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Plant Physiol. (1982) 70, 517-5230032-0889/82/70/05 17/07/$00.50/0

Photosynthesis and Inorganic Carbon Usage by the MarineCyanobacterium, Synechococcus sp.

Received for publication October 26, 1981 and in revised form April 9, 1982

MURRAY R. BADGER AND T. JOHN ANDREWSDepartment ofEnvironmental Biology, Research School of Biological Sciences, Australian National University,Canberra City 2601 (M. R. B.); and Australian Institute of Marine Science, PMB No. 3, MSO, Townsville,Queensland 4810 (T. J. A.) Australia

ABSTRACT

The marine cyanobacterium, Synechococcus sp. Nageli (strain RRIMPN1) changes its affinity for external inorganic carbon used in photosyn-thesis, depending on the concentration of CO2 provided during growth. Thehigh affinity for CO2 + HCO- of air-grown cells (K1/2 < 80 nanomoles[pH 8.21) would seem to be the result of the presence of an induciblemechanism which concentrates inorganic carbon (and thus C02) within thecells. Silicone-oil centrifugation experiments indicate that the inorganiccarbon concentration inside suitably induced cells may be in excess of1,000-fold greater than that in the surrounding medium, and that thisaccumulation is dependent upon light energy. The quantum requirementsfor 02 evolution appear to be some 2-fold greater for low CO0-grown cells,compared with high C02-grown cells. This presumably is due to thediversion of greater amounts of light energy into inorganic carbon transportin these cells.A number of experimental approaches to the question of whether CO2

or HCO3- is primarily utilized by the inorganic carbon transport system inthese cells show that in fact both species are capable of acting as substrate.C02, however, is more readily taken up when provided at an equivalentconcentration to HCO3-.This discovery suggests that the mechanisticbasis for the inorganic carbon concentrating system may not be a simpleHC03- pump as has been suggested. It is clear, however, that duringsteady-state photosynthesis in seawater equilibrated with air, HC03-uptake into the cell is the primary source of internal inorganic carbon.

The utilization of inorganic carbon by cyanobacteria has re-cently been the subject of several investigations (6, 9). It is clearthat these photosynthetic organisms are able to change theirrelative affinities for external inorganic carbon, depending on thelevel in the external medium, and that there is some activeaccumulation mechanism which allows CO2 to be concentratedinside the cell. The concentrating mechanism has been proposedto be an HCO3 pump (3), rather than a CO2 utilizing system,which would function most efficiently at alkaline pH where alarge portion of the inorganic carbon is present as HCO3 . Anenvironment where a HCO3 transport mechanism could be ex-pected to play a major role in the fixation of carbon from theaqueous medium is seawater, where a more or less constantalkaline pH of around 8.2 exists, providing a constant high levelof HCO3 . Experiments described here were aimed at character-izing the photosynthetic properties of a marine cyanobacterium,Synechococcus sp., and assessing to what extent HCO3 ratherthan CO2 is used for photosynthesis.We have used a combination of techniques, including silicone

oil centrifugation and mass spectrometric monitoring of gas ex-

change, to show that this cyanobacterium does possess an induc-ible active CO2 concentrating system. In exploring the question ofactive species used by this concentrating mechanism, we are forcedto conclude that both CO2 and HC03- can both act as substrates;however, during steady state photosynthesis in seawater HC03-will predominately be taken up.

MATERIALS AND METHODS

Materials. The unicellular marine cyanobacterium, classifiedaccording to Rippka et al. (13) as Synechococcus sp. Nageli (strainRRIMP N1) was kindly supplied in axenic culture by Dr L. J.Borowitzka. This organism is an oval rod, 1.5 x 3 tim, and Rippkaet al. (13) consider Agmenellum quadruplicatum and Coccochloriselabens to belong to the same group. NaH14CO3, [U-_4C]sorbitoland [3H]H20 were from New England Nuclear.Growth of Synechococcus. The cells were grown to late log

phase in 300-ml batches contained in 500-ml conical flasks.Growth medium was a 0.2 ,um filter-sterilized seawater mediumbased on the 'f' medium of Guillard and Ryther (5), and bufferedat pH 8.2 with 50 mm Bicine. Cultures were shaken in a temper-ature-controlled water bath (27-31 °C) and bubbled with either air(low-CO2 grown) or air enriched with 2 to 4% CO2 (high C02-grown). Light (400 ,uE m-2 s-1 at the surface of the culture vessels)was provided by a mercury vapour lamp suspended over the bath.

Preparation and Assay of Cells. At the end of the growth period,cells were harvested by centrifugation at 5,000g for 10 min, andresuspended in 2 to 3 ml of C02-free culture media. The cells werestored in the dark on the bench and slowly bubbled with C02-freeair prior to their use in experiments. All assays were performed inseawater medium at pH 8.2, 25°C, unless otherwise specified.

Silicone Oil Centrifugation. Measurement of the internal inor-ganic carbon pool and fixation of CO2 in acid stable products wasas described previously (2, 6).

Photosynthetic 02 Evolution. This was measured in an 02electrode chamber (Rank Bros., Bottisham, Cambridge), unlessotherwise specified.Mass Spectrometric Studies. Monitoring of dissolved CO2 and

02 in aqueous algal suspensions was achieved through the use ofan aqueous inlet system attached to a mass spectrometer similarto that described previously by Radmer and Kok (11). For thispurpose, a VG Micromass 6 mass spectrometer system was used.This is a single collector instrument, with multiple ion monitoringachieved through stepping of accelerating voltage. Calibration ofthe instrument to CO2 was achieved by injecting known amountsof NaHCO3 into solution of known pH and calculating theexpected CO2 increase with the Henderson-Hasselbach equation.02 was calibrated as for an 02-electrode.

Inorganic Carbon Terminology. At the pH of seawater (8.2), theinorganic carbon species exist primarily as HCO3 ions. Given the

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BADGER AND ANDREWS

pK' and pK2 of H2CO3 to be around 6 and 10 in seawater, CO2and CO3 - will exist in about the same concentrations, being lessthan 2% of the total species. For the purposes of the discussion inthis 2paper, total inorganic carbon refers to CO2 + HC03- +CO3 -. Where references are made to CO2 or HCO3- seecifically,HCO3- refers to HCO3- plus the small amount of CO3

RESULTS

Response of Photosynthesis to Inorganic Carbon. From thedata presented in Figure 1, it appears that photosynthesis in air-grown cells (low CO2 cells) shows a very high affinity for externalinorganic carbon (K1/2 = 6-8 uM [CO2 + HCO3-1) and especiallyCO2 (K1/2 = 56-74 nm [CO2]). Cells grown at 5% CO2 (high CO2cells) showed at least an order of magnitude lower affinity (K1/2= 130 /LM [CO2 + HCO3j or 1.21yM [CO2]). Thus, Synechococcussp. show the ability to photosynthesize at very low levelsof CO2and their capacity for doing this varies with CO2 levels duringgrowth.

Light Responses of High and Low C02-Grown CeOls. Evidencepresented here (see later results) and for other cyanobacteria (6, 9)indicate that the efficient utilization of external CO2 bylow-CO2grown cells is based on the induction of an inorganic carbontransport mechanism which results in CO2 being concentratedwithin the cell. This mechanism appears to be strongly dependentupon energy produced by photosynthetic electron transport. Ifthis is the case, then there may be significant differences in thelight response of photosynthesis of high andlow C02-grown cells.High andlow C02-grown cells presumably differ in their abilityto concentrate inorganic carbon via active transport (6) and hencewould divert differing amounts of light energy to inorganic carbontranspc02-evolin FigurespiralinitialihighersaturatiThe dillightinpresumby the igrownthan thenergy

4001

300

-C

U

2 200E

EM-

100

0

FIG.

bon, in

wererurfor low

240

-CI 180

CY)E 120

E_a

60

-o

a)

+

0 f ,

I-fl-60 - /

ol12o 20 300IA "̂%^^ I%^^0

lw l-Uw 2grwn .ce ll,high C 02 grown cells

/e --o

/~~~~~~~~~~~~~~~~~~~~~~~~~~

//

0

15)5) 200 3500

pE mT2 s1400 500

FIG. 2. The response to lightof02-evolution in either high or low C02-grown cells of Synechococcus sp. 02-evolution was measured in a flat-fronted, water jacketed chamber, constructed in-house. Chl was keptconstant at 13 to 14,Ig Chl/ml for both high and low CO0-grown cells.

Temperature was 28'C and inorganic carbon was 2.5 mm. Data is pre-

sented as the meansplus SE for four experiments. Cells were added to thecuvette and light intensity increased to maximum levels. After ratesof 02evolution had stabilized, the intensity was decreased to darkness by theuse of neutral density filters. Light was provided by a quartz-iodideprojector lamp. If the initial saturating light was given after darkness hadbeen reached, then the initial ratesof 02 evolution were restored.

,rt as opposed to C02 fixation. Light response curves of net More accurate estimates of quantum yields were not made becauselution, at nonlimiting levels of inorganic carbon, are shown of the difficulty of directly measuring absorbance in the samples.ire 2. High C02-grown cells showed higher levels of dark All curves were run, however, at the same Chl concentration intion than was seen for low C02-grown cells; however, the the cuvette. It was not, however, possible to estimate the amountincrease in02-evolution at low light levels was markedly to which accessory pigments differed between the cell types. Ifin high-CO2 cells. Maximum ratesof 02 evolution, at high CO2 cells had more accessory light receptors per Chl thaning light, on a Chl basis were not significantly different. did the CO2 cells, then the difference in initial slopes seen

fferences in the slopes of the light response curves at low here could be attributed to this. As both cell types were grown inidicate that the quantum yields for net02-evolution and the samelight environment, this does not seem likely.iably netCO2 fixation are significantly different. As judged The light compensation point of both cell types do not differ as

measured initial slopes, the energy requirementof C02- greatly as do the initial slopes. This seems to be a result of an

cells for net02-evolution may be 2-fold or more greater adjustment in dark respiration rates. These are significantly lowerat of high C02-grown cells. This is consistent with a greater in low C02-grown cells. Light levels required to saturate each cellrequirement for netCO2 fixation in CO2 grown cells. type are also very similar. This appears to be due to the apparently

sigmoidal response of low C02-grown cells. Where the initial slopeincreases rather than decreases after the light compensation point,

High CO2 synechococcus high C02-grown cells consistently show a more or less hyperbolicresponse to light, whereas lowCO 2 cells are sigmoidal in nature.

Evidence forCO2 Concentration. Previous evidence for theactive concentration of inorganic carbon, and hence C02, insidecyanobacteria has come from the use ofsiliconeoil centrifugation

LowCO2 synechococcus techniques (6, 9). Similar experiments were conducted with lowC02-grown cells of Synechococcus sp. and the results are presentedin Figure 3a. Clearly, inorganic carbon within the cells reachedlevels in excess of 30mm whereas the extern al level declined from40 to around 10,Mm during the time course. Fixation of inorganiccarbon into acid stable products showed aslight lag during theinitial period but remained essentially linear for most of theincubation period.DCMU at l0-5M almost completely inhibited fixationofCO2.

______________________________________ _, , .into acid stable products and substantially eliminated the accu-005 1. 0 1. 5 220 2.5 mulation of inorganic carbon with thecell s. Both the concentration

Inorganic carbon, mM gradient measured and the effect of DCMU indicate that there isI. The response of photosynthetic 02 evolution to inorganic car- light energized active accumulation of inorganic carbon with thesehigh and lOW C02-growncelll of Synechococcus sp. Responses cells.n at30° C (pH 8.22), and Chl concentrations of 2.5 and3. 8 jig/ml Active Species Accumulated and Used for Photosynthesis. Pre-and highCO 2 cells, respectively. Light was at 450 uEMm 2Ss2- vious studies have implicated HC03- as the inorganic carbon

II" 1-- (-rl- ,,r%-n ralic

518

Plant Physiol. Vol. 70, 1982

41-o

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PHOTOSYNTHESIS IN SYNECHOCOCCUS SP.

0

EM

0to>o-

o )0 'U-

0

v)

:6o

Ec

-

oU..0

Ec

Time, sec Time, secFIG. 3. Measurement of internal inorganic carbon pools (acid unstable) in low C02-grown cells of Synechococcus sp. by silicone oil centrifugation.

Cells were incubated in an illuminated 02 electrode, 30°C at 3.9/pg Chl-ml, until 02 evolution had ceased and they had fixed the available inorganiccarbon. Aliquots (300 ul) were taken from this and subjected to silicone oil centrifugation experiments (at 26-28°C) as described in "Materials andMethods." Incubations were performed in the presence (--- -) and absence ( ) of carbonic anhydrase (0.1 mg/ml). Inorganic carbon (40 pM) wasadded to the silicone oil centrifugation tubes to initiate incubation time courses either in the form of HC03- (a) or CO2 (b). The CO2 solution wasobtained by preparing 4 mm NaH14CO3 (5,500 cpm/nmol) in 10 mm acetic acid (pH 4.0). This was kept in a serum stoppered 1 ml vial and used asneeded. The HC03- solution was prepared as 4 mm NaH'4CO3 (5,500 cpm nmol) in H20. For the DCMU treatment (minus carbonic anhydrase)DCMU was added to the cells in the incubation tube 10 s before addition of NaH'4CO3. Acid stable (A) and inorganic carbon pools (A) are presented.

Table I. Response of Km[CO2] and K,[HC03l topH in low C02-grownSynechococcus sp.

Oxygen evolution was measured in an illuminated (450 IE m-2 s-') 02electrode at 30°C. To obtain the desired pH, seawater medium wasbuffered with 50 mm Mes (pH 6), 50 mm Hepes (pH values 7 and 8) and50 mm Bicine (pH values 8 and 9). Cells (2.1 ,ug Chl ml-') were added intoCOrfree medium and allowed to photosynthesize until 02 evolutionceased. At this stage, additions of NaHCO3 were made and rates of 02evolution determined.

pH Vmaxa KmICO2 + HCO3-1 KmICO2IC Km[HCO3-]IM

6 258 30 ± 3 15 157 428 80± 10 7 738 383 42±8 0.40 429 390 76 ± 1 0.07 76

a Rates in ,umol mg Chl-' h-'.h Km and SE determined from statistical method of Wilkinson (14).cKml[CO21 and KmIHCO3-] determined using Henderson-Hesselback

equation and a pK' of 5.98 at 30°C.

species used in photosynthesis by cyanobacterial cells. Evidencefor this has been derived from experiments looking at the effect ofpH on photosynthesis, and comparing acutal rates of photosyn-thesis at alkaline pH values with theoretical rates of uncatalyzedconversion of HCO3 to CO2 (10). To look at this question inSynechococcus sp., we adopted a number of approaches.

Response to pH. Table I shows the response of V , Km (CO2+ HCO3 ), and Km (CO2) for photosynthetic 02-evolution frompH 6 to 9. Vma. was somewhat lower at pH 6 and increased toremain fairly constant between pH values 7 to 9. Km (CO2)declined from 21 AM at pH 6 to 0.18 AM at pH 9, whereas therewas no general trend in Km (HCO3-) being about 70,M at pHvalues 7 and 9 and some 2-fold lower at pH 8. These data supportthe use of HCO3- as an active species; however, they are not

conclusive.Internal Inorganic Carbon Accumulation with either C02 or

HC03 as Substrate. Inasmuch as it is clear (Fig. 3a) that Syne-chococcus sp. accumulates inorganic carbon within the cells andthat it is this pool which is in fact the substrate for photosynthesis,investigations were made to determine whether CO2 or HCO3was the primary species being accumulated into this pool. Anexperiment was performed in conjunction with that described inFigure 3a, where either CO2 or HCO3 was injected into theincubation medium to initiate the time course. This was doneeither in the presence or absence of carbonic anhydrase. Theresults are presented in Figures 3,a and b. When the inorganiccarbon (40 pM) was injected as CO2 (Fig. 3b), there was little lagin the build up of the internal inorganic carbon pool and as theinorganic carbon in the medium became depleted then the pooldecreased and acid stable incorporation began to decline. Withcarbonic anhydrase present, the result was somewhat different.There was a slow build up of the inorganic carbon pool to a levellower than that seen without carbonic anhydrase and fixation intoacid-stable products proceeded at a slower rate. When HCO3 wasinjected as the inorganic carbon species, the results were similarto CO2 plus carbonic anhydrase, with a slow build up of internalpool being noted and then a decline as external inorganic carbonwas depleted. There was essentially no difference between plusand minus carbonic anhydrase. A difference between the twoexperiments is in the absolute size of the pools accumulated. Withcarbonic anhydrase, the inorganic carbon pool in Figure 3b is lessthan that in Figure 3a, and so is the acid-stable product pool.Reasons for this are not clear, but it is probably due to somerelease of CO2 from the acid solution in which the CO2 solutionwas prepared, even though it was stored in a stoppered vial. Theimportant aspect of the experiment is the difference between theplus and minus carbonic anhydrase treatments. This experimentcertainly shows that, when provided at similar concentrations,CO2 was more rapidly accumulated into the inorganic carbon pool

519



BADGER AND ANDREWS

than was HC03-.Isotopic Disequilibrium during Steady-State Photosynthesis. This

approach to determine the active inorganic carbon species usedfor photosynthesis by algae has been employed previously by

,,) 24I0 0

20

c3 16-0u

-~12-

8-

4-';;'CO2 plus CA

0 20 40 60 80 100

Time, sec

FIG. 4. Time courses of inorganic carbon fixation during isotope dis-equilibrium experiments. Low C02-grown cells (3.2 ug Chl ml-') wereincubated in the light (600 MuE m-2 s-', 25°C) in a stoppered 02-electrodechamber, and allowed to fix the available inorganic carbon (as monitoredby the cessation of net 02 evolution). NaHCO3 was then added sufficientto bring total inorganic carbon to 192 ,UM. The cells were allowed to reacha linear rate of 02 evolution (21 nmol min-'ml-') prior to the injection of['4C1C02 or ['4C]HC03-, I min after the first injection. The HCO3- wasadded as I 1 [1'4C]NaHCO3 (58.8 tLCi/Mumol, 33 mm [pH 9.0]). The 1'4C]CO2 was prepared by taking I M,l [14C]NaHCO3 and I ,lI 1 M Na-acetate(pH 4.0) into a microsyringe, and injecting the mixture into the suspension.Aliquots (100 ,l) were taken at 5 s intervals following injection (time = 0)and fixation stopped by injection into vials containing 0.5 ml 2 N formicacid. The vials were dried under a stream of air and counted by liquidscintillation. The lines presented are the best fits to the data pointsobtained.

1.5lllow CO2 grownsynechococcus

//1.0 0

In~~ ~ ~~~~0jectioRaeCnmlsAn~l

0.5-

001- 10 ~~20 30 40

Injection Rote, (nmoles min1imh1)FIG. 5. The effect of injected inorganic carbon species (CO2 or HCO:-)

on the CO2 level during steady-state photosynthesis in low C02-grownSynechococcus sp. Inorganic carbon was injected either as 10 mM NaHCO:s(HCO:1-), or 10 mM NaHCO:1 in 30 mm Mes (pH 5.0) (CO2). The effect ofcarbonic anhydrase (0.1 mg/ml) during injection of CO2 is also presented.Experiments were run at 300C (pH 8.2) and 12.5 jig Chl/ml. Injection ofinorganic carbon began after the cells had used the available CO2 andHCO:- in solution. At this stage, 02 was 300 to 350 MM.

Lehman (7) and Findenegg (4) and Lehman (7) gives a detailedaccount of the theory associated with this experimental technique.

Figure 4 shows the time course of acid-stable 14C fixation bylow C02-grown Synechococcus cells following the injection of asmall amount of highly labeled CO2 or HC03- (6.3 nmol/ml).These cells were photosynthesizing at a steady-state rate of pho-tosynthesis in the presence of an excess of unlabeled (compared tothe labeled species injected) inorganic carbon (192 nmol/ml).Interpretation of these time courses is dependent on the fact thatin the absence of carbonic anhydrase, it takes at least 60 s forequilibration to occur between the labeled inorganic carbon spe-cies.

Injection of ['4C]HC03-, in the absence of carbonic anhydrase,results in a time course of fixation with a distinct lag, with linearrates of fixation being reached after 40 to 60 s. Addition ofcarbonic anhydrase does little to alter this except for enhancingfixation slightly during the early part of the time course, and it issignificant that the lag period remains. The theory behind thistechnique would argue that the presence of this lag period isindicative that the cells use CO2 for photosynthesis; however, thepersistence of it in the presence of carbonic anhydrase is incontradiction to this. A possible reason for this time course is thatin fact HCO3- is primarily taken up from the medium into thecell; however, as seen in Figure 3, due to the existence of a largeinternal pool of unlabeled inorganic carbon inside the cells it willtake a finite time for this pool to reach isotopic equilibrium withthe external pool. As this internal pool is the immediate substratefor photosynthesis, then a lag in fixation will be involved with theisotopic equilibration between inside and outside pools, ratherthan between external CO2 and HCO3 . Hence this time course isstill consistent with HCO3- being taken up from the externalmedium into the cell and used in photosynthesis.

Injection of 14CO2 gives a completely different picture anddifferent considerations must be made in the interpretation. In theabsence of carbonic anhydrase, much higher initial rates of fixa-tion are seen with CO2 than with HCO3 injection, and these ratesdecline over the first 60 s to reach a linear rate comparable to thatseen with HCO3-. Addition of carbonic anhydrase results asexpected in a time course with kinetics very similar to that withHC03- plus carbonic anhydrase. The only difference lies in theabsolute value of the counts fixed. The smaller number of countsfixed when CO2 is injected is probably due to diffusion of CO2out of the acidified sample prior to injection into the cell suspen-sion. Unlike the situation with HC03- injection, CO2 injection, atthis pH, will cause a considerable change in the actual concentra-tion of CO2 species in solution. The CO2 concentration in the cellsuspension prior to injection would be expected to be around 2Mm, and thus the 6 Mm concentration of labeled species injectedwill be significant in relation to this. It could be argued that directfixation of CO2 may occur from the medium by RuP2 carboxylaseinside the cells under these conditions and that this was the reasonfor the time course. If one takes a Km (CO2) for this enzyme of 200ltM (1), a Vm, of 21 nmol min-' ml-' and uses the initial rate offixation over the first 10 s after injection then the following can becalculated. Given 8 Mm C02 species externally, then direct fixationfrom this pool would support a rate of 0.8 nmol min-' ml-'. Theobserved rate over the first 10 s, assuming that an average isotopicequilibrium of 20%o has occurred, corresponds to around 4 nmolmin-' ml-' of CO2 species fixed. Thus, direct fixation couldprobably not support the observed rate of "'C incorporation.Direct fixation from the outside pool is also unlikely, if it isconsidered as before that a large pool of unlabeled inorganiccarbon (both CO2 + HC03-) exists within the cell, and isotopicdilution will be much greater here than calculated for the externalsituation. The only feasible explanation for the time course is thatCO2 is taken up rapidly into the internal inorganic carbon pooland fixed from this into acid-stable products. Thus, overall, this

520 Plant Physiol. Vol. 70, 1982




experiment can be interpreted to implicate both CO2 and HC03-as being taken up into the cell and used as primary substrates forfilling the internal inorganic carbon pool.

Steady-State Injection of CO2 or HCO3 while Measuring CO2.Use was made ofan aqueous inlet attached to a mass spectrometer,with which monitoring of both CO2 and 02 changes in solutionwhile the cyanobacteria and photosynthesizing can be achieved.

Again, as the interconversion between CO2 and HCO3 is aslow process in the absence of carbonic anhydrase, it is possible touse this to distinguish between CO2 or HCO3 as an active species.In the experiment described in Figure 5, inorganic carbon eitherin the form of CO2 or HCO3 was injected at a constant rate intoa concentrated suspension of Synechococcus in the light. At eachinjection rate, the cells were allowed to reach a steady-state CO2concentration with the external CO2 measured mass spectromet-rically at each point. At this stage, it is assumed that the inorganiccarbon injection rate is equal to net CO2 fLxation by the cells. IfCO2 were the active species being used, then regardless ofwhetherCO2 or HC03- were injected, the steady-state CO2 reached foreach injection rate should be the same, only the HC03- (which isnot measured) should change. This is clearly not the case. WhenCO2 is injected, CO2 in solution is considerably higher than whenHCO3 is the species entering solution. Addition of carbonicanhydrase to the CO2 injection treatment gives a response whichis slightly above that for HCO3 without carbonic anhydrase.These results are consistent with HCO3 being the species used insteady-state photosynthesis. They are not inconsistent, however,with the possibility that CO2 and HCO3 may both be taken up.

Monitoring of External C02 during Photosynthesis. Anothermeans ofobtaining information about the inorganic carbon speciesused during photosynthesis is to monitor the level of free CO2 insolution while making various changes to the environmental con-ditions. Such experiments have been performed previously withScenedesmus sp. by Radmer and Ollinger (12) and indicate thatHCO3 is taken up by this organism during photosynthesis. Figure6 shows typical traces of changes in CO2 levels in a suspension ofSynechococcus that occur when the light is turned on and off.Without carbonic anhydrase present in the external medium, thereis a slow interconversion between CO2 and HC03-, and resultsobtained with and without carbonic anhydrase are very different.Without carbonic anhydrase (trace a) switching the light on resultsin a sharp decline in C02, followed by an apparent rise in CO2

level over a period of 2 min and then a gradual decline to zeroCO2 and zero inorganic carbon. At this point, 02 evolution ceases(data not shown). If during this period of photosynthesis the lightis switched off (trace b), there is a sharp increase in CO2 to a levelin excess of the initial concentration followed by a decline to asomewhat lower steady-state value. If the light is switched off afterthe cells have fixed all the inorganic carbon, then there is a muchsmaller rise in CO2. If carbonic anhydrase is added (trace b), thesharp decline in CO2 following illumination is not seen. There isonly a steady decrease in CO2 level down to zero.The rise in CO2 following the initial decline after the start of

illumination may be much larger with highly active cells or withdenser suspensions of cells in the chamber (Fig. 7). Here, in theabsence of carbonic anhydrase, there was an initial decline in CO2at the light-on point to almost zero level but over the following 2min, CO2 levels rose to about 8 times the initial level. Afterreaching the peak value, there was a steady decline to zero CO2 atwhich stage all inorganic carbon in the suspension was fixed. Thispattern was not seen when carbonic anhydrase was present, butrather there was a steady decline in CO2 levels down to zero.During the period that CO2 levels were being monitored, 02evolution was simultaneously measured (lower traces). 02 evolu-tion showed the same pattern both with and without carbonicanhydrase present, with an initial lag after the light was turned onfollowed by a linear increase in 02 until all inorganic carbon wasused up. The time taken to use up the inorganic carbon was alsothe same both with and without carbonic anhydrase.The time courses for the change in CO2 levels following the

onset of illumination (Figs. 6 and 7) both indicate that there isevolution of CO2 into the medium following an initial rapiduptake. This evolution is occurring at a time when both net 02evolution and net CO2 fixation are proceeding at a linear rate.Evidence that this CO2 efflux is coming from a pool of concen-trated CO2 within the cell is obtained from changes which occurafter the light is switched off. In Figure 6 (trace b), when the lightis switched off, there is a rapid evolution of CO2 followed by aslow decline to a steady-state level. Carbonic anhydrase eliminatesthis rapid burst and suggests that it is CO2 and not HC03- whichis being evolved into the medium. A more obvious example ofthis is seen in Figure 8. Here, a constant rate of photosynthesiswas being maintained in the light by the constant injection ofHCO3 . The injection was stopped and this led to a slow decline

FIG. 6. Changes in external CO2 concentration during photosynthesis time courses. Cells (low C02-grown) were at 3.7 ,jg Chl/mi and 30°C and lightwas provided at 350 ME m-2 s-'. Carbonic anhydrase was added at 0.1 mg/ml. CO2 was measured mass spectrometrically as described in "Materials andMethods."

light on

2.5uMC02-

light on(b) i2.5uMC02- ---m

1min

521

C02



BADGER AND ANDREWS

FIG. 7. Changes in external CO2 and 02 concentrations followingillumination. Cells (low COrgrown) were at 14.5 ,ug Chl/ml and 30°C.Carbonic anhydrase was added at 0. mg/ml, and light was 350 ME m-2s-'. CO2 and 02 were measured mass spectrometrically as described in"Materials and Methods."

injectionoff

1 uMC02I

_ zeroC02

FIG. 8. Changes in external CO2 concentration after switching off thelight following a period of steady-state photosynthesis. Prior to darkeningcells (13.3 jig Chl ml-') were maintained at a constant photosynthesis rateby the injection of NaHCO3- at 43 nmol min-' ml-'. Injection ceasedapproximately I minute prior to the light-off treatment. CO2 was measuredmass spectrometrically as described in "Materials and Methods."

in the CO2 level. When the light was turned off, there was amassive efflux of CO2 into the medium followed by a slow declineto a steady-state level that was nearly twice the level before thelight was switched off. This was not accompanied by any visibleuptake of 02 (data not shown). When the light was switched on

again at this stage, a similar time course to that in Figure 7 wasseen. These results strongly indicate that there is a concentratedpool of CO2 associated with the cell in the light which is releasedto the medium when the light is switched off. The requirement forlight energy to cause the initial CO2 uptake as well as retention ofa CO2 pool inside the cell is further seen by the effects of DCMUon the observed changes in CO2 levels (Fig. 9). DCMU at levelsused here substantially eliminated the initial CO2 uptake as well

light off

FIG. 9. Effect of DCMU (10-5 M) on the change in external CO2concentration following illumination in low C02-grown Synechococcus sp.

as the CO2 release upon darkening.In the CO2 monitoring experiments described in Figure 6 and

7, it is superficially conceivable that the initial uptake of CO2 is aresult of RuP2 carboxylase taking up CO2 directly from themedium at a rate which causes disequilibrium between CO2 andHCO3 externally. This direct uptake would eventually be re-

placed by indirect uptake from the internal CO2 pool resultingfrom HCO3 transport. The switch from CO2 to HCO3 utilizationcould lead to a subsequent rise in CO2 as CO2 and HCO3- reachequilibrium externally. However, it is not possible in this mannerto increase the CO2 concentration to levels higher than the initialones (as seen in Fig. 7). Anyway, uptake of CO2 by RuP2 carbox-ylase at these low levels of external CO2 would be expected to besmall based on the measured Km (CO2) of this enzyme in excess

of200 ,UM (1) and its efficient functioning is presumably dependenton the accumulation of CO2 internally.

DISCUSSION

The marine cyanobacterium, Synechococcus sp., changes itsaffinity for extemal inorganic carbon used in photosynthesis,depending on the level during growth (Fig. 1). Cells grown at airlevels of CO2 possess an extremely high affinity for free CO2(K1/2 < 80 nm [pH 8.21) and this would seem to be the result ofthe presence of a mechanism which concentrates inorganic carbon(and hence CO2) within the cells. Concentrations of inorganiccarbon inside the cell in excess of 1,000-fold over those existing inthe external medium, are readily measured (Fig. 3). This concen-

trating mechanism is active in nature and utilizes energy producedby whole chain electron transport as evidenced by the effect ofDCMU on its operation (Fig. 3a).Comparison of the light-response of net 02 evolution (Fig. 2)

indicates that the quantum yield for net 02 evolution (and presum-ably CO2 fixation) in low C02-grown cells is approximately halfthat ofhigh C02-grown cells. This is consistent with a considerableamount of light energy being used to concentrate CO2 within thecell as well as to fix CO2 via the photosynthetic carbon reductioncycle. Further estimation of the absolute energy costs are ratherpremature due to the limitations of the comparison as discussedin the results.

Previous work with cyanobacteria (6, 9) has suggested thatinorganic carbon concentrating mechanisms found in these orga-nisms functioned around the operation of a HCO3 transportsystem. In examining the utilization of inorganic carbon speciesby Synechococcus, both for photosynthesis and for accumulationinto the internal inorganic carbon pool, we have been confrontedwith a number of seemingly confficting results which need expla-nation. From the steady-state injection studies described in Figure5, and the pH response of Km (C02) and Km (HCO3) (Table I) itseems clear that during steady-state photosynthesis HC03- ratherthan CO2 is the major species taken up from the external medium.The monitoring of external CO2 levels during photosynthesis inFigures 6 and 7 also supports this conclusion in that CO2 in the

light on Alight on

C02X f-

5pC2 light off

C02

L 1]minmlight off '

522 Plant Physiol. Vol. 70, 1982




external solution can be increasing (rather than decreasing) at atime when net 02 evolution is proceeding at a linear rate. Additionof carbonic anhydrase to the external medium (Fig. 7) had noeffect on the rate of 02 evolution or the time taken to fix all of theinorganic carbon from the external medium. An effect would beexpected if photosynthesis was limited by conversion of HCO3 toCO2. In some conflict with these observations is the evidence thatCO2 can be taken up at an apparently faster rate into the internalinorganic carbon pool than can HCO3 (Fig. 3) and that uponillumination, CO2 is rapidly removed by the cells from the externalmedium (Figs. 6 and 7). The isotope disequilibrium experiments(Fig. 4) can, in the midst of this, be interpreted to indicate thatboth CO2 and HCO3 can be taken up from the external solution.There is one explanation which readily unifies this set of data,

and this is that as indicated by the isotope disequilibrium experi-ment, both CO2 and HCO3 are taken up from the externalmedium into the internal inorganic carbon pool. If this is the case,then the relative contribution by each flux to supplying this poolwill be dependent upon the rate at which each species is trans-ported for a given external concentration. To explain the data inFigure 3, it must be inferred that, at similar external concentra-tions, CO2 is transported into the cells much more rapidly thanHCO3-. The word transport and its inference of being an activeprocess is justified as the internal concentration of inorganiccarbon, whether CO2 or HCO3 is supplied is much higher thanany passive distribution can explain (Fig. 3).The actual mechanism by which this can occur is not at all

clear. It is possible that CO2 and HCO3 are alternate substratesfor the one transport system in these cells and that when suppliedat similar concentrations, the system is more active with CO2. Thispossibility does not seem likely as HCO3 and CO2 are notstructurally analogous. A second alternative is that there is arelatively unstirred layer around the cells which becomes alkalineupon illumination. Such alkalization associated with HCO3 trans-port has been proposed for other algal systems (8). In this case,HCO3 would be the species taken up and the high OH concen-tration in the unstirred layer would act to catalyze the conversionof CO2 to HCO3 . This is unlikely for several reasons. First,physical calculations based on unstirred layer sizes and possibleefflux rates for OH- suggest that at pH 8.2, the cell surface wouldbe unlikely to be any higher than half a pH unit higher (calcula-tions not shown). This would not be enough to increase the rateof conversion ofCO2 to HCO3 sufficiently to explain the observedrates ofCO2 uptake. Second, if there were an alkaline layer aroundthe cell, it is hard to see how CO2 could diffuse back out throughit again, as would be necessary to explain the CO2 burst seen afterthe light is switched on (Figs. 6-8). Third, measurements ofinternal pH in cyanobacteria have been made previously usingweak acids and the silicone oil centrifugation technique (6, 9).These have estimated that at an external pH of 8.0, the internalpH is 7.5 to 8.0. As these measurements include a considerableamount of extracellular fluid, then no alkalinity of the outer layersurrounding the cell is indicated. At this stage we cannot speculatefurther on the basis for uptake of both CO2 and HCO3-

Regardless of what the explanation is for both CO2 and HCO3,it is clear that HCO3 rather than CO2 is the predominant inor-ganic carbon species being accumulated inside the cells. Thisarises primarily because at these alkaline pH values, CO2 is asmall proportion of the total inorganic carbon (at pH 8.2 it is<2%). The cells do take up what CO2 there is available; however,they rapidly become limited by the rate at which HCO3 can beconverted to CO2. The HCO3 in equilibrium with atmosphericCO2 is sufficient to support uptake rates which presumably lead

to saturating levels of CO2 within the cells and HC03- uptakealone can support the observed photosynthetic rates. As has beendiscussed previously (6), it is impossible to estimate during pho-tosynthesis what proportion of the internal inorganic carbon isHCO3 and what is CO2. This stems largely from the fact that dueto the lack of carbonic anhydrase activity in cyanobacteria, it isreasonable to assume that HCO3 and CO2 concentration are notin rapid equilibrium during steady-state photosynthesis. Undersuch conditions, which may be achieved by constant injection ofHCO3 (Fig. 8), the flux of HCO3 and CO2 transported into thecell will equal CO2 efflux from the cells plus CO2 fixed inphotosynthesis by RuP2 carboxylase (presuming the membrane isimpermeable to HC03-). The actual concentration gradient ofCO2 between inside and out will be dependent upon the physicalresistances to diffusion involved. This will mean, however, that ifHCO3 is being taken up from the medium and concentratedinside the cell in addition to the CO2 uptake, then CO2 will becontinuously effluxing in a net manner. The efflux following theaccumulation of HCO3 could be responsible for the rise in CO2seen after the initial uptake following illumination (Figs. 6 and 7).Likewise, the rapid efflux of CO2 into the medium when the lightis switched off (Figs. 6 and 8) could be a result of the loss of theinternal CO2 pool to the external medium.The results obtained here are probably generally applicable to

inorganic carbon usage by other cyanobacteria. The adaptation tolow CO2 and the inorganic carbon concentration achieved bySynechococcus is very similar to that seen in both A. variabilis (6)and C. peniocystis (9). Data have also been obtained (Badger,unpublished) showing that A. variabilis has very similar CO2 timecourse transients following illumination. Clearly then, what hasbeen implicated as a HCO3 transport mechanism responsible forthe accumulation of inorganic carbon within cyanobacteria mustbe reassessed mechanistically to take account of the fact that bothCO2 and HCO3 act as substrates for this system.

LITERATURE CITED

1. ANDREWS TJ, KM ABEL 1981 Kinetics and subunit interactions of ribulosebis-phosphate carboxylase-oxygenase from the cyanobacterium, Synechococcus sp.J Biol Chem 256: 8445-8451

2. BADGER MR, A KAPLAN, JA BERRY 1980 Internal inorganic carbon pool ofChlamydomonas reinhardtit evidence for a carbon-dioxide concentrating mech-anism. Plant Physiol 66: 407-413

3. FINDENEGG GR 1974 Carbonic anhydrase and the driving force of light depend-ent uptake of C I - and HCO.,- by Scenedesmus. In U Zimmermann, J Dainty,eds, Membrane Transport in Plants. Springer, Berlin, pp 192-196

4. FINDENEGG GR 1980 Inorganic carbon transport in microalgae. II. Uptake ofHC03- ions during photosynthesis of five microalgae species. Plant Sci Lett18: 289-297

5. GUILLARD RRL, JH RYTHER 1962 Studies of marine planktonic diatoms. Can JMicrobiol 8: 229-239

6. KAPLAN A, MR BADGER, JA BERRY 1980 Photosynthesis and the intracellularinorganic carbon pool in the bluegreen alga Anabaena variabilis response toexternal CO2 concentration. Planta 149: 219-226

7. LEHMAN JT 1978 Enhanced transport of inorganic carbon into algal cells and itsimplications for the biological fixation of carbon. J Phycol 14: 33-42

8. LuCAS WJ 1976 Plasmalemma transport of HCO3 and OH- in Chara corallina:non-antiporter systems. J Exp Bot 27: 19-31

9. MILLER AG, B COLMAN 1980 Active transport and accumulation of bicarbonateby a unicellular cyanobacterium. J Bacteriol 143: 1253-1259

10. MILLER AG, B COLMAN 1980 Evidence for HCO3 transport by the blue-greenalga (Cyanobacterium) Coccochloris peniocystis. Plant Physiol 65: 397-402

11. RADMER RJ, B KOK 1976 Photoreduction 02 primes and replaces CO2 assimila-tion. Plant Physiol 58: 336-340

12. RADMER RJ, 0 OLLINGER 1980 Light-driven uptake of oxygen, carbon dioxide,and bicarbonate by the green alga Scenedesmus. Plant Physiol 65: 723-729

13. RIPPKA R, J DERUELLES, JB WATERBURY, M HERDMAN, RY STAINER 1979Generic assignments, strain histories, and properties of pure cultures of cyano-bacteria. J Gen Microbiol 111: 1-61

14. WILKINSON GN 1961 Statistical estimations in enzyme kinetics. Biochem J 80:324-332

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