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Calcium uptake by symbiosomes and the peribacteroid membrane vesicles isolated from yellow lupin root nodules
• JOUR.AL OF • PI.M PII'I~.I..,
© 1998 by Gustav Fischer Verlag, lena
Calcium Uptake by Symbiosomes and the Peribacteroid Membrane Vesicles Isolated from Yellow Lupin Root Nodules IGOR
M.
ANDREEV, PAVEL
N.
DUBROVO, VALERIA
V.
KRYLOVA,
and
STANISLAV
F.
IZMAILOV
Ttmiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia Received November 5, 1997 . Accepted March 25, 1998
Summary
In order to test whether the symbiosomes of infected cells are able to actively take up calcium ions, preparations of these nitrogen-foong units and the PBM vesicles isolated from yellow lupin (Lupinus luteus L.) root nodules were investigated to this end. Ca2+ uptake was recorded with the use of the metallochromic Ca2+ indicator arsenazo III added to the incubation medium. It was found that the addition of ATP to symbiosomes suspended in the presence of Mg2+ and Ca2+ initiated a gradual removal of calcium from the incubation medium. This process was rapidly inhibited with addition of vanadate, but was resistant to protonophores, the Ca2+ ionophore A23187, valinomycin in the presence of potassium ions, and erythrosin B, and was greatly stimulated by nitrate anions. Qualitatively similar results were obtained with preparations of the PBMs, except that in this case the Ca2+ ionophore A23187 effectively facilitated the calcium release from the PBM vesicles afrer the uptake. The data obtained demonstrategrimary active transport of calcium across the PBM, which is most likely caused by the activity of the Mg"+ -dependent ci+ -ATPase associated with this membrane.
Key words: Lupinus luteus L., peribacteroid membrane (PBM), symbiosomes, vesicle PBM preparations, ATP-dependent ctl+ transport, Cr/+ -ATPase. Abbreviations: PBM = peribacteroid membrane; PBS = peribacteroid space; BTP = bis-tris-propane (1,3-bis( tris(hydroxymethyl}methylamino)propane); HEPES = N -2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; DCCD = N,N'-dicyclohexylcarbodiimide; FCCP = carbonyl cyanide 4-trifluoromethoxyphenylhydrazone. Introduction
Root nodules of plants of the family Leguminosae are able to reduce atmospheric dinitrogen to ammonia (Bergersen, 1982). This process takes place within the infected cells of these nodules, namely in bacteroids enveloped in a membrane of plant origin called the peribacteroid membrane (PBM). This membrane together with bacteroids forms the symbiosome that is the basic nitrogen-foong unit of the nodule. It has been established that the metabolic interaction between the plant and the bacteroids is regulated by a series of transporters and channels on the PBM and the bacteroid membrane (Udvardi and Day, 1997).
J Plant PhysioL
~L
153. pp. 610-614 (1998)
There are several lines of evidence that calcium is of particular importance in nitrogen-foong organs, and bacteroids need to be supplied with this cation in functioning nodules. Thus, it has been found that calcium is involved in the regulation of both malate and ammonium transport across the PBM. It is required for activity of the protein kinase, associated with the PBM from soybean nodules (Bassarb and Werner, 1987). The symbiosomes isolated from these nodules exhibit a marked stimulation of malate uptake as a result of phosphorylation of certain PBM-Iocated proteins (Ou Yang et al., 1991; Weaver et al., 1991). Calcium also inhibits ion movement through the ammonium channel in the PBM on the bacteroid side in soybean (Tyerman et al., 1995; White-
Calcium Uptake in Membrane Vesicles
head et al., 1995), and, as shown previously by us (Andreevet al., 1997), the transport activity of the PBM H+ -ATPase from yellow lupin root nodules. Available data indicating the high total calcium content in symbiosomes and bacteroids (unpublished results mentioned by Tyerman et al., 1995; Andreeva et al., 1995; Vincent and Humphrey, 1963) suggest that the symbiosomes may be stores of calcium and involved in calcium homeostasis in infected cells. Lastly, it is interesting to note in this regard that root hairs of soybeans, target cells for infection by Rhizobium japonicum, accumulate calcium more than 7-fold as well as iron and cobalt (more than lO-fold and 8-fold, respectively) compared to the other parts of the root system in accordance with a special large requirement of rhizobia for these elements (Werner et al., 1985; Trinick, 1982). While all of these findings argue for an important role of calcium in functioning of symbiosomes, until now, however, Ca2+ transport systems in the PBM responsible for sequestering this cation into these nitrogen-fIXing units remain unknown. Earlier, in our studies carried out on the PBM preparations from yellow lupin root nodules Ca2+, Mi+ -stimulated ATP hydrolytic activity associated with the PBM was detected (Andreev et al., 1997). However, any reliable evidence for the presence of Ca2+-translocating ATPase on this membrane was not obtained. In the present work based on the use of Ca2+ -indicator arsenazo III we have found and partly characterized for the first time the ATP-driven uptake of calcium by purified symbiosomes and by the PBM vesicles isolated from yellow lu~in root nodules. The data obtained suggest that Mg +dependent Ca2+ -pumping ATPase is responsible for this process.
Materials and Methods
Plant material and p"parations ofsymbiosom~s and of th~ PBM fraction Root nodules of Lupinus lut~ L. were produced under controlled conditions as previously described (Andreev et al., 1997). The preparation of symbiosome and the PBM fractions was done according to the methods described by Andreev et al. (1997). A bacteroid fraction was obtained by osmotic shock treatment of washed symbiosomes as was used for preparation of the PBMs (Andreev et al., 1997).
Assays Ca2+ uptake by symbiosomes and the PBM vesicles was measured by continuously monitoring differential (720-650 nm) absorption changes undergone by the metallochromic Ca2+ indicator arsenazo III (Scarpa, 1979). The reaction was carried out in an unstirred I-em light-path cuvette of a Hitachi-557 double wavdength spectrophotometer at room temperature (20-22 ·C). In most cases the assay medium (2 ml) contained 0.4 mol/l sorbitol, 20 mmol/l HEPES-BTp, pH 7.2, 1 mmol/l Na2-ATP, 27~mol/l arsenazo III and about 100-15011g of symbiosomal protein. eaCh (20-30I1mol/l) was added to the cuvette afrer obtaining the absorption baseline, and the reaction was started by addition of 3 mmol/l MgS04 or 1mmol/l NarATP-BTP (pH 7.0). The latter was added to the in-
611
cubation mixture containing 3 mmol/l MgS04 instead of ATP. Other details are described in the corresponding figure legends. Protein determination was carried out using the method of Bradford (1976) with bovine serum albumin as a standard. All experiments were repeated at least twice on three separate preparations of symbiosomes and the PBM vesicles. Ch~icals
Sorbitol was obtained from Calbiochem (USA). ATP disodium salt, BTP, HEPES, valinomycin and DCCD were provided by Sigma (USA). Arsenazo III was purchased from Serva (Germany). All other chemicals were of the highest quality available.
Results
In the first series of our experiments, we examined a capacity of symbiosomes having intact PBM in its native orientation to take up calcium ions in the presence of ATP and Mg2+ in the incubation mixture. As follows from Fig. 1 (trace 1), the addition of ATP to symbiosomes suspended in the presence of Mg2+ and Ca2+ initiates a relatively slow absorbance change of arsenazo III, suggesting a gradual removal of Ca2+ (added and contaminating) from the incubation mixture. It can be seen that the response of the Ca2+ indicator does not achieve a steady value even in about 20 min after addition of ATP, and is not revealed in the absence of ATP (traces 1 and 2) or Mi+ (traces 3 and 4) in the incubation mixture. Initial rapid upward and downward deflections of the Ca2+ level trace have been observed upon addition of ATP or Mg2+, respectivelr: These effects can reflect artificial responses of the Ca + indicator induced by ATP or Mi+ ion addition. The essential point is that these artificial responses are complete within the mixing time and do not interfere with the kinetics of Ca2+ uptake. Also, Fig. 1 shows that the MgATP-induced Ca2+ uptake by symbiosomes is rapidly stopped by addition of vanadate, a well-known inhibitor of P-type ATPases (trace 3), and does not occur when the assay medium initially contains this inhibitor (trace 2). Another important observation is that the time course of the process in question remains unaltered after addition of the protonophore FCCP (trace 1), (NH4hS04 (trace 3) or nigericin to the symbiosomes (data not shown), i.e. compounds abolishing the apH across the PBM. It should be noted that the Ca2+ ionophore A23187 and erythrosin B also had no effect on the kinetics of the process (trace 1). On the other hand, the latter was greatly accelerated upon addition to symbiosomes of nitrate anions (trace 4). Although such an action of these anions may be suggested to be a result of dissipation of the membrane potential on the PBM, a similar stimulatory effect was not practically observed by us in the presence of valinomycin and potassium ions. The latter agents did not change, in fact, the MgATPinitiated spectral response of the Ca2+-indicator (data not shown). A slight increase in the rate of the process was also observed in the presence of KSCN and DCCD (data not shown). Since bacteroids are able to contaminate isolated symbiosome preparations, we decided to examine whether they con-
612
IGOR M. AND1U!EV, PAVEL N. DUBllOVO, VALERIA V. KRYWVA, and STANlSLAV F. IZMAlWV
bact.
ca2+
,
vanadate
A2318'l
erythr . B
ATP
'-_------2 FJg_ 1: A time course of MgATP-induced absorbance change of arsenazo III in symbiosomes. A representative experiment is shown. Conditions are given in «Materials and Methods». At indicated times 20l1mollL CaCI 2, 1 mmollL Na2ATP, 3 mmol/L MgS04' 2 J1mollL FCCp, I J1mollL A23187, 511mollL erythrosin B, 100 I1mollL vanadate, 30 mmollL KN03 and 5 mmollL (NH4hS04 were added. In the case of trace 2 the assay medium was supplemented with 100 I1mollL vanadate prior to the addition of Na2ATP. A part of trace 4 associated with the nitrate effect has been corrected for dilution artefact. In the case of trace 5 bacterioids were added to the assay medium at the same protein concentration as symbiosomes.
tribute to the observed Ca2 +-accumulating ability of syrnbiosomes. As indicated in Fig. 1 (trace 5), bacteroids added to the assay mixture at the same protein concentration as syrnbiosomes resulted in a spectral response of arsenazo III that is consistent with continuous release of calcium ions in the assay mixture. ATP only slighdy influenced the kinetics of this process, but the latter was to some extent impeded by subsequent addition of Although an origin of these absorption changes in unclear, they likely cannot account for the MgATP-initiated effect observed in the case of syrnbiosomes (traces 1, 3, 4). Most of the above results regarding the detected MgATPdependent Ca2+ -translocating capacity of the PBM are con-
Mi+.
firmed by the data obtained in the experiments with isolated PBMs. These experiments were carried out under the same conditions as in the case of syrnbiosomes, and the ci+indicator was added again into the outer assay medium. All of the tested characteristics of the process in question, as revealed here, were qualitatively similar to those established for syrnbiosomes (data not shown) except that the Ca2 + ionophore A23187 reversed almost completely the Mg ATPinitiated spectral response of arsenazo III, and its change with time, as expected, exhibited more profound saturable behaviour. This is demonstrated in Fig. 2, where only some characteristics of the Mg ATP-induced absorption signal of the Ca2+ -indicator in the PBM vesicles are presented. These
613
Calcium Uptake in Membrane Vesicles A2318'l
vanadate
+
'--2
-t__~?-----~..vesicles 1112+
+
valinomycin ~720-650
= 0.005
1
2 ain
t---+I FIg. 2: MgATP-energized calcium uptake by the PBM vesicles as a function of time. A representative experiment is shown. The PBM vesicles were loaded with 50 mmol/L K2S04 and placed into the same medium as used for symbiosomes and bacteroids. This medium was supplemented with 1 mmol/L Na2ATP and 30 ~mol/L CaCI2, and in the case of trace 2, additionally, with 30 mmol/L BTP-N03' At indicated times 3 mmol/L MgS04' 2 ~mol/L valinomycin, 30 mmol/L BTP-N03 (adjusted to pH 7.0), 1 ~mol/L A23187 and 100 ~mol/L vanadate were added. A part of trace 1 associated with the nitrate effect has been corrected for dilution artefact.
characteristics indicate that such a response is due to accumulation of Ca2+ inside the vesicles. All of the experiments described above were performed at pH 7.2, i.e. in the pH range where the activity of some known ci+ -ATPases in plant cell membranes achieves maximal values (Evans et al., 1991). Is this also true for the MgATP-induced ci+ -translocating activity detected on the PBM? We attempted to answer this question by investigating the effect of pH of the assay medium on the Ca2+ uptake by the PBM vesicles. According to our preliminary data this process is to a great extent inhibited at pH 8.0 and is completely blocked at pH 5.5 where the transport activity of yellow lupin PBM H+ -pumping ATPase achieves its maximal value (Andreev et al., 1997). Therefore, it is to be expected that the pH range selected for studies in the present work provides conditions favorable for a high Ca2 + uptake activity of symbiosomes and the PBM vesicles.
Discussion
As considered above, calcium is an essential ion for symbiotic nitrogen ftxation occurring inside bacteroids of infected nodule cells. The only way for the bacteroid in the symbiosome to be supplied with calcium is to take it up from the host cell cytosol. To our knowledge, this is the ftrst repon characterizing the transport of calcium through the PBM. The results presented here clearly show that Ca2+ is actively absorbed by symbiosomes and the PBM vesicles. This uptake process appears to require ATP and Mi+, it is sensitive to vanadate, but insensitive to agents that collapse ~pH and/or ~'" on the PBM. These features strongly suggest a direct coupling of the Ca2+ transport to ATP hydrolysis rather than secondary transport, such as, for example, ~pH mediated. Taken to~ether, the results obtained are consistent with a primary Ca +-translocating ATPase that mediates Ca2+ transpon from the host cell cytosol to the PBS.
The effect of vanadate suggests that this enzyme belongs to P-type ATPases. As noted in «Results», there are some reasons to believe that the pH optimum for the activity of the Ca2 +transport system revealed here differs signiftcandy from that of the H+ -ATPase associated with the same PBM, and is likely in neutral pH range. According to our results the enzyme in question transports Ca2 + ions electroneutrally across the PBM. In the light of these ftndings a great stimulatory effect of nitrate anions on the kinetics of this process is unexpected, and the mechanism of such an action of nitrate is unclear. In this connection, it is of interest to note that a similar stimulatory effect of nitrate was also observed in the case of Ca2 +-ATPase of endoplasmic reticulum from zucchini hypocotyls where Ca2 + transpon was shown to be not electrogenic as well (Lew et al., 1986). It is evident that the mechanism underlying electro neutral transmembrane transfer of Ca2+ by the detected Ca2+ translocating system in the PBM requires funher studies. In the fresent work we failed to detect the Ca2+ efflux from Ca2 -loaded symbiosomes afrer the addition of the Ca2+ ionophore A23187 in the incubation mixture (Fig. 1, trace 1). This ionophore, however, effectively facilitated such a process in the case of the PBM vesicles (Fig. 2, trace 1). These observations suggest that calcium is not stored in the PBS but immediately absorbed by the microsymbiont. Although other possible explanations for these results cannot be ruled out, such a conclusion is consistent with the ftndings (Tyerman et al., 1995) that most of the calcium in the symbiosomes is likely to be bound with bacteroids. Therefore, in accordance with the data presented by Tyerman et al. (1995) there is some reason to expect that free calcium concentration in the PBS is relatively low and is tighdy regulated. The fact that no Ca2+ uptake activity was detected by us in isolated bacteroids, at least in the absence of ATP and Mi+ in the incubation mixture (Fig. 1, trace 5), does not contradict with such a possibility because their native environment in situ, i.e. inside symbiosomes, may be signiftcandy different from that in our assay medium.
614
IGOR M. ANOREEV, PAVEL N. DUBROVO, VALERIA V. KRYLOVA, and STANISLAV F. IZMAIWV
The revealed capacity of symbiosomes to take up calcium ions indicates that these nitrogen-fIXing units may be involved in calcium homeostasis in infected nodule cells. In this respect symbiosomes may functionally replace vacuoles in such cells in accordance with the idea offered by Mellor (1989). As already noted above, some literature data also argue in favour of this suggestion. In conclusion, our results throw new light upon the transport phenomena on the PBM and may appear to be essential for our understanding of their participation in the control of symbiotic nitrogen fixation in the root nodules of leguminous plants. Further studies will be required for detailed characterization of the identified Ca2 +-transport system in the PBM, and these are currendy in progress. Acknowledgements
We thank Dr. G. Ya. Zhiznevskaya from our Institute for critically reading the manuscript. This work was supported by a grant from the Russian Foundation for Fundamental Research. References ANOREl!V, 1., P. DU1)ROVO, V. KRYLOVA, 1. N. ANOREEVA, V. KoREN'KOV, E. M. SOROKlN, and S. F. IZMAIwv: Characterization of ATP-hydrolyzing and ATP-driven proton-translocating activities associated with the peribacteroid membrane from root nodules of Lupinus luteus L. J. Plant Physiol. 151,563-569 (1997). ANOREEVA, 1. N., G. M. KOZHARlNOVA, and S. F. IZMAIwv: Calcium compartmentation in root nodules of leguminous plants: electron microscope investigation. Dokl. Akad. Nauk 344, 402406 (1995). BASSARB, S. and D. WERNER: Ca2+ -dependent protein kinase activity in the peribacteroid membranes from soybean nodules. J. Plant Physiol. 130,233-241 (1987). BERGERSEN, F. J.: In: Root Nodules: Structure and Function, Research Studies Press, Chichester, pp. 23-50 (1982).
BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248-254 (1976). EVANS, D. E., S.-A. BRIARS, and L. E. WILLIAMS: Active calcium transport by plant cell membranes. J. Exp. Bot. 236. 285-303 (1991). LEW, R. R., D. P. BRISKIN, and R. E. WYSE: Ca2+ uptake byendoplasmic reticulum from zucchini hypocotyls. The use of chlorotetracycline as a probe for Ca2 + uptake. Plant Physiol. 82,47-53 (1986). MELWR, R. B.: Bacteroids in the Rhizobium-legume symbiosis inhabit a plant internal lytic compartment: implications for other microbial endosymbioses. J. Exp. Bot. 40, 831-839 (1989). Ou YANG, L.-J., J. WHELAN, C. D. WEAVER, D. M. ROBERTS, and D. A. DAY: Protein phosphorylation stimulates the rate of malate uptake across the peri bacteroid membrane of soybean nodules. FEBS Lett. 293, 188-190 (1991). SCARPA, A.: Measurement of cation transport with metallochromic indicators. Methods Enzymol. 56. 301-337 (1979). TRiNICK, M. J.: Nitrogen Fixation. In: BROUGHTON, W. J. (ed.): Biology, Vol. 2. Clarendon Press, Oxford, p. 76-146 (1982). TYERMAN, S. D., L. F. WHITEHEAD, and D. A. DAY: A channel-like transporter for NH4 + on the symbiotic interface of NrHxing plants. Nature 378, 629-632 (1995). UOVARDI, M. K. and D. A. DAY: Metabolite transport across symbiotic membranes of legume nodules. Annu. Rev. Plant Physiol. Plant Mol. BioI. 48,493-523 (1997). VINCENT, J. M. and B. A. HUMPHREY: Partition of divalent cations between bacterial wall and cell content. Narure 199, 149-151 (1963). . WEAVER, c. D., B. CROMBIE, G. STACEY, and D. M. ROBERTS: Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fIxing soybean nodules. Plant Physiol. 95, 222-227 (1991). WERNER, D., K. P. KUHLMANN, F. GWYSTEIN, and F. W. RiCHTER: Calcium, iron and cobalt accumulation in root hairs of soybean. Z. Naturforschung 40c, 912-913 (1985). WHITEHEAD, L. F., S. D. TYERMAN, C. L. SAWM, and D. A. DAY: Transport of fixed nitrogen across symbiotic membranes of legume nodules. Symbiosis 19, 141-154 (1995).
© 1998 by Gustav Fischer Verlag, lena
Calcium Uptake by Symbiosomes and the Peribacteroid Membrane Vesicles Isolated from Yellow Lupin Root Nodules IGOR
M.
ANDREEV, PAVEL
N.
DUBROVO, VALERIA
V.
KRYLOVA,
and
STANISLAV
F.
IZMAILOV
Ttmiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia Received November 5, 1997 . Accepted March 25, 1998
Summary
In order to test whether the symbiosomes of infected cells are able to actively take up calcium ions, preparations of these nitrogen-foong units and the PBM vesicles isolated from yellow lupin (Lupinus luteus L.) root nodules were investigated to this end. Ca2+ uptake was recorded with the use of the metallochromic Ca2+ indicator arsenazo III added to the incubation medium. It was found that the addition of ATP to symbiosomes suspended in the presence of Mg2+ and Ca2+ initiated a gradual removal of calcium from the incubation medium. This process was rapidly inhibited with addition of vanadate, but was resistant to protonophores, the Ca2+ ionophore A23187, valinomycin in the presence of potassium ions, and erythrosin B, and was greatly stimulated by nitrate anions. Qualitatively similar results were obtained with preparations of the PBMs, except that in this case the Ca2+ ionophore A23187 effectively facilitated the calcium release from the PBM vesicles afrer the uptake. The data obtained demonstrategrimary active transport of calcium across the PBM, which is most likely caused by the activity of the Mg"+ -dependent ci+ -ATPase associated with this membrane.
Key words: Lupinus luteus L., peribacteroid membrane (PBM), symbiosomes, vesicle PBM preparations, ATP-dependent ctl+ transport, Cr/+ -ATPase. Abbreviations: PBM = peribacteroid membrane; PBS = peribacteroid space; BTP = bis-tris-propane (1,3-bis( tris(hydroxymethyl}methylamino)propane); HEPES = N -2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; DCCD = N,N'-dicyclohexylcarbodiimide; FCCP = carbonyl cyanide 4-trifluoromethoxyphenylhydrazone. Introduction
Root nodules of plants of the family Leguminosae are able to reduce atmospheric dinitrogen to ammonia (Bergersen, 1982). This process takes place within the infected cells of these nodules, namely in bacteroids enveloped in a membrane of plant origin called the peribacteroid membrane (PBM). This membrane together with bacteroids forms the symbiosome that is the basic nitrogen-foong unit of the nodule. It has been established that the metabolic interaction between the plant and the bacteroids is regulated by a series of transporters and channels on the PBM and the bacteroid membrane (Udvardi and Day, 1997).
J Plant PhysioL
~L
153. pp. 610-614 (1998)
There are several lines of evidence that calcium is of particular importance in nitrogen-foong organs, and bacteroids need to be supplied with this cation in functioning nodules. Thus, it has been found that calcium is involved in the regulation of both malate and ammonium transport across the PBM. It is required for activity of the protein kinase, associated with the PBM from soybean nodules (Bassarb and Werner, 1987). The symbiosomes isolated from these nodules exhibit a marked stimulation of malate uptake as a result of phosphorylation of certain PBM-Iocated proteins (Ou Yang et al., 1991; Weaver et al., 1991). Calcium also inhibits ion movement through the ammonium channel in the PBM on the bacteroid side in soybean (Tyerman et al., 1995; White-
Calcium Uptake in Membrane Vesicles
head et al., 1995), and, as shown previously by us (Andreevet al., 1997), the transport activity of the PBM H+ -ATPase from yellow lupin root nodules. Available data indicating the high total calcium content in symbiosomes and bacteroids (unpublished results mentioned by Tyerman et al., 1995; Andreeva et al., 1995; Vincent and Humphrey, 1963) suggest that the symbiosomes may be stores of calcium and involved in calcium homeostasis in infected cells. Lastly, it is interesting to note in this regard that root hairs of soybeans, target cells for infection by Rhizobium japonicum, accumulate calcium more than 7-fold as well as iron and cobalt (more than lO-fold and 8-fold, respectively) compared to the other parts of the root system in accordance with a special large requirement of rhizobia for these elements (Werner et al., 1985; Trinick, 1982). While all of these findings argue for an important role of calcium in functioning of symbiosomes, until now, however, Ca2+ transport systems in the PBM responsible for sequestering this cation into these nitrogen-fIXing units remain unknown. Earlier, in our studies carried out on the PBM preparations from yellow lupin root nodules Ca2+, Mi+ -stimulated ATP hydrolytic activity associated with the PBM was detected (Andreev et al., 1997). However, any reliable evidence for the presence of Ca2+-translocating ATPase on this membrane was not obtained. In the present work based on the use of Ca2+ -indicator arsenazo III we have found and partly characterized for the first time the ATP-driven uptake of calcium by purified symbiosomes and by the PBM vesicles isolated from yellow lu~in root nodules. The data obtained suggest that Mg +dependent Ca2+ -pumping ATPase is responsible for this process.
Materials and Methods
Plant material and p"parations ofsymbiosom~s and of th~ PBM fraction Root nodules of Lupinus lut~ L. were produced under controlled conditions as previously described (Andreev et al., 1997). The preparation of symbiosome and the PBM fractions was done according to the methods described by Andreev et al. (1997). A bacteroid fraction was obtained by osmotic shock treatment of washed symbiosomes as was used for preparation of the PBMs (Andreev et al., 1997).
Assays Ca2+ uptake by symbiosomes and the PBM vesicles was measured by continuously monitoring differential (720-650 nm) absorption changes undergone by the metallochromic Ca2+ indicator arsenazo III (Scarpa, 1979). The reaction was carried out in an unstirred I-em light-path cuvette of a Hitachi-557 double wavdength spectrophotometer at room temperature (20-22 ·C). In most cases the assay medium (2 ml) contained 0.4 mol/l sorbitol, 20 mmol/l HEPES-BTp, pH 7.2, 1 mmol/l Na2-ATP, 27~mol/l arsenazo III and about 100-15011g of symbiosomal protein. eaCh (20-30I1mol/l) was added to the cuvette afrer obtaining the absorption baseline, and the reaction was started by addition of 3 mmol/l MgS04 or 1mmol/l NarATP-BTP (pH 7.0). The latter was added to the in-
611
cubation mixture containing 3 mmol/l MgS04 instead of ATP. Other details are described in the corresponding figure legends. Protein determination was carried out using the method of Bradford (1976) with bovine serum albumin as a standard. All experiments were repeated at least twice on three separate preparations of symbiosomes and the PBM vesicles. Ch~icals
Sorbitol was obtained from Calbiochem (USA). ATP disodium salt, BTP, HEPES, valinomycin and DCCD were provided by Sigma (USA). Arsenazo III was purchased from Serva (Germany). All other chemicals were of the highest quality available.
Results
In the first series of our experiments, we examined a capacity of symbiosomes having intact PBM in its native orientation to take up calcium ions in the presence of ATP and Mg2+ in the incubation mixture. As follows from Fig. 1 (trace 1), the addition of ATP to symbiosomes suspended in the presence of Mg2+ and Ca2+ initiates a relatively slow absorbance change of arsenazo III, suggesting a gradual removal of Ca2+ (added and contaminating) from the incubation mixture. It can be seen that the response of the Ca2+ indicator does not achieve a steady value even in about 20 min after addition of ATP, and is not revealed in the absence of ATP (traces 1 and 2) or Mi+ (traces 3 and 4) in the incubation mixture. Initial rapid upward and downward deflections of the Ca2+ level trace have been observed upon addition of ATP or Mg2+, respectivelr: These effects can reflect artificial responses of the Ca + indicator induced by ATP or Mi+ ion addition. The essential point is that these artificial responses are complete within the mixing time and do not interfere with the kinetics of Ca2+ uptake. Also, Fig. 1 shows that the MgATP-induced Ca2+ uptake by symbiosomes is rapidly stopped by addition of vanadate, a well-known inhibitor of P-type ATPases (trace 3), and does not occur when the assay medium initially contains this inhibitor (trace 2). Another important observation is that the time course of the process in question remains unaltered after addition of the protonophore FCCP (trace 1), (NH4hS04 (trace 3) or nigericin to the symbiosomes (data not shown), i.e. compounds abolishing the apH across the PBM. It should be noted that the Ca2+ ionophore A23187 and erythrosin B also had no effect on the kinetics of the process (trace 1). On the other hand, the latter was greatly accelerated upon addition to symbiosomes of nitrate anions (trace 4). Although such an action of these anions may be suggested to be a result of dissipation of the membrane potential on the PBM, a similar stimulatory effect was not practically observed by us in the presence of valinomycin and potassium ions. The latter agents did not change, in fact, the MgATPinitiated spectral response of the Ca2+-indicator (data not shown). A slight increase in the rate of the process was also observed in the presence of KSCN and DCCD (data not shown). Since bacteroids are able to contaminate isolated symbiosome preparations, we decided to examine whether they con-
612
IGOR M. AND1U!EV, PAVEL N. DUBllOVO, VALERIA V. KRYWVA, and STANlSLAV F. IZMAlWV
bact.
ca2+
,
vanadate
A2318'l
erythr . B
ATP
'-_------2 FJg_ 1: A time course of MgATP-induced absorbance change of arsenazo III in symbiosomes. A representative experiment is shown. Conditions are given in «Materials and Methods». At indicated times 20l1mollL CaCI 2, 1 mmollL Na2ATP, 3 mmol/L MgS04' 2 J1mollL FCCp, I J1mollL A23187, 511mollL erythrosin B, 100 I1mollL vanadate, 30 mmollL KN03 and 5 mmollL (NH4hS04 were added. In the case of trace 2 the assay medium was supplemented with 100 I1mollL vanadate prior to the addition of Na2ATP. A part of trace 4 associated with the nitrate effect has been corrected for dilution artefact. In the case of trace 5 bacterioids were added to the assay medium at the same protein concentration as symbiosomes.
tribute to the observed Ca2 +-accumulating ability of syrnbiosomes. As indicated in Fig. 1 (trace 5), bacteroids added to the assay mixture at the same protein concentration as syrnbiosomes resulted in a spectral response of arsenazo III that is consistent with continuous release of calcium ions in the assay mixture. ATP only slighdy influenced the kinetics of this process, but the latter was to some extent impeded by subsequent addition of Although an origin of these absorption changes in unclear, they likely cannot account for the MgATP-initiated effect observed in the case of syrnbiosomes (traces 1, 3, 4). Most of the above results regarding the detected MgATPdependent Ca2+ -translocating capacity of the PBM are con-
Mi+.
firmed by the data obtained in the experiments with isolated PBMs. These experiments were carried out under the same conditions as in the case of syrnbiosomes, and the ci+indicator was added again into the outer assay medium. All of the tested characteristics of the process in question, as revealed here, were qualitatively similar to those established for syrnbiosomes (data not shown) except that the Ca2 + ionophore A23187 reversed almost completely the Mg ATPinitiated spectral response of arsenazo III, and its change with time, as expected, exhibited more profound saturable behaviour. This is demonstrated in Fig. 2, where only some characteristics of the Mg ATP-induced absorption signal of the Ca2+ -indicator in the PBM vesicles are presented. These
613
Calcium Uptake in Membrane Vesicles A2318'l
vanadate
+
'--2
-t__~?-----~..vesicles 1112+
+
valinomycin ~720-650
= 0.005
1
2 ain
t---+I FIg. 2: MgATP-energized calcium uptake by the PBM vesicles as a function of time. A representative experiment is shown. The PBM vesicles were loaded with 50 mmol/L K2S04 and placed into the same medium as used for symbiosomes and bacteroids. This medium was supplemented with 1 mmol/L Na2ATP and 30 ~mol/L CaCI2, and in the case of trace 2, additionally, with 30 mmol/L BTP-N03' At indicated times 3 mmol/L MgS04' 2 ~mol/L valinomycin, 30 mmol/L BTP-N03 (adjusted to pH 7.0), 1 ~mol/L A23187 and 100 ~mol/L vanadate were added. A part of trace 1 associated with the nitrate effect has been corrected for dilution artefact.
characteristics indicate that such a response is due to accumulation of Ca2+ inside the vesicles. All of the experiments described above were performed at pH 7.2, i.e. in the pH range where the activity of some known ci+ -ATPases in plant cell membranes achieves maximal values (Evans et al., 1991). Is this also true for the MgATP-induced ci+ -translocating activity detected on the PBM? We attempted to answer this question by investigating the effect of pH of the assay medium on the Ca2+ uptake by the PBM vesicles. According to our preliminary data this process is to a great extent inhibited at pH 8.0 and is completely blocked at pH 5.5 where the transport activity of yellow lupin PBM H+ -pumping ATPase achieves its maximal value (Andreev et al., 1997). Therefore, it is to be expected that the pH range selected for studies in the present work provides conditions favorable for a high Ca2 + uptake activity of symbiosomes and the PBM vesicles.
Discussion
As considered above, calcium is an essential ion for symbiotic nitrogen ftxation occurring inside bacteroids of infected nodule cells. The only way for the bacteroid in the symbiosome to be supplied with calcium is to take it up from the host cell cytosol. To our knowledge, this is the ftrst repon characterizing the transport of calcium through the PBM. The results presented here clearly show that Ca2+ is actively absorbed by symbiosomes and the PBM vesicles. This uptake process appears to require ATP and Mi+, it is sensitive to vanadate, but insensitive to agents that collapse ~pH and/or ~'" on the PBM. These features strongly suggest a direct coupling of the Ca2+ transport to ATP hydrolysis rather than secondary transport, such as, for example, ~pH mediated. Taken to~ether, the results obtained are consistent with a primary Ca +-translocating ATPase that mediates Ca2+ transpon from the host cell cytosol to the PBS.
The effect of vanadate suggests that this enzyme belongs to P-type ATPases. As noted in «Results», there are some reasons to believe that the pH optimum for the activity of the Ca2 +transport system revealed here differs signiftcandy from that of the H+ -ATPase associated with the same PBM, and is likely in neutral pH range. According to our results the enzyme in question transports Ca2 + ions electroneutrally across the PBM. In the light of these ftndings a great stimulatory effect of nitrate anions on the kinetics of this process is unexpected, and the mechanism of such an action of nitrate is unclear. In this connection, it is of interest to note that a similar stimulatory effect of nitrate was also observed in the case of Ca2 +-ATPase of endoplasmic reticulum from zucchini hypocotyls where Ca2 + transpon was shown to be not electrogenic as well (Lew et al., 1986). It is evident that the mechanism underlying electro neutral transmembrane transfer of Ca2+ by the detected Ca2+ translocating system in the PBM requires funher studies. In the fresent work we failed to detect the Ca2+ efflux from Ca2 -loaded symbiosomes afrer the addition of the Ca2+ ionophore A23187 in the incubation mixture (Fig. 1, trace 1). This ionophore, however, effectively facilitated such a process in the case of the PBM vesicles (Fig. 2, trace 1). These observations suggest that calcium is not stored in the PBS but immediately absorbed by the microsymbiont. Although other possible explanations for these results cannot be ruled out, such a conclusion is consistent with the ftndings (Tyerman et al., 1995) that most of the calcium in the symbiosomes is likely to be bound with bacteroids. Therefore, in accordance with the data presented by Tyerman et al. (1995) there is some reason to expect that free calcium concentration in the PBS is relatively low and is tighdy regulated. The fact that no Ca2+ uptake activity was detected by us in isolated bacteroids, at least in the absence of ATP and Mi+ in the incubation mixture (Fig. 1, trace 5), does not contradict with such a possibility because their native environment in situ, i.e. inside symbiosomes, may be signiftcandy different from that in our assay medium.
614
IGOR M. ANOREEV, PAVEL N. DUBROVO, VALERIA V. KRYLOVA, and STANISLAV F. IZMAIWV
The revealed capacity of symbiosomes to take up calcium ions indicates that these nitrogen-fIXing units may be involved in calcium homeostasis in infected nodule cells. In this respect symbiosomes may functionally replace vacuoles in such cells in accordance with the idea offered by Mellor (1989). As already noted above, some literature data also argue in favour of this suggestion. In conclusion, our results throw new light upon the transport phenomena on the PBM and may appear to be essential for our understanding of their participation in the control of symbiotic nitrogen fixation in the root nodules of leguminous plants. Further studies will be required for detailed characterization of the identified Ca2 +-transport system in the PBM, and these are currendy in progress. Acknowledgements
We thank Dr. G. Ya. Zhiznevskaya from our Institute for critically reading the manuscript. This work was supported by a grant from the Russian Foundation for Fundamental Research. References ANOREl!V, 1., P. DU1)ROVO, V. KRYLOVA, 1. N. ANOREEVA, V. KoREN'KOV, E. M. SOROKlN, and S. F. IZMAIwv: Characterization of ATP-hydrolyzing and ATP-driven proton-translocating activities associated with the peribacteroid membrane from root nodules of Lupinus luteus L. J. Plant Physiol. 151,563-569 (1997). ANOREEVA, 1. N., G. M. KOZHARlNOVA, and S. F. IZMAIwv: Calcium compartmentation in root nodules of leguminous plants: electron microscope investigation. Dokl. Akad. Nauk 344, 402406 (1995). BASSARB, S. and D. WERNER: Ca2+ -dependent protein kinase activity in the peribacteroid membranes from soybean nodules. J. Plant Physiol. 130,233-241 (1987). BERGERSEN, F. J.: In: Root Nodules: Structure and Function, Research Studies Press, Chichester, pp. 23-50 (1982).
BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248-254 (1976). EVANS, D. E., S.-A. BRIARS, and L. E. WILLIAMS: Active calcium transport by plant cell membranes. J. Exp. Bot. 236. 285-303 (1991). LEW, R. R., D. P. BRISKIN, and R. E. WYSE: Ca2+ uptake byendoplasmic reticulum from zucchini hypocotyls. The use of chlorotetracycline as a probe for Ca2 + uptake. Plant Physiol. 82,47-53 (1986). MELWR, R. B.: Bacteroids in the Rhizobium-legume symbiosis inhabit a plant internal lytic compartment: implications for other microbial endosymbioses. J. Exp. Bot. 40, 831-839 (1989). Ou YANG, L.-J., J. WHELAN, C. D. WEAVER, D. M. ROBERTS, and D. A. DAY: Protein phosphorylation stimulates the rate of malate uptake across the peri bacteroid membrane of soybean nodules. FEBS Lett. 293, 188-190 (1991). SCARPA, A.: Measurement of cation transport with metallochromic indicators. Methods Enzymol. 56. 301-337 (1979). TRiNICK, M. J.: Nitrogen Fixation. In: BROUGHTON, W. J. (ed.): Biology, Vol. 2. Clarendon Press, Oxford, p. 76-146 (1982). TYERMAN, S. D., L. F. WHITEHEAD, and D. A. DAY: A channel-like transporter for NH4 + on the symbiotic interface of NrHxing plants. Nature 378, 629-632 (1995). UOVARDI, M. K. and D. A. DAY: Metabolite transport across symbiotic membranes of legume nodules. Annu. Rev. Plant Physiol. Plant Mol. BioI. 48,493-523 (1997). VINCENT, J. M. and B. A. HUMPHREY: Partition of divalent cations between bacterial wall and cell content. Narure 199, 149-151 (1963). . WEAVER, c. D., B. CROMBIE, G. STACEY, and D. M. ROBERTS: Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fIxing soybean nodules. Plant Physiol. 95, 222-227 (1991). WERNER, D., K. P. KUHLMANN, F. GWYSTEIN, and F. W. RiCHTER: Calcium, iron and cobalt accumulation in root hairs of soybean. Z. Naturforschung 40c, 912-913 (1985). WHITEHEAD, L. F., S. D. TYERMAN, C. L. SAWM, and D. A. DAY: Transport of fixed nitrogen across symbiotic membranes of legume nodules. Symbiosis 19, 141-154 (1995).