Accumulation of calcium and its involvement in the control of nitrogenase activity in the symbiosomes of broad bean root nodules

Nitrogenase activity and calcium level assayed cytochemically in situ with potassium pyroantimonate were investigated in root nodule symbiosomes as affected by 2-h and 4-h incubation of broad bean (Vicia faba L.) roots in a Ca-deficient medium (buffered tap water in the absence and in the presence of EGTA and the Ca2+-ionophore A23187). Nitrogenase activity distinctly declined as a result of the root treatment, and the strongest effect was observed after the roots were incubated in the medium containing EGTA and the ionophore A23187. The observed decrease in the nitrogenase activity of the symbiosomes was accompanied by considerable calcium depletion. The presence of substantial Ca2+ pool inside the symbiosomes was additionally confirmed in the experiments with the preparations of isolated symbiosomes and bacteroids using the fluorescence Ca probe chlortetracycline. According to cytochemical assay for calcium in isolated symbiosomes, calcium uptake by the symbiosomes was considerably enhanced in the presence of ATP and Mg2+ ions and significantly inhibited by orthovanadate. The results obtained indicate that: (I) the symbiosomes in the infected cells of the broad bean root nodules behave as intracellular Ca-storing compartments; (2) most calcium inside the symbiosomes is within the bacteroids; (3) calcium uptake by the symbiosomes is an energy-dependent process, which is most likely catalyzed by Mg2+-dependent Ca2+-translocating ATPase located in the peribacteroid membrane; (4) calcium in the symbiosomes is involved in the control of their nitrogenase activity, however, the mechanism of such a control is not clear.