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The effects of malate, succinate, and glutamate on the kinetics of changes in the pH gradient (delta pH) and membrane potential (delta psi) on the peribacteroid membrane (PBM) of the symbiosomes of bean root nodules varying in age were recorded spectrophotometrically. Addition of all the tested metabolites to potassium-free incubation medium stimulated a passive acidification of the peribacteroid space (PBS) and dissipation of delta psi in PBM of young developing nodules in the presence of the K+/H+ antiporter nigericin in the medium. However, in mature nodules with a high nitrogen-fixing activity, only malate and succinate (but not glutamate) increased delta pH during both passive and ATP-dependent PBS acidification. Dicarboxylates also caused dissipation of both delta pH in the presence of nigericin in the medium and delta psi generated on PBM by H+-ATPase. A decrease in the effects of metabolites on delta pH and the absent activity of the PBM H+ pump were observed in the aging nodules. The obtained data on the changes in deltapH and dlta psi caused by the metabolites in question suggest that PBM is permeable for all these metabolites only in young nodules. Only malate and succinate (but not glutamate) are transported through PBM in mature nodules; and the rate of metabolite translocation through PBM in aging nodules is decreased.

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron microscopy demonstrates a considerable calcium depletion in symbiosomes of the infected nodule cells by EGTA and A23187. Ca2+ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca2+ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached the baseline in symbiosomes. The experiments on isolated symbiosomes and arsenazo III demonstrated that verapamil inhibited Ca2+ transport induced by valinomycin at the background of K+ ions. These data suggest the presence of a verapamil-sensitive transporter on the peribacteroid membrane responsible for Ca2+ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of the root nodules is discussed.

The effect of exogenous calmodulin (CaM) on the transport and catalytic activities of Ca2+-ATPase in the symbiosome membrane (SM) from broad bean root nodules was studied at various pH values and concentration of free calcium in the medium (pCa). It was shown that the concentration of H+ ions, which determines the effective interaction of CaM with Ca2+-ATPase, corresponds to pH 7.0, which coincides with the pH value that ensures the optimal functioning of this enzyme. Manifestation of the calmodulin effect was observed only at low Ca2+ concentrations in the medium. The minimum Ca2+ concentration at which CaM initiates stimulation of the ATP-dependent transport of this cation through the SM catalyzed by Ca2+-ATPase is 0.05 μM. Activation of transport activity of Ca2+-ATPase increases with Ca2+-sensor concentration from 20 µg/mL to 80 µg/mL and is at its maximum at a free calcium concentration of 0.118 μM, while hydrolytic activity of the enzyme—at 0.351 µM, when CaM level is 40 µg/mL, which corresponds to half of its saturation dose. Thus, optimal conditions have been identified for the functioning of CaM as a Ca2+-ATPase stimulator, which makes it possible to study the role of this enzyme as an important determinant of Ca2+ signals in the implementation of processes associated with the nitrogen-fixing function of symbiosomes. [ABSTRACT FROM AUTHOR]

On the preparations of symbiosomes isolated from broad bean (Vicia faba L.) root nodules, the transport activity of symbiosome membrane (SM) Ca2+-ATPase expressed as ITP- or Ca2+-dependent alkaline pH shift inside these structures as a function of pH of their incubation medium and calcium concentration in it, respectively, was investigated. The found pH and calcium concentration dependences are considered as novel evidence for close coupling of catalytic and transport activities of Ca2+-ATPase and its high affinity for Ca2+ ions. It was also shown that Cd2+ and Mn2+ ions added to symbiosome incubation medium instead of Ca2+ but at the same concentration do not result in dissipation of ∆pH on the SM generated by the symbiosome H+-ATPase providing evidence for incapability of the calcium pump to perform their translocation through the SM. All these data are discussed in the light of the results obtained earlier by the authors and others. [ABSTRACT FROM AUTHOR]

On the preparations of symbiosomes isolated from broad bean (Vicia faba L.) root nodules, the transport activity of symbiosome membrane (SM) Ca2+-ATPase expressed as ITP- or Ca2+-dependent alkaline pH shift inside these structures as a function of pH of their incubation medium and calcium concentration in it, respectively, was investigated. The found pH and calcium concentration dependences are considered as novel evidence for close coupling of catalytic and transport activities of Ca2+-ATPase and its high affinity for Ca2+ ions. It was also shown that Cd2+ and Mn2+ ions added to symbiosome incubation medium instead of Ca2+ but at the same concentration do not result in dissipation of ∆pH on the SM generated by the symbiosome H+-ATPase providing evidence for incapability of the calcium pump to perform their translocation through the SM. All these data are discussed in the light of the results obtained earlier by the authors and others. [ABSTRACT FROM AUTHOR]

A temporal pattern of the peribacteroid membrane (PBM) transport function was studied. Spectrophotometric recording was used for establishing the effect of carbon-and nitrogen-containing substrates (malate, succinate, and glutamate) on the acidification of the peribacteroid space and the intensity of light scattering in the symbiosome suspension from broad bean ( Vicia faba L.) root nodules of different age. At the early stages of nodule formation and functioning, PBM is permeable not only for malate and succinate, but also for glutamate, and this permeability fully provides for the active bacteroid division and the nitrogenase complex synthesis in the bacteroids at the expense of the carbon-and nitrogen-containing substrates. Mature nodules are characterized by the greatest nitrogen-fixing activity. In these nodules, PBM is selectively permeable for malate and succinate, but constitutes a barrier for glutamate. Thereby, mutually beneficial relations between the symbiotic partners are achieved. In senescent nodules, a rearrangement of symbiotic interactions is directed toward a minimization of both carbon and nitrogen metabolite consumption by the bacteroids. It is concluded that, in the course of the development of the legume-rhizobia symbiosis, the PBM transport function is changed. This function determines a qualitatively different pattern of symbiotic partner interactions in the following sequence: parasitism-mutualism-commensalism. [ABSTRACT FROM AUTHOR]