Cytoenzymological analysis of adenylyl cyclase activity and 3′:5′‐cAMP immunolocalization in chloroplasts of Nicotiana tabacum

Abstract

In this study a combination of cytoenzymological and immunocytochemical techniques was used in order to demonstrate the presence of cyclic nucleotide metabolism in chloroplasts of higher plants. Catalytic cytochemistry was used to localize adenylyl cyclase activity by means of electron microscope investigation on Nicotiana tabacum cv. Petit Havana leaf fragments. Various immunocytochemical techniques were explored to visualize the presence of the second messenger adenosine 3':5'-cyclic monophosphate. Making use of adenylyl imidodiphosphate as a substrate, the enzyme activity was predominantly located at the intermembrane space of the chloroplast envelope. In order to provide further topographical information, intact, isolated chloroplasts were submitted to the same cytoenzymological procedure and revealed stromal adenylyl cyclase activity. Using high-pressure freezing as a physical fixative to obtain an instantaneous metabolic arrest the cellular vitrified water phase was sublimed under ultra-high vacuum by means of molecular distillation drying, avoiding recrystallization and hence redistribution of small highly diffusible molecules. This sequential combination preserved 3':5'-cAMP epitope retention in chloroplasts as was demonstrated by immunogold labelling. These results further substantiate in a unique way the growing evidence of the presence of an organelle-specific cAMP metabolism in higher plants. Furthermore the data presented support the status of chloroplasts as an excellent model to further investigate cAMP metabolism and to correlate it with a variety of physiological functions.