyclonal antibody was raised against NR purified from G. polyedra cells and used as a probe in immunogold labelling experiments on cell thin sections, comparing day- and night-phase cells. The enzyme localizes to chloroplasts in day-phase cells, while the enzyme is active, and is largely absent in night-phase cells. Counts of gold particle distribution in day- versus night-phase cells show an approximate three-fold increase in enzyme labelling in day-phase plastids. These results closely approximate the four-fold differences shown for NR activity between day and night Gonyaulax cells by biochemical studies. We conclude from the diurnal difference in labelling that NR is localized in Gonyaulax chloroplasts during the day phase and is absent (broken down) in night-phase cells. Thus NR in Gonyaulax is compartmentalized in the chloroplasts and is therefore subject to similar circadian control mechanisms exhibited for other plastid rhythms.