Planta

Planta 149, 219-226 (1980)

9 by Springer-Verlag 1980

Photosynthesis and the lntracellular Inorganic Carbon Pool in the Bluegreen Alga Anabaena variabilis: Response to External CO2 Concentration* Aaron Kaplan**, Murray R. Badger***, and Joseph A. Berry Department of Plant Biology, Carnegie Institution of Washington, 290 Panama Street, Stanford, CA 94305, USA

Abstract. The apparent photosynthetic affinity of A. variabilis to CO2 is greatly affected by the CO2 concentration in the medium during growth. Halfmaximal rate of photosynthetic 0 2 evolution is achieved at 10 ~tM and 100 gM inorganic carbon (Cinorg) in cells grown at low-COa (air) and high CO2 (5% v/v CO2 in air), respectively, whilst the maximum rate of photosynthesis is similar in both cases. Both high- and low-CO2-grown Anabaena accumulate Cinorg within the cell; however, the rate of accumulation and the steady-state internal Ci.org concentration reached is much higher in low as compared with highCO2-grown cells. It is suggested that Anabaena cells actively accumulate Cinorg- Measurements of the kinetics of Ci,org transport indicate that the affinity of the transport mechanism for Ci.o~g is similar (K,n(Cinorg(-~150 gM) in both high- and l o w - C O 2g r o w n cells. However, Vm,xis 10-fold higher in the latter case. It is suggested that this higher Vma~for transport is the basis of the superior capability to accumulate Ci,org and the higher apparent photosynthetic affinity for external C~norgin low-CO2-grown Anabaena. Carbonic anhydrase activity was not detectable in Anabaena, yet both photosynthetic affinity to C~,org in the medium (but not V~,x) and the rate of accumulation of C~.o~g were inhibited by the carbonic-anhydrase inhibitor ethoxyzolamide. Key words: Anabaena - Carbonic anhydrase - Carbon (inorganic) pool Photosynthesis (inorganic C pool).

*

CIW-DPB Publication No. 682 Present address: Department of Botany, Hebrew University, Jerusalem, Israel *** P r e s e n t address: Research School of Biological Sciences, P.O. Box 475, Canberra City, A.C.T. 2601, Australia **

Abbreviations: Cinorg= inorganic carbon; PEP=phosphoenol pyrurate; RuBP = ribulose- 1,5-bisphosphate

Introduction

The photosynthetic affinity of green algae to C O 2 is strongly dependent on the CO2 concentration in the medium during growth (Berry et al. 1976; Findenegg 1976; Hogetsu and Miyachi 1977). The apparent photosynthetic KIn(CO/) of Chlamydomonas reinhardtii grown under low CO2 concentration is 10-fold lower than that observed in high COz-grown cells (Berry et al. 1976). The previous growth conditions also affect the rate of glycolate excretion following transfer of the algae to low CO2 conditions. Highand low-COz-adapted cells do not differ in their photosynthetic carbon fixation pathway. Neither the first products of photosynthesis nor the relative activity of the carboxylating enzymes, ribulosebisphosphate (RuBP) carboxylase and phosphoenol-pyruvate (PEP) carboxylase, in green algae are altered by the CO2 concentration during growth (Berry et al. 1976; Reed and Graham 1977). Low-COz-grown Chlamydomonas, however, exhibit a greatly enhanced ability to concentrate COz from the medium as compared with high CO2-adapted cells (Badger et al. 1977). This ability to concentrate free CO2 can explain the higher apparent affinity of these cells for CO2 in the medium and the decrease of glycolate excretion during the course of adaptation from high to low-CO2 conditions (Berry et al. 1976; Badger et al. 1977). Badger et al. (1978) attributed the ability of low CO2 grown Chlamydomonas to concentrate CO2 within the cell to their increased capability to utilize HCO~ from the medium, which indicated an active transport mechanism for HCO3. A metabolic influx pump of HCO3 was also suggested in Hydrodictyon (Raven 1970) and Chara (Lucas and Smith 1973; Lucas 1975, 1976; Walker and Smith 1977). Elucidation of a CO2-concentrating mechanism is complicated by the fact that the cells of green algae consist of several compartments. Evaluation of the 0032-0935/80/0149/0219/$ 01.60

A. Kaplan et aI. : Inorganic Carbon Pool in Anabaena

220 c o n c e n t r a t i o n o f i n o r g a n i c c a r b o n (C~,o~g) a n d t h e p H w i t h i n a g i v e n c o m p a r t m e n t is e s s e n t i a l i n f o r m a tion for the understanding of the mechanism involved. Bluegreen algae (cyanobacteria) have the advant a g e t h a t t h e y c o n s i s t o f v i r t u a l l y a single c o m p a r t m e n t , w h i c h s i m p l i f i e s e v a l u a t i o n o f t h e i n t e r n a l Cinorg c o n c e n t r a t i o n . T h e g l y c o l a t e e x c r e t i o n r a t e in b l u e g r e e n a l g a e is a f f e c t e d by t h e C O 2 c o n c e n t r a t i o n d u r ing g r o w t h in a m a n n e r s i m i l a r t o t h a t o b s e r v e d in g r e e n a l g a e ( I n g l e a n d C o l m a n 1976). It is t h u s possible t h a t b l u e g r e e n a l g a e e x h i b i t t h e s a m e p h e n o m e n o n of induction, by low CO2 concentration, of a capability to c o n c e n t r a t e C O 2 w i t h i n t h e cells. W e t h e r e f o r e s t u d i e d v a r i a t i o n s in t h e Cinorg p o o l w i t h i n t h e cells o f Anabaena a n d o f p h o t o s y n t h e s i s in r e s p o n s e t o t h e e x t e r n a l Cinorg c o n c e n t r a t i o n .

Material and Methods Growth Conditions. Anabaena variabilis cells, strain M-3 from the collection of Tokyo University, Japan, were grown at 30 ~ C in 300-ml shake flasks containing Kratz and Myers (1955) medium C. The cultures were aerated with air (low-CO2 ceils) or 5% CO2 (v]v) in air (high-CO2 cells). Continuous iIlumination was provided by VHO fluorescent lamps (F48 T12-CW-VHO, GTE; Sylvania, Danvers, Mass., USA) at a fluence of 8.9 mW .cm- 2 (400 700 nm). Light intensity was measured by Licor LI 185A (Lambda, Lincoln, Neb., USA). Units in mW-cm 2 were calculated using a conversion factor obtained from the manufacturer.

Measurement of 02 Evolution. Cells were harvested by centrifugation at room temperature at 700.g, resuspended in 50 mM N-2hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES) buffer, pH 8.0, and placed in the chamber of an oxygen electrode (Rank Broth., Bottisham, Cambridge, U.K.) modified to an increased sensitivity as described in Berry and Bowes (1973). The rate of oxygen evolution in response to Cinorg concentration in the medium was measured at 30~ C, 8.0 mW.cm -2 (400-700 rim). Known amounts of NaHCO3 were added to the cell suspension following depletion of the medium from Cinorgas determined by the cessation of COz-dependent 02 evolution (see Badger et al. 1977). Accumulation of Acid-Stable and -Labile 14C was followed by using the filtering centrifugation technique as described for our studies with Chlamydomonas(Badger et al. 1977). Anabaenacells were preincubated in the closed Os-electrode chamber to allow for COs-dependent 02 evolution to cease. This minimized the dilution of NaH~4CO3 by an unknown amount of unlabelled COs. Samples of cell suspension, 300 gl each, were then transferred into 400-gl plastic sample tubes containing (from bottom to top) 20 gl 1 M glycine, pH 10.0, 0.75 % (w/v) sodium dodecyl sulfate (SDS) (killing medium); 65pl silicone oil (1:4, v/v) SF 96(50): versilube F50 (from General Electric, Silicone Products Division, Waterford, N.Y., USA). Incubations were initiated in the light (7.5 mW. cm- 2, 400-700nm) at 30~ for 10 s followed by the addition of Nail ~4CO3 (time zero). Incubations were terminated by centrifugation (microfuge B; Beckman Instruments, Palo Alto, Cal., USA). Sodium hydroxide, 3 M, 15 gl, was then injected into the top layer to minimize diffusion of CO2 through the silicone oil. This diffusion was smaller than 2% of the acid-labile ~4C over the entire range of C~no~gconcentrations. The tubes were frozen in liquid nitrogen and the pellet removed with a razor blade and placed in a closed

vial containing 430 gl 0.1 NaOH. Two samples of 170 gl each were then taken from this vial, and one of them placed into an equal volume of 0.1 N NaOH while the other was placed into equal volume of 0.5 N HC1, and left at room temperature for 30 rain with frequent flushing with CO2 to remove the acid-labile 14C. Radioactivity in the samples was measured by liquid scintillation counting (LKB-Wallac 81000; LKB, Pleasant Hill, Cal., USA). Photosynthesis was estimated from the acidified sample whereas the acidlabile 14C was estimated from the difference in counts between the alkaline and acidified samples. The volume of the cells taken down through the silicone layer was estimated from the radioactivity (3H or 1~C) in the pellet as compared to that in 10 gl of supernatant following incubation for 10 s of the ceil suspension with 2 gl tritiated water or [14C]sorbitol in the sample tubes. The volume occupied by the tritiated water presents the intra- and intercellular volume whereas that occupied by the sorbitol is the intercellular one. The internal volume of the cells (sorbltol-impermeable-space = SIS) was thus calculated from the difference between the water-permeable and sorbitolpermeable volumes (see Werdan et al. 1972; Badger et al. i977). Sorbitol volume was 3040% of the tritiated-water volume. The concentration of Cinorg within the cell was calculated from the acid-labile ~4C on the basis of SIS, taking into account the acidlabile ~C in the sorbitol volume of the pellet.

Enzyme Essays. Cells were broken in a French press in a medium containing 100 mM HEPES, pH 8.05, 20 mM MgCI2 'and 5 mM dithiothreitol. RuBP-carboxylase activity and PEP-carboxylase activity were measured in the supernatant following centrifugation at 10,000-g. Ribulosebisphosphate-carboxylase activity was measured after activation (30 min at 0~ in the presence of 5 mM NaHCO3) in a medium containing 20 mM MgC12, 0.4 mM RuBP and 10 mM NaH~4CO3 for 1 rain at 30~ C. Ten pl enzyme was added to a final volume of 0.4 ml to start the assay. The assay was terminated by the addition of 50 ~tl 6N acetic acid (see Lorimer et al. 1976). Phosphoeno-pyruvate-carboxylase activity was determined by the method of Bj6rkman and Gaul (1969). Carbonic anhydrase activity was measured at I ~ on both supernatant and resuspended pellet after centrifugation at 10,000 .g. The activity was determined from the time taken to lower the pH of 1,5 ml, 5 mM HEPES buffer, pH 8.15, by the addition of 2.0 ml distilled water saturated with CO2 at 1~ C, to pH 7.4, in the presence or absence of cell-free homogenate.

Results The Photosynthetic Affinity for Inorganic Carbon in the Medium. T h e p h o t o s y n t h e t i c p e r f o r m a n c e o f l o w a n d h i g h - C O z , g r o w n Anabaena variabilis cells in res p o n s e t o t h e c o n c e n t r a t i o n o f C~norg in t h e m e d i u m is s h o w n in Fig. 1. T h e h a l f - m a x i m u m r a t e o f p h o t o s y n t h e s i s f o r l o w - a n d h i g h - C O 2 cells was o b t a i n e d at 10 a n d 100 p M H C O ~ - , r e s p e c t i v e l y . T h i s c o r r e s p o n d s to a p p r o x . 0.25 a n d 2.5 p M C O 2 , r e s p e c t i v e l y , at t h e p H u s e d (8.0). T h e m a x i m u m r a t e o f p h o t o s y n thesis, h o w e v e r , was s i m i l a r in b o t h t y p e s o f cells. T h e s e results a g r e e w i t h o b s e r v a t i o n s in g r e e n a l g a e r e p o r t e d by B e r r y et al. (1976), H o g e t s u a n d M i y a c h i (1977) a n d F i n d e n e g g (1976) w h o s h o w e d t h a t t h e p h o t o s y n t h e t i c r e s p o n s e o f t h e s e a l g a e t o C02 c o n c e n t r a t i o n in t h e m e d i u m is a f f e c t e d b y t h e C O 2 c o n centration during growth.

A. Kaplan et al. : Inorganic Carbon Pool in Anabaena

221 1. The activity of RuBP carboxylase (RuBPCase) and PEP carboxylase(PEPCase)in Anabaena variabilis adapted to high (5%, v/v) or low (0.03%, v/v) CO2 concentration

Table 300

IY•kow

C O 2 grown

Y

Growth conditions

grown

200

PEPCase (gmolCO2 rag-1 Chl. h 1

C ::L

Low CO2

I00

High CO 2

44.4 45.6

RuBPCase (txmol CO2 mg- 1 KIn(CO2) Chl.h ~) (gM) 480 582

330+ 35a 204_+24

Standard error of the mean

0'2

o.'4 s

o18 [CO 2 + HC03- ] ,

ola

f'o

mM

Fig. 1. The response of photosynthetic 0 2 evolution in Anabaena variabilis to C~norgconcentration in the medium, Cells grown at low (0.03%, v/v) or high (5%, v/v) CO2 in air 30~ C, 50 mM HEPES buffer, pH 8.0, 8 mW.cm 2 (400-700nm), cell density corresponding to 7.2 gg Chl/ml were used The photosynthetic affinity to CO2 observed here is among the highest reported for any organism including C# species of higher plants (see review by Hatch and Osmond 1976). It was therefore important to measure the activity of the carboxylating enzymes in these cells. The activity of RuBP carboxylase and PEP carboxylase in crude extracts of high- and lowCO2 A. variabilis is given in Table 1. In both type of cells RuBP-carboxylase activity, at rate-saturating CO2 concentration, was 10 times higher than PEPcarboxylase activity, and slightly higher than the maximum rates of photosynthesis observed with intact cells. The activity of the primary CO2 fixation enzymes in Anabaena was not altered by the different CO2 concentration during growth and the relative activity of PEP and RuBP carboxylase was similar to that observed in other species which utilize the C3 mechanism of CO2 fixation (Hatch and Osmond 1976). Ribulosebisphosphate carboxylase from highand low-CO2 cells appeared to be identical. The affinity of the enzyme for CO2 is quite low (/s to 300 gM CO2) in comparison to the enzyme from higher plants (Lorimer et al. 1976), the one observed in green algae (Berry et al. 1976), or to the apparent affinity of the intact cells for CO2 (Fig. 1). Thus while there is sufficient RuBP carboxylase present in the cells to account for the rates of CO2 fixation observed, higher concentrations of CO2 are required to express that activity with the isolated enzyme than with the intact cells. This anomaly might be explained if the concentration of CO2 at the site of RuBP carboxylase were higher than in the surrounding medium. We therefore examined the amount of C~norg present in intact cells during photosynthesis according to the technique of Werdan et al. 1972).

Accumulation of Inorganic Carbon Within the Cells. The time dependence of accumulation of acid-labile and acid-stable 14C in response to the external concentration of Cinorg is shown in Fig. 2. Both lowand high-CO2 Anabaena contained higher levels of acid-labile 14C (Cinorg) within the cells than was present in the medium. The extent of accumulation of Cinorg depended both on time and on the external Cinorg concentration. Steady-state internal Cinorg concentration was reached within 0.5-2 rain (at the various external C~norg concentration) and was higher in low-CO2 than in high-CO2 cells. This difference was especially pronounced in the lower range of external C~norg concentration. The rate of photosynthetic CO2 fixation (accumulation of acid-stabile 1r also increased with time, especially in the lower external Cinorg range (Fig. 2b and d, for low- and high-CO2grown cells, respectively). The lag in photosynthesis rate was more pronounced in high-CO2 cells where the rate of accumulation and the pool sizes of Cinorg were lower. The internal Cinorg pool in low-CO2 Anabaena reached a concentration of 50 mM at an external concentration of 1 m M and at lower external concentrations the ratio of internal to external concentration approached 500 : 1. The time dependence of photosynthesis and the level of Cinorg pool following addition of H C O ; to a relatively dense suspension of low-CO2 cells which had previously depleted their CO2 supply are shown in Fig. 3. The flow of carbon in this system appears to proceed initially from the external pool into the internal inorganic carbon pool. As the internal pool builds up, the rate of 14C fixation increases. When the external Cinorg supply is exhausted, the internal pool begins to be diminished, and the rate of 1r fixation declines. These characteristics are consistent with internal acid-unstable ~r being a pool of (CO2 + HCO~-) which is an intermediate in photosynthetic carbon assimilation for this alga. This dynamic relationship between the inorganic carbon pool and photosynthesis would not be expected if for example the accumulation of C~norg were on a side pathway

222

A. Kaplan et al. : Inorganic Carbon Pool in Anabaena

50

C~

'r

'~

I,O

b

D

4o

'0~ 30 + C L). 2c o 05

E

~

o

o

o

3b

~o

,;o

,~o

30

Time, s

60 Time, s

90

120

25 C

IO

Fig. 2a-d. The time course of

20

accumulation of acid-stable (b and d) and -labile (a and e) 14C as affected by the external C~,o~g concentration, in low-(a and b) and high-(e and d) COz-grown A. variabilis. Numbers at right o f a and e give the C~no~g concentration in the medium (raM). The symbols in b and d denote C~,org concentration in the medium as in a and e. Sorbitol-impermeable space (SIS) was 1.02 and 0.2 gl, chlorophyll concentration was 2.56 and 4.2 gg Chl/gl SIS in high- and low-CO2 grown cells, respectively

v)

o~

15

'~

:

?

Ck 5

005

o

002 o

ao

o

8;

~o

o,o i

0

L2o

30

Time, s

IO - o.

20 ~ Exfernal

I~

:~

.

.

~

u')

~,,

u. u. ,,,

..c

_

0

30

60

O0

120

Time, s

60

9o

120

o

Time, s

Fig. 3. The time dependence of accumulation of acid-stable and acid-labile t4C of low-COz A. variabilis, t0 pM NaH~4CO3 were

added at time zero. The external concentration values are calculated by subtracting the amount of C~no~g taken up by the cells from that added. 0.4 ~tl sorbitol-impermeable space (SIS), 3.9 gg Chl/~tl SIS. Note the difference in scale between the internal and external C i n o r g concentration

such as precipitation of CaCO3 (see review by Golubic 1973). Further, under the experimental conditions used here (data presented in Fig. 3), Ca z + concentration inside the cells is 0.6 gM, indicating that CaCO3 could not precipitate within the cell.

Kinetic Analysis of the Accumulation of Acid-Stable and -Labile 14C. The rate of Cinorg flux into the cells (influx) as affected by the external concentration of Cinorg (Fig. 4) was determined from the initial rate of accumulation of acid-stable and -labile ~4C (Fig. 2). It is assumed that during the initial period the efflux of Cinorg from the cell is negligible. Analysis of the data in Fig. 4 indicates that the affinity of the transport system to Cinor~ is similar in both type of cells (Km(Cinorg)= 141 + 11 and 166_+22 gM Cino~g for low- and high-CO2 cells, respectively). The maximum reaction velocity (Vmax), however, is about 10 times higher in low-COa cells (305 + 9 and 27 _+ 1 nmol C~no~ggl- 1 SIS min- 1, respectively, which correspond

A. Kaplan et al. : Inorganic Carbon Pool in Anabaena

223

I Transpor't

300 300

f_________----o T -c T

E ~'00

200

g "5 E

0 2 evolution

f

-6

I00

E ::k 100

High COz g r o w n

O.l

012

01,3 0149

IiO

50

Exfernal [CO2 + HCOa-], rnM

1

i

i

iO0

150

200

1

Exfernal [CO 2 + HCOs- ] , ]aM

Fig. 4. The initial rate of accumulation of acid-stable and acid-labile t4C in response to the external concentration of Ci.o~g in highand low-CO2 A. variabilis. Initial rates are calculated from the data of Fig. 2 assuming that the efflux of C~.org is negligible

i

i

,

aoo

to 3848+ 121 and 402_+20 pmol Cinorg mg -1 chlorophyll H-*, respectively). The higher Vm,~ of the C~.org influx into low-CO2 Anabaena is probably responsible for the higher steady-state concentration of intracellular C~no~g (Fig. 2a and c) and the shorter lag time in photosynthesis (accumulation of acid-stable 14C) observed in these cells (Fig. 2b, d). Implicit in this suggestion is that C i n o r g transport must exceed the rate of photosynthesis in Anabaena. This was confirmed by comparing the rate of photosynthetic O2 evolution (Fig. 1) with that of C~,o~g influx (kinetic data from Fig. 4) in response to external C~.o~g concentration (Fig. 5a and b, for low- and high-CO2 Anabaena, respectively). The rate of Ci.o~g influx in low-CO2 cells exceeded the rate of photosynthesis at all but the lower range of external Q.o,.g concentration. This also occurred with high-CO2 cells but the rate of influx of Cino~g did not exceed that of photosynthesis at high external C~,org concentration by such a large margin as occurred with low-CO2 cells. This probably accounts for the lower apparent affinity of the high-CO2 cells for CO2 and for the lower stead-state concentration ofintracellular C~,o~gobserved with high- as compared with l o w - C O 2 cells. Response of Acid-Stable and -Labile 14C Pool to a Carbonic-Anhydrase Inhibitor. In the case of Chlamydomonas we found that inhibition of carbonic anhydrase lowered the apparent affinity of l o w - C O 2 cells to Ci.org (Berry et al. 1976) and altered the rate of accumulation of C i n o r g within the cells (Badger et al: 1978). Carbonic-anhydrase activity was not detectable in cell homogenates of either high- or low-COz A. variabilis. Nevertheless, addition of the carbonic-anhydrase inhibitor, ethoxyzolamide (10-5 M), lowered

200

ZL

lO0

b o12

o4

o'.6

oi~

,io

Ex+ernal [CO 2 + HCOa- ] ,mM

Fig. 5a and b. Comparison of the rates of photosynthetic 02 evolution with that of influx of Cjnorg in response to the Cinorg concentration in the medium, in low- (a) and high- (b) COg A. variabilis. Data for photosynthesis rate are those depicted in Fig. I. The rate of influx of C~.org is calculated from the kinetic data obtained from Fig. 4. At external concentrations of 20 and 50 gM C~norg the rate of its influx in low-CO2 cells (a) were 478 and 1000 ~tmol- mg- ~ Chl. h - l, respectively

the photosynthetic affinity to Cinorg of intact, l o w - C O 2 Anabaena (Fig. 6). The maximum rate of photosynthesis was not affected by the inhibitor. It is therefore unlikely that the concentration of inhibitor used here altered the photosynthetic rate by reducing the rate of electron transport (Swader and Jacobson 1972; Graham et al. 1974; Longerman and Sargent 1978). The same inhibitor also reduced the ability of Anabaena cells to accumulate C i n o r g within the cells (Fig. 7). The degree of inhibition was time dependent and it is possible that exposing the cells to the inhibitor for longer periods of time prior to the addition of HCO~ would result in larger inhibition of accumulation of Ci,org. The rate of accumulation of acidstable 14C was also affected by the inhibitor as predicted from O2-evolution measurements (Fig. 6).

224

A. Kaplan et al. : Inorganic Carbon Pool in Anabaena i

~g

i

i

i

B

8

o"

/c

i

0

[ z.S,,r~ol~,~l-

i

2

3 Time,min

i

~

q

5

4

6

Fig. 6. R e s p o n s e of photosynthetic 0 2 evolution by low-CO 2 A.

variabilis to ethoxyzolamide(10 ~ M). Data obtained from the O2-electrode chart. Curve A control following addition of NaHCO3 to final concentrationof 20 gM in the absence of ethoxyzolamide; B=control with saturating concentration of NaHCO3 (60 gM); C=20 gM NaHCO3 in the presence of ethoxyzolamide; D=500 gM NaHCO3 which in the presence of ethoxyzolamide is the concentration required to saturate the rate of Oz evolution

i

2C

i

In~ernalICOn+H

4o


2 mM CO2 (not shown). This is 10-fold higher than that of the RnBP carboxylase of Anabaena (Table 1) and it is therefore suggested that the different species of Cinorg inside Anabaena cells may not be in rapid equilibrium. Therefore free COz concentration may be lower than that expected under equilibrium conditions. Nevertheless this 50- to 500 fold potential concentration gradient of CO2 between the inside and outside of these cells must contribute to generating a higher COz concentration at the site of the RuBP-carboxylase reaction and may explain the kinetics of photosynthesis (Table 1). It is concluded that Anabaena cells are capable of concentrating CO2 within their volume, as is the case in Chlamydomonas (Badger et al. 1977, 1978). This seems to be the result of an active transport process, but the mechanism is unclear.

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D6hler, G. (1974) Carboanhydrase-Aktivit~it und Enzyme des Glykolatweges in der Blaualge Anacystis nidulans. Planta 117, 97 99 Falkner, G., Horner, F., Werdan, K., Heldt, H.W. (1976) pH changes in the cytoplasm of the blue-green algae Anacystis nidulans caused by light-dependent proton flux into the thylakoid space. Plant Physiol. 58, 717 718 Findenegg, G.R. (1974) Carbonic anhydrase and the driving force of light dependent uptake of C1- and HCO3 by Scenedesmus. In: Membrane transport in plants, pp. 192-196, Zimmermann, U., Dainty, J., eds. Springer, Berlin Heidelberg New York Findenegg, G.R. (1976) Correlation between accessibility of carbonic anhydrase for external substrate and regulation of photosynthetic use of CO2 and HCO~ by Scenedesmus obliquus. Z. Pflanzenphysiol. 79, 428-437 Golubic, S. (1973) The relationship between blue-green algae and carbonate deposits. In: The biology of blue-green algae, pp. 434-472, Carr, N.G., Whitton, B.A., eds. Blackwell Scientific Pnbt., Oxford

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