Immunogold Localization of Ribulose-1,5-Bisphosphate Carboxylase with Reference to Pyrenoid Morphology in Chloroplasts of Synchronized Euglena gracilis Cells

Abstract

Euglena gracilis strain (Z) cells were synchronized under photoautotrophic conditions using a 14 hour light:10 hour dark regimen. The cells grew during the light period (growth phase) and divided during the following 10 hour period either in the dark or in the light (division phase). Changes in morphology of the pyrenoid and in the distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within the chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. Epon-embedded sections were labeled with an antibody to the holoenzyme followed by protein A-gold. The immunoreactive proteins were concentrated in the pyrenoid, and less densely distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. Toward the end of the division phase, the pyrenoid began to form in the center of a chloroplast, and the immunoreactive proteins started to concentrate over that rudimentary pyrenoid. During the growth phase, small areas rich in gold particles, called `satellite pyrenoid,' were observed, in addition to the main pyrenoid. From a comparison of photosynthetic CO₂-fixation with the total carboxylase activity of Rubisco extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO₂-fixation in photosynthesis.