Passive Entry of CO2 and Its Energy-dependent Intracellular Conversion to HCO 3 − in Cyanobacteria Are Driven by a Photosystem I-generated ΔμH+
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m101973200
ISSN1083-351X
AutoresDan Tchernov, Yael Helman, Nir Keren, Boaz Luz, Itzhak Ohad, Leonora Reinhold, Teruo Ogawa, Aaron Kaplan,
Tópico(s)Spectroscopy and Quantum Chemical Studies
ResumoCO2 entry intoSynechococcus sp. PCC7942 cells was drastically inhibited by the water channel blocker p-chloromercuriphenylsulfonic acid suggesting that CO2 uptake is, for the most part, passive via aquaporins with subsequent energy-dependent conversion to HCO3− . Dependence of CO2uptake on photosynthetic electron transport via photosystem I (PSI) was confirmed by experiments with electron transport inhibitors, electron donors and acceptors, and a mutant lacking PSI activity. CO2 uptake was drastically inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and ammonia but substantially less so by the inhibitors of ATP formation arsenate and N, N,-dicyclohexylcarbodiimide (DCCD). Thus a ΔμH+ generated by photosynthetic PSI electron transport apparently serves as the direct source of energy for CO2 uptake. Under low light intensity, the rate of CO2 uptake by a high-CO2-requiring mutant ofSynechococcus sp. PCC7942, at a CO2concentration below its threshold for CO2 fixation, was higher than that of the wild type. At saturating light intensity, net CO2 uptake was similar in the wild type and in the mutant IL-3 suggesting common limitation by the rate of conversion of CO2 to HCO3− . These findings are consistent with a model postulating that electron transport-dependent formation of alkaline domains on the thylakoid membrane energizes intracellular conversion of CO2 to HCO3− . CO2 entry intoSynechococcus sp. PCC7942 cells was drastically inhibited by the water channel blocker p-chloromercuriphenylsulfonic acid suggesting that CO2 uptake is, for the most part, passive via aquaporins with subsequent energy-dependent conversion to HCO3− . Dependence of CO2uptake on photosynthetic electron transport via photosystem I (PSI) was confirmed by experiments with electron transport inhibitors, electron donors and acceptors, and a mutant lacking PSI activity. CO2 uptake was drastically inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and ammonia but substantially less so by the inhibitors of ATP formation arsenate and N, N,-dicyclohexylcarbodiimide (DCCD). Thus a ΔμH+ generated by photosynthetic PSI electron transport apparently serves as the direct source of energy for CO2 uptake. Under low light intensity, the rate of CO2 uptake by a high-CO2-requiring mutant ofSynechococcus sp. PCC7942, at a CO2concentration below its threshold for CO2 fixation, was higher than that of the wild type. At saturating light intensity, net CO2 uptake was similar in the wild type and in the mutant IL-3 suggesting common limitation by the rate of conversion of CO2 to HCO3− . These findings are consistent with a model postulating that electron transport-dependent formation of alkaline domains on the thylakoid membrane energizes intracellular conversion of CO2 to HCO3− . carbonic anhydrase water channel blocker photosystem I photosystem II electron transport chlorophyll iodoacetamide 3- (3,4-dichlorophenyl)-1,1-dimethylurea 2,5-dimethyl-p-benzoquinone methyl viologen carbonyl cyanide m-chlorophenylhydrazone On illumination, many photosynthetic microorganisms maintain the concentration of dissolved CO2 ([CO2(dis)]) in their surrounding medium below that expected at chemical equilibrium with HCO3− (1Tu C.K. Spiller H. Wynns G.C. Silverman D.N. Plant Physiol. 1987; 85: 72-77Crossref PubMed Google Scholar, 2Badger M.R. Palmqvist K. Yu J.W. Physiol. Plant. 1994; 90: 529-536Crossref Scopus (122) Google Scholar, 3Miller A.G. Espie G.S. Canvin D.T. Plant Physiol. 1988; 86: 677-683Crossref PubMed Google Scholar, 4Brechignac F. Andre M. Plant Physiol. 1985; 78: 551-554Crossref PubMed Scopus (14) Google Scholar, 5Espie G.-S. Miller A.-G. Canvin D.-T. Plant Physiol. 1991; 97: 943-953Crossref PubMed Scopus (30) Google Scholar). This displacement of [CO2(dis)] from equilibrium can be observed in the absence of CO2 fixation and is largely due to CO2 uptake, intracellular conversion to HCO3−, and release of the latter into the medium (5Espie G.-S. Miller A.-G. Canvin D.-T. Plant Physiol. 1991; 97: 943-953Crossref PubMed Scopus (30) Google Scholar, 6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar). The reverse phenomenon has been described inSynechococcus WH 7803 (7Tchernov D. Hassidim M. Luz B. Sukenik A. Reinhold L. Kaplan A. Curr. Biol. 1997; 7: 723-728Abstract Full Text Full Text PDF PubMed Google Scholar) andNannochloropsis sp. (8Sukenik A. Tchernov D. Huerta E. Lubian L.M. Kaplan A. Livne A. J. Phycol. 1997; 33: 969-974Crossref Scopus (53) Google Scholar, 9Huertas I.M. Colman B. Espie G.S. Lubian L.M. J. Phycol. 2000; 36: 314-320Crossref Scopus (66) Google Scholar) where HCO3− uptake, internal conversion to CO2, and efflux of the latter result in elevated [CO2(dis)] in the medium. HCO3− transport systems, in Cyanobacteria, are probably located at the cytoplasmic membrane and are believed to be driven by ATP either directly (10Omata O. Price D.G. Badger M.R. Okamura M. Gohta S. Ogawa T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13571-13576Crossref PubMed Scopus (198) Google Scholar) or possibly indirectly (11Reinhold L. Volokita M. Zenvirth D. Kaplan A. Plant Physiol. 1984; 76: 1090-1092Crossref PubMed Scopus (32) Google Scholar, 12Bonfil D.J. Ronen-Tarazi M. Sultemeyer D. Lieman-Hurwitz J. Schatz D. Kaplan A. FEBS Lett. 1998; 430: 236-240Crossref PubMed Scopus (56) Google Scholar, 13So A.K.C. Kassam A. Espie G.S. Can. J. Bot. 1998; 76: 1084-1091Google Scholar). CO2 uptake has been observed to result in HCO3− accumulation in the cytoplasm where [CO2(dis)] is maintained below that expected at chemical equilibrium, and it has been inferred that a CA1-like activity is involved in its uptake and intracellular conversion to HCO3− (6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar, 14Volokita M. Zenvirth D. Kaplan A. Reinhold L. Plant Physiol. 1984; 76: 599-602Crossref PubMed Scopus (111) Google Scholar, 15Abe T. Tsuzuki M. Miyachi S. Plant Cell Physiol. 1987; 28: 671-677Crossref Scopus (16) Google Scholar, 16Price G.D. Badger M.R. Plant Physiol. 1989; 91: 505-513Crossref PubMed Google Scholar, 17Price G.D. Sültemeyer D. Klughammer B. Ludwig M. Badger M.R. Can. J. Bot. 1998; 76: 973-1002Google Scholar). The location of the CA-like activity has not been identified and the mode of energization of the active HCO3− accumulation is not understood. Active transport of CO2 across the plasmalemma has also been suggested (5Espie G.-S. Miller A.-G. Canvin D.-T. Plant Physiol. 1991; 97: 943-953Crossref PubMed Scopus (30) Google Scholar,18Miller A.G. Espie G.E. Canvin D.T. Can. J. Bot. 1991; 69: 925-935Crossref Google Scholar), but it is difficult to distinguish this from diffusion of CO2 across the plasma membrane with subsequent energy-dependent conversion to HCO3− (6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar,19Fridlyand L. Kaplan A. Reinhold L. Biosystems. 1996; 37: 229-238Crossref PubMed Scopus (67) Google Scholar). Passive entry of CO2 across the membrane may occur via aquaporins (20Tyerman S.D. Bohnert H.J. Maurel C. Steudle E. Smith J.A.C. J. Exp. Bot. 1999; 50: 1055-1071Google Scholar), a possibility examined here by the application of a water channel blocker (WCB), p-chloromercuriphenylsulfonic acid. Use of this WCB prevented changes in cell volume and inactivation of PSI and PSII following osmotic stress in Synechococcussp. strain PCC7942 (21Allakhverdiev S.I. Sakamoto A. Nishiyama Y. Murata N. Plant Physiol. 2000; 122: 1201-1208Crossref PubMed Scopus (139) Google Scholar). Until recently, it was widely accepted that cyclic PSI activity plays the major role in energization of CO2 uptake in Cyanobacteria (22Ogawa T. Miyano A. Inoue Y. Biochim. Biophys. Acta. 1985; 808: 77-84Crossref Scopus (83) Google Scholar, 23Li Q.L. Canvin D.T. Plant Physiol. 1998; 116: 1125-1132Crossref PubMed Scopus (40) Google Scholar). Some recent observations appear to conflict with this conclusion. In the ΔndhD1/D2 mutant ofSynechocystis sp. strain PCC6803 lacking components of NAD(P)H dehydrogenase (NDH-1), oxidation of P700 was depressed, but CO2 uptake was only slightly affected (24Ohkawa H. Pakrasi H.B. Ogawa T. J. Biol. Chem. 2000; 275: 31630-31634Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). On the other hand, in the ΔndhD3/D4 mutant (25Ohkawa H. Price D.G. Badger M.R. Ogawa T. J. Bacteriol. 2000; 182: 2591-2596Crossref PubMed Scopus (93) Google Scholar) and the ΔndhD3 mutant of Synechococcus sp. strain PCC7002 (26Klughammer B. Sultemeyer D. Badger M.R. Price G.D. Mol. Microbiol. 1999; 32: 1305-1315Crossref PubMed Scopus (96) Google Scholar) CO2 uptake was drastically depressed with only a small effect on P700 oxidation (i.e. PSI cyclic electron transport, ET). We have therefore reexamined the involvement of PSI in CO2 uptake and suggest how the former data may be reconciled. We have recently suggested a working hypothesis according to which CO2 uptake by Cyanobacteria and its intracellular conversion to HCO3− may be energized by photosynthetic electron transport via the formation of alkaline domains on the stromal face of the thylakoid membrane (6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar). Catalyzed conversion of CO2 to HCO3− in these domains would maintain an inward diffusion gradient for CO2. Results presented are consistent with a prediction of this model,i.e. that CO2 uptake would depend on a ΔμH+ rather than on ATP hydrolysis. Synechococcus sp. PCC7942,Synechocystis PCC6803, and mutants thereof were grown on BG-11 medium (27Stanier R.Y. Kunisawa R. Mandel M. Cohen Bazire G. Bacteriol. Rev. 1971; 35: 171-205Crossref PubMed Google Scholar) supplemented with 20 mm Hepes-NaOH, pH 8.0, and with 5 mm glucose in the case of theSynechocystis PCC 6803 mutant ΔpsaA/B (28Vermaas W.F. Shen G. Styring S. FEBS Lett. 1994; 337: 103-108Crossref PubMed Scopus (43) Google Scholar). The cultures were aerated with either high or low CO2concentration (5% CO2 in air or 1:1 mixture of air and CO2-free air, respectively), at 30 °C and light intensity of 100 μmol photons m−2 s−1. The cells were harvested during the log phase of growth and resuspended in growth media. Gas exchange measurements were performed with a membrane inlet mass spectrometer (Balzers QMG 421) as described earlier (7Tchernov D. Hassidim M. Luz B. Sukenik A. Reinhold L. Kaplan A. Curr. Biol. 1997; 7: 723-728Abstract Full Text Full Text PDF PubMed Google Scholar). Changes in the concentration of one gas affects the signal obtained for the others and, if not corrected for, may lead to erroneous interpretation of the results. Simultaneous measurements of argon and nitrogen concentrations were therefore used to correct for variations or drifts in the system due to biological formation or consumption of O2 and CO2, small changes in the rate of stirring or temperature, and in the gas consumption by the mass spectrometer. The latter was minimized by using silicon tubing with a small surface area (7Tchernov D. Hassidim M. Luz B. Sukenik A. Reinhold L. Kaplan A. Curr. Biol. 1997; 7: 723-728Abstract Full Text Full Text PDF PubMed Google Scholar). The cells were placed in a temperature-controlled chamber (2.8 ml) at 30 °C and illuminated with two optic fibers at the desired light intensity. The various chemicals supplied to the cell suspension were introduced via a special injection port. Fig. 1 may serve as an example of the correct interpretation of curves obtained using the closed membrane introduction mass spectrometry chamber. Upon illumination of Synechococcus sp. strain PCC7942 cells the dissolved CO2 concentration ([CO2(dis)]) in the medium declined steeply even prior to the onset of net O2 evolution (see Fig. 1, panels A and B). Note that the slope of the curve relating external [CO2(dis)] to time (Panel B) cannot be taken as a direct indication of the initial rate of CO2removal by the cells because CO2 is being formed continuously in the solution by net dehydration of HCO3−. Moreover, even though HCO3− concentration is virtually constant under the conditions of this experiment, net dehydration rate is not constant but rises because of the decline in CO2 hydration rate as [CO2(dis)] drops. Consequently, the further [CO2(dis)] deviates from chemical equilibrium with HCO3− the lower the rate of CO2hydration, and therefore the higher the rate of net dehydration. At plateaus in the curve, where the CO2 concentrations are relatively constant, the net rate of CO2 uptake by the cells will be equal to the net rate of CO2 formation by dehydration of HCO3− in the medium. The extent of [CO2(dis)] displacement from equilibrium was strongly affected by light intensity (Figs. 1 and 3). Raising the latter from 85 μmol photons m−2 s−1 (Fig. 1,panel B) to 750 μmol photons m−2s−1 (panel C) led to an immediate drop in the ambient [CO2(dis)] suggesting a higher rate of net CO2 uptake. The rate of O2 evolution also increased from 130 to 310 μmol O2 mg−1 Chl h−1. Return to the light intensity of 85 μmol photons m−2 s−1 (panel D) caused the [CO2(dis)] curve to rise again to a level higher than in the preceding light period at this intensity (compare panels B and D). The upward slope of the [CO2(dis)] curve at high light intensity (Fig. 1,panel C) and the following downward slope at low light (panel D) most likely reflect changes in the light-driven ET rate via the photosystems due to adjustments in the efficiency of energy transfer from the phycobilisomes. 2D. Tchernov, Y. Helman, B. Luz, I. Ohad, L. Reinhold, and A. Kaplan, manuscript in preparation. Upon darkening (Fig. 1, panel E), the [CO2(dis)] rose rapidly to the equilibrium value as the net rate of CO2 uptake fell below the dehydration rate. The [CO2(dis)] frequently rose transiently above that expected at equilibrium probably due to formation of CO2from HCO3− in the intracellular Ci pool and leak of the former to the medium (2Badger M.R. Palmqvist K. Yu J.W. Physiol. Plant. 1994; 90: 529-536Crossref Scopus (122) Google Scholar). Note that in the second half of the period of high illumination (panel C), the rate of O2 evolution rose as the ambient concentration of CO2 increased, i.e. slower net CO2uptake. Moreover, while the ambient [CO2(dis)] declined (during the second half of panel D) indicating a rising rate of CO2 uptake, the rate of O2 evolution remained constant. If CO2 fixation accounted for alterations in [CO2(dis)], one would have expected that changes in the CO2 uptake curve to be the mirror image of those in the O2 evolution curve, but that is not the case. These data provide supporting evidence for the conclusion that CO2 uptake does not solely reflect CO2 fixation and may occur even in its absence (5Espie G.-S. Miller A.-G. Canvin D.-T. Plant Physiol. 1991; 97: 943-953Crossref PubMed Scopus (30) Google Scholar, 6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar). To distinguish between the effects of light intensity on CO2uptake and on CO2 fixation, we used the high-CO2-requiring mutant of SynechococcusPCC7942, IL-3 (32Ronen-Tarazi M. Bonfil D.J. Schatz D. Kaplan A. Can. J. Bot. 1998; 76: 942-948Google Scholar) which maintains the [CO2(dis)] below CO2/HCO3− equilibrium even at CO2 concentrations lower than its threshold for net CO2 fixation (6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar). High-CO2-grown cells ofSynechococcus PCC 7942 and of mutant IL-3 were exposed to a range of light intensities in the membrane introduction mass spectrometry chamber. The cells were provided with 1 mmCi, sufficient to saturate photosynthesis in the case of the wild type but too low to enable CO2-dependent O2 evolution in the case of the mutant. At light intensities below 200 μmol photons m−2 s−1, CO2 uptake by IL-3 was considerably faster than in the wild type (Fig.2). At higher light intensities, the rates of net CO2 uptake by the mutant andSynechococcus PCC7942 were similar (Fig. 2,inset). The rates of net CO2 uptake declined when cells of Synechococcus or mutant IL-3 were exposed to light intensity higher than 600 μmol photons m−2s−1 (Fig. 2, inset) probably due to photoinhibition. Addition of the WCB p-chloromercuriphenylsulfonic acid to a cell suspension of high-CO2-grown Synechococcus PCC7942 resulted in severe, almost complete, inhibition of net CO2 uptake by over 90% (as calculated from the CO2 concentration at the plateau attained after the addition of the WCB, Fig.3 A). Photosynthetic O2 evolution was also severely depressed. To distinguish between a direct effect of the WCB on CO2 uptake and a possible indirect effect due to the decline in CO2fixation, we applied iodoacetamide (IAC) that completely inhibits CO2 fixation (5Espie G.-S. Miller A.-G. Canvin D.-T. Plant Physiol. 1991; 97: 943-953Crossref PubMed Scopus (30) Google Scholar, 6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar, 29Miller A.G. Canvin D.T. Plant Physiol. 1989; 91: 1044-1049Crossref PubMed Google Scholar, 30McGinn P.J. Coleman J.R. Canvin D.T. Can. J. Bot. 1997; 75: 1913-1926Crossref Scopus (5) Google Scholar, 31Salon C. Li Q.L. Canvin D.T. Can. J. Bot. 1998; 76: 1-11Google Scholar). We also made use of the high-CO2-requiring mutant of SynechococcusPCC7942, IL-3 in which the light-saturated rate of CO2uptake is similar to that of its wild type (Fig. 2) even at CO2 concentrations lower than its threshold for net CO2 fixation (6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar). In the presence of IAC the rate of net CO2 uptake only declined by about 20% (Fig.3 B), although O2 evolution was completely suppressed (not shown) providing further evidence that displacement of [CO2(dis)] from equilibrium may occur irrespective of whether or not CO2 is fixed. Addition of the WCB either to IAC-treated wild type or to IL-3 cells inhibited CO2 uptake almost completely (Fig. 3 B). The possibility that WCB inhibition of CO2 uptake and fixation reflected severe unspecific damage to the cells was examined by raising the concentration of Ci in the medium. Normal photosynthetic rates (306 μmol O2 evolved mg−1 Chl h−1) were observed when WCB-treatedSynechococcus cells were supplemented with 20 mmCi. These data indicate that a reduced availability of CO2 to otherwise fully functional photosynthetic machinery led to the inhibition of CO2 fixation byp-chloromercuriphenylsulfonic acid. Interestingly,Synechocystis PCC6803 is far less inhibited by the WCB thanSynechococcus PCC7942 for a reason yet unknown. The functional linkage between photosynthetic ET and CO2 uptake was examined with the aid of electron acceptors, donors, and electron transfer inhibitors. In addition toSynechococcus PCC7942 we also examinedSynechocystis PCC6803, which exhibits a similar displacement of [CO2(dis)] from equilibrium upon illumination and where mutants impaired in PSI activity are available. As previously reported (33Kaplan A. Zenvirth D. Marcus Y. Omata T. Ogawa T. Plant Physiol. 1987; 84: 210-213Crossref PubMed Google Scholar), inhibition of linear electron flow by DCMU abolished CO2 uptake (Fig.4 A). However, addition of duroquinol that donates electrons to plastoquinol and reduces cytochrome b6f thus priming light-driven PSI electron flow, reestablished CO2 uptake but not CO2 fixation (completely inhibited by DCMU). These results provide further evidence that generation of CO2/HCO3− disequilibrium is not compulsorily linked to CO2 removal by the carboxylation reactions but does require PSI activity (Fig.4 A). Direct evidence for the role of PSI in CO2 uptake was obtained by use of a ΔpsaA/B mutant ofSynechocystis PCC6803 that lacks a functional PSI (28Vermaas W.F. Shen G. Styring S. FEBS Lett. 1994; 337: 103-108Crossref PubMed Scopus (43) Google Scholar). The mutant was unable to displace [CO2(dis)] from equilibrium even when supplied with DMBQ, an efficient electron acceptor of PSII (Fig. 4 B). The high DMBQ-dependent O2 evolution (290 μmol O2 mg−1Chl h−1, Fig. 4 B) indicated significant PSII activity in the mutant. This mutant obviously is neither able to fix CO2 nor to perform cyclic electron flow in the light. Thus linear electron flow from PSII via plastoquinone to DMBQ, which does not involve PSI, is not sufficient to drive light-dependent CO2 uptake. Addition of the electron acceptors DMBQ or methyl viologen (MV) that draw electrons from PSII and PSI, respectively thus inhibiting cyclic electron flow via PSI, resulted in net CO2 extrusion inSynechococcus. The [CO2(dis)] at steady state was higher than expected at CO2/HCO3− equilibrium (Fig. 4, C and D). Addition of CA to the medium reestablished the chemical equilibrium value (Fig.4 C). Similar results were obtained when MV was supplied to mutant M55 of Synechocystis PCC6803 in whichndhB, the encoding subunit II of NAD(P)H dehydrogenase NDH-1, was inactivated (not shown). Net CO2 efflux of the magnitude observed in the presence of MV is highly likely to be the consequence of net HCO3− uptake followed by intracellular conversion to CO2 and leak of the latter to the medium (7Tchernov D. Hassidim M. Luz B. Sukenik A. Reinhold L. Kaplan A. Curr. Biol. 1997; 7: 723-728Abstract Full Text Full Text PDF PubMed Google Scholar, 23Li Q.L. Canvin D.T. Plant Physiol. 1998; 116: 1125-1132Crossref PubMed Scopus (40) Google Scholar, 34Salon C. Mir N.A. Canvin D.T. Plant Cell Environ. 1996; 19: 260-274Crossref Scopus (18) Google Scholar). These results suggest potential reversibility of the direction of the Ci cycling. The ΔpsaA/B mutant did not evolve CO2 in the presence of DMBQ (Fig. 4 B) for a reason not understood. To distinguish between ATP hydrolysis and ΔμH+ as the direct source of energy for CO2 uptake, we have examined the effects of drugs that specifically inhibit ATP synthesis as well as uncouplers that dissipate the ΔμH+. Arsenate and DCCD inhibit the formation of ATP at the substrate level and proton gradient driven synthesis respectively while hardly affecting ΔμH+ (35Cortes P. Castrejon V. Sampedro J.G. Uribe S. Biochim. Biophys. Acta. 2000; 1456: 67-76Crossref PubMed Scopus (21) Google Scholar, 36Letellier L. Howard S.P. Buckley J.T. J. Biol. Chem. 1997; 272: 11109-11113Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The uncouplers CCCP and ammonia, on the other hand, abolish the generation of ΔμH+. Photosynthetic O2 evolution gradually ceased following addition of arsenate to a cell suspension of SynechococcusPCC7942 (Fig. 5 A). The slowness of the response probably reflects the rate of arsenate uptake. Although the ambient [CO2(dis)] rose after about 100 s, it was still below the HCO3− equilibrium value after photosynthesis had halted completely. The further rise in [CO2(dis)] after addition of CA confirmed the lack of HCO3− equilibrium due to net CO2 uptake. Addition of DCCD brought about a rapid rise in [CO2(dis)] (Fig. 5 B), but as in the case of arsenate treatment, [CO2(dis)] was still well below the equilibrium value after O2 evolution (CO2fixation) had ceased. Following the DCCD treatment, the rate of CO2 uptake, calculated from the plateaus in the curve before and after the addition of DCCD, declined by 54% (from 240 to 112 μmol CO2 absorbed mg−1 Chl h−1). At the same time, net O2 evolution (235 μmol O2 evolved mg−1 Chl h−1) was replaced by respiratory O2 uptake (67 μmol O2 absorbed mg−1 Chl h−1). Supply of MV led to a slight rise in [CO2(dis)] but the rate of increase was much lower than that observed in the absence of DCCD (Fig.5 B) possibly because of lack of sufficient ATP to drive HCO3− uptake (and consequently CO2extrusion). Subsequent CA supply sharply increased the [CO2(dis)] again indicating that the latter was below the equilibrium value due to net CO2 uptake. On the addition of the proton conductor CCCP, the rate of O2 evolution rose transiently (Fig. 5 C), most probably because of stimulation of electron transport due to dissipation of thetrans-thylakoid ΔμH+. Photosynthetic O2 evolution then ceased, but O2 uptake at a rate similar to that of dark respiration was detectable. The [CO2(dis)] rose almost immediately upon addition of CCCP, briefly overshooting equilibrium value. The [CO2(dis)] level trace resembles that observed when photosynthetic ET is halted by darkening (see Fig. 1). Similar results were obtained using ammonium chloride (not shown). The [CO2(dis)] was below CO2/HCO3− equilibrium even when the internal ATP was largely exhausted as indicated by the cessation of O2 evolution following the arsenate or DCCD treatments (Fig. 5, A and B). On the other hand, the uncouplers abolished both CO2 uptake and fixation (Fig.5 C). These data suggest that CO2 uptake depends on a ΔμH+ and that direct involvement of ATP hydrolysis in the displacement of [CO2(dis)] below equilibrium is therefore unlikely. Severe inhibition of CO2 uptake by the aquaporin blocker (Fig. 3) suggests that these channels form a major route for CO2 entry to high-CO2-grownSynechococcus cells. Because passage of CO2 through aquaporins is presumably passive either by diffusion or by mass flow together with water molecules, CO2 transport mediated by specific membrane entities would appear to play a minor role, if any, unless the aquaporin blocker also specifically inhibits these entities. Results presented in this work confirm that net CO2 uptake by Cyanobacteria is not directly linked to CO2 fixation and may proceed in its absence (Figs. 1, 2, and 4). Under low, but not high light intensity, CO2 uptake by mutant IL-3, which had been maintained at CO2 concentration lower than its threshold for CO2 fixation, was faster than in the wild type (Fig.2). This may reflect consumption of NADPH and/or dissipation of ΔμH+ to support CO2 fixation in the wild type. As discussed below, CO2 uptake may well be driven by electron transport-dependent ΔμH+ (6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar). At saturating light intensity, net CO2 uptake was similar in the wild type and in mutant IL-3 suggesting common limitation by the rate of conversion of CO2 to HCO3− . In view of the queries recently raised as to the role of PSI as the major energy source for CO2 uptake (24Ohkawa H. Pakrasi H.B. Ogawa T. J. Biol. Chem. 2000; 275: 31630-31634Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), we summarize the evidence in favor of this role obtained in the present investigation as follows. 1) CO2/HCO3− disequilibrium consequent on net CO2 uptake was formed even in the presence of DCMU when reduced duroquinone (Fig. 4 A) or dithiothreitol (not shown) was added. 2) Ci cycling was absent in the ΔpsaA/B mutant, lacking PSI activity even in the presence of DMBQ, which enabled a high flow of electrons via PSII (Fig. 4 B); 3) Synechocystis PCC6803 mutant M55 or the Synechococcus PCC7942 mutant N5 in which ndhBhad been inactivated (37Ogawa T. Plant Physiol. 1992; 99: 1604-1608Crossref PubMed Scopus (90) Google Scholar, 38Marco E. Ohad N. Schwarz R. Lieman-Hurwitz J. Gabay C. Kaplan A. Plant Physiol. 1993; 101: 1047-1053Crossref PubMed Scopus (39) Google Scholar) are defective in cyclic PSI ET. Although they exhibit photosynthetic carbon fixation when supplied with elevated CO2 levels (38Marco E. Ohad N. Schwarz R. Lieman-Hurwitz J. Gabay C. Kaplan A. Plant Physiol. 1993; 101: 1047-1053Crossref PubMed Scopus (39) Google Scholar), they are unable to displace the CO2/HCO3− equilibrium. 4) Draining electrons from PSI by means of artificial acceptors switchedSynechococcus PCC7942 from net CO2 uptake to net HCO3− uptake (Fig. 4, C and D, see also Ref. 23Li Q.L. Canvin D.T. Plant Physiol. 1998; 116: 1125-1132Crossref PubMed Scopus (40) Google Scholar). These observations together with those reported elsewhere (23Li Q.L. Canvin D.T. Plant Physiol. 1998; 116: 1125-1132Crossref PubMed Scopus (40) Google Scholar, 25Ohkawa H. Price D.G. Badger M.R. Ogawa T. J. Bacteriol. 2000; 182: 2591-2596Crossref PubMed Scopus (93) Google Scholar, 39Ohkawa H. Sonoda M. Katoh H. Ogawa T. Can. J. Bot. 1998; 76: 1035-1042Crossref Scopus (49) Google Scholar) provide a very strong case for the central role of PSI ET in driving CO2 uptake. The question is, therefore, how these observations can be reconciled with the contrasting effects of inactivation of NDH-1 components on CO2 uptake and on P700 oxidation in Synechocystis PCC6803 (24Ohkawa H. Pakrasi H.B. Ogawa T. J. Biol. Chem. 2000; 275: 31630-31634Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Another problem is that the results presented here indicate that a ΔμH+generated by photosynthetic ET serves as the direct source of energy for CO2 uptake (Fig. 5). This finding is consistent with the predictions of our model (6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar), but the exclusive dependence of CO2 uptake on PSI ET has to be reconciled with the accepted notion that both linear and cyclic PSI ET lead to the formation of a ΔμH+ across the thylakoid membrane. Both problems are addressed in the following discussion. To account for the effect of mutation in a component of NDH-1 on CO2 uptake and on P700 oxidation, Klughammer et al., (26Klughammer B. Sultemeyer D. Badger M.R. Price G.D. Mol. Microbiol. 1999; 32: 1305-1315Crossref PubMed Scopus (96) Google Scholar) suggested that in Synechococcus PCC7002 one type of NDH-1 essential for cyclic ET is located on the thylakoid, whereas another type engaged in CO2 uptake is located on the cytoplasmic membrane. However, immunolocalization studies have demonstrated that a component of NDH-1, NdhB, critical for CO2 uptake is exclusively located in the thylakoid membrane (25Ohkawa H. Price D.G. Badger M.R. Ogawa T. J. Bacteriol. 2000; 182: 2591-2596Crossref PubMed Scopus (93) Google Scholar). The possibility should be considered that only a small fraction of the PSI population is engaged in CO2 uptake. Depression of CO2 uptake in the ΔndhD3/ΔndhD4 mutant (24Ohkawa H. Pakrasi H.B. Ogawa T. J. Biol. Chem. 2000; 275: 31630-31634Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) was in fact associated with some decline in cyclic PSI activity, possibly reflecting this small fraction of PSI. In the ΔndhD1/ΔndhD2 mutant where cyclic PSI activity was largely depressed, CO2 uptake was little affected presumably because the PSI units engaged in CO2uptake were still operating. The marked dependence of the PSI/PSII ratio on the growth conditions, particularly CO2concentration (22Ogawa T. Miyano A. Inoue Y. Biochim. Biophys. Acta. 1985; 808: 77-84Crossref Scopus (83) Google Scholar) and salinity (40Hagemann M. Jeanjean R. Fulda S. Havaux M. Joset F. Erdmann N. Physiol. 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Different routes for electron flow might also be the basis of the differences in CO2 uptake between the variousndhD mutants (24Ohkawa H. Pakrasi H.B. Ogawa T. J. Biol. Chem. 2000; 275: 31630-31634Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Uptake of CO2 was observed in each of the single mutants ΔndhD3 and ΔndhD4but not in the double mutant (24Ohkawa H. Pakrasi H.B. Ogawa T. J. Biol. Chem. 2000; 275: 31630-31634Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Ogawa et al. 3M. Shibata, H. Ohkawa, T. Kaneko, H. Fukuzawa, S. Tabata, A. Kaplan, and T. Ogawa, submitted for publication. concluded that two discrete systems for CO2 uptake operate inSynechocystis PCC6803. This is based on the differing kinetic parameters for CO2 uptake between mutants ΔndhD4 and ΔndhD3 and the inducibility of CO2 uptake by low CO2 conditions in the wild type and in mutant ΔndhD4 but not in mutant ΔndhD3. The NdhD3- and NdhD4-dependent CO2 uptake systems may constitute alternative PSI-dependent routes for electron flow. Quantitative consideration shows that the residual rate of P700 oxidation (i.e. the PSI cyclic electron flux) in the ΔndhD1/ΔndhD2 (24Ohkawa H. Pakrasi H.B. Ogawa T. J. Biol. Chem. 2000; 275: 31630-31634Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) was too low to account for the rate of CO2 uptake in this mutant. This may indicate the presence of an alternative acceptor of electrons from the NdhD3- and NdhD4-dependent CO2 uptake systems such as succinate:quinol oxidoreductases (44Cooley J.W. Howitt C.A. Vermaas W.F.J. J. Bacteriol. 2000; 182: 714-722Crossref PubMed Scopus (84) Google Scholar), a possibility currently being examined. It has recently been proposed (6Kaplan A. Reinhold L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 539-570Crossref PubMed Scopus (596) Google Scholar) that CO2 is converted to HCO3− in alkaline domains on the stromal face of the thylakoid membrane (see the Introduction). Conversion of CO2 to HCO3− in such domains maintains the inward diffusion gradient for CO2 and a cytoplasmic CO2 concentration below that of equilibrium with HCO3− . Withdrawal of electrons from plastoquinone by either DMBQ or MV (Fig. 4) would prevent the formation of these alkaline domains and thus also of CO2 uptake. The exclusive reliance of CO2 uptake on PSI-generated ΔμH+ probably involves an electron carrier yet to be identified. In view of the finding that NdhD3 and NdhD4 are essential components of two CO2 uptake systems (Ogawa et al., submitted) but not of the respiratory electron path they are likely candidates for the formation of the alkaline domains during PSI ET. Another candidate is ferredoxin-NADPH reductase. Recent studies (43van Thor J.J. Jeanjean R. Havaux M. Sjollema K.A. Joset F. Hellingwerf K.J. Matthijs H.C.P. Biochim. Biophys. Acta. 2000; 1457: 129-144Crossref PubMed Scopus (79) Google Scholar) demonstrated that linear ET was functional but that the plastoquinone-cytochrome b6f complex reductase step of cyclic PSI was defective in a mutant where the N-terminal of ferredoxin-NADPH reductase was truncated preventing its association with the thylakoids). We thank Dr. W. Vermaas who kindly provided us with mutant ΔpsaA/B ofSynechocystis PCC 6803. The water channel blocker,p-chloromercuriphenylsulfonic acid, was generously provided by Prof. N. Murata, Okazaki, Japan.
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