Photosynthesis Research 12:25-33 (1987) © Martinus Nijhoff Publishers, Dordrecht--Printed in the Netherlands

25

Regular paper

Effect of dissolved inorganic carbon on oxygen evolution and uptake by Chlamydomonas reinhardtii suspensions adapted to ambient and C02-enriched air DIETER

F. S O L T E M E Y E R ,

KLAUS

KLUG

& HEINRICH

P. F O C K *

Fachbereich Biologie, Universitgtt Kaiserslautern, Postfach 3049, D-6750 Kaiserslautern, Federal Republic of Germany; (*author for correspondence) Received 10 March 1986; accepted in revised form 16 July 1986

Key words: Chlamydomonas reinhardtii, CO2/HCO 3 concentrating mechanism, Mehler reaction, oxygen evolution, oxygen uptake, pseudocyclic photophosphorylation Abstract. Mass spectrometric measurements of 1602and 1802isotopes were used to compare the rates of gross 02 evolution (E0), 02 uptake (U0) and net O2 evolution (NET) in rela~on to different concentrations of dissolved inorganic carbon (DIC) by Chlamydomonasreinhardtii cells grown in air (air-grown), in air enriched with 5% CO2 (CO2-grown) and by cells grown in 5% CO2 and then adapted to air for 6 h (air-adapted). At a photon fluence rate (PFR) saturating for photosynthesis (700#mol photons m -2 s-l), pH = 7.0 and 28 °C, U 0 equalled E 0 at the DIC compensation point which was 10/~M DIC for CO2-grown and zero for air-grown cells. Both E 0 and U0 were strongly dependent on DIC and reached DIC saturation at 480 pM and 70 #M for CO 2-grown and air-grown algae respectively. U0 increased from DIC compensation to DIC saturation. The U0 values were about 40 (CO2-grown), 165 (air-adapted) and 60gmolO2mgChl ~h -1 (air-grown). Above DIC compensation the U0/E 0 ratios of air-adapted and air-grown algae were always higher than those of CO2-grown cells. These differences in O2 exchange between CO2- and air-grown algae seem to be inducable since air-adapted algae respond similarly to air-grown cells. For all algae, the rates of dark respiratory O2 uptake measured 5 min after darkening were considerably lower than the rates of 02 uptake just before darkening. The contribution of dark respiration, photorespiration and the Mehler reaction to U0 is discussed and the energy requirement of the inducable COz/HCO; concentrating mechanism present in air-adapted and air-grown C. reinhardtii cells is considered. Abbreviations: DIC - dissolved inorganic carbon, DCMU - 3-(3,4-dichlorophenyl)-l,l-dimethylurea, E0 - rate of photosynthetic gross 02 evolution, PCO - photosynthetic carbon oxidation; PFR - photon fluence rate, PS ! - photosystem I, PS II photosystem II, U0 - rate of O2 uptake in the light; MS - mass spectrometer Introduction

The simultaneous evolution and uptake of 02 in the light is a well known feature of photosynthesizing cells (for a review see [4]). While 02 production is only associated with the photolysis of water [11], several reactions including mitochondrial respiration [16, 20], photorespiration [4, 7, 8] and photoreduction of Oz (Mehler reaction; [13, 25]) may contribute to O2 consumption in the light. When grown at ambient CO2 (0.034% CO2) cyanobacteria and green algae have been shown to actively accumulate bicarbonate [2, 3, 21]. Photosynthesis

26 supplies energy for the concentrating mechanism [2, 21], although the reactions involved in providing the energy for this process are not known. Some authors favour PS I driven cyclic electron flow (cyclic photophosphorylation) as the source of energy [14, 22]. However, D C M U inhibits the accumulation of bicarbonate suggesting a requirement for linear electron transport-mediated ATP generation [2, 3]. In Order to investigate the reactions which may be involved in providing energy for the CO2/HCO3 concentrating mechanism we compared the rates of gross 02 uptake (U0) and evolution (E0) for CO2-grown, air-adapted, and air-grown Chlamydomonas cells in relation to different concentrations of DIC.

Materials and methods

Chlamydomonas reinhardtii cells (strain 137c) were grown axenically in batch cultures in sterile nutrient solution as described previously [25]. The growth medium contained 10raM NH + instead of N O ; in order to prevent electron flow to nitrate reduction. The cultures were bubbled with air (0.034% CO2; air-grown algae) for 4 days or with air enriched with 5% CO2 (CO2-grown algae) for 2 days. Both gas streams (201 h- 1) were passed through sterile filters before entering the algal suspensions. For studies on the 02 gas exchange during adaptation to air, CO2-grown cells were transferred to ambient air and adapted for 6 h (air-adapted algae). After harvesting, the algae were concentrated to a Chl concentration of 600-1000/~gml i and bubbled with sterile CO2-free air in the dark for up to three hours [25]. This treatment had no effect on the 02 gas exchange of the algae [9, 25]. The rates of 1602 evolution and 1802 uptake were measured using a closed system consisting of an assimilation chamber, a MS (GD 150/4, Varian MAT, Bremen) and a Clark-type oxygen electrode [25]. All these experiments were carried out in sterile CO2-free nutrient solution (pH = 7.0, t = 28 °C) with an initial total 02 concentration of about 21% which contained low 1602and high I802 partial pressures. Light was provided by a 250 W projector lamp (model Prado Universal, Leitz GmbH, D-6330 Wetzlar). It was adjusted to a photon fluence rate of 700 ~mol photons m -z s-1 which saturated photosynthetic net Oz evolution [25]. The assimilation chamber was filled with 40 ml nutrient solution and connected to the MS. 02 consumption by both MS and the O2 electrode was less than 4nmol 02 h -~ (blank). After reading the blank for 10-15rain the light was switched on and an aliquot of concentrated algal suspension was introduced into the chamber to give a final Chl concentration of 1-3/~g ml- 1. The cells were allowed to reach DIC compensation and then, every third minute, the required volume (2 to 21/A) of an appropriate KHCO3 stock solution (0.1 to 3 M) was injected into the algal suspension to produce the desired DIC concentration.

27 When DIC saturation was reached, the cells were darkened and 5 rain after darkening the rate of dark respiratory 02 uptake was measured. The rates of gross 02 (E0) and uptake (U0) were calculated from the MS signals (m/e = 32 for 1602 and m/e = 36 for 1802) using expressions reported by Radmer and Kok [17]. The sum of E 0 and U0 did not deviate by more than 5% from NET calculated from the 02 electrode signal at light saturation and at DIC concentrations between 0 and 80/~M DIC. Chlorophyll was extracted overnight at 4 °C in 80% acetone in the presence of a trace of CaCO3 and determined according to Arnon [1]. All the chemicals used were reagent grade. Solutions were prepared almost CO2 and O2 free by bubbling with sterile C O 2 - f r e e N 2. Labelled oxygen (180, 99.88 Atom%) was supplied by Kontron GmbH, D-7500 Karlsruhe.

Results

The responses of E0, U0 and net O2 exchange by CO2-grown, air-adapted and air-grown C. reinhardtii cells in relation to DIC in the medium are shown in Figs 1-3. At a saturating PFR (700#mol photons m-2s-1), CO2-grown cells exhibited photosynthetic net 02 exchange with a compensation point of 10#M DIC, an apparent KM (DIC) of 64 #M and maximal net rates of O2 evolution at DIC concentrations higher than 480/~M (Fig. 1). Air-adapted and air-grown cells showed similar net O: exchange kinetics, namely an undetectable DIC compensation point, an apparent KM (DIC) of 8-13/~M and saturation between 60 and 80#M DIC (Figs 2 and 3). For all algal cultures, maximal net 02 evolution was near 300 #mol mg Chl- 1h- t. 400

L._~']-

x

< -T-

NET " 200

X Lu ._.5 i

100

2oo

3oo

DIE [,uH]

4oo

Uo

560 '

4,

660

Fig. 1. Short-term effect of increasing DIC-concentrations on gross 02 evolution (E0) , 02 uptake (U0) and net 02 exchange (NET) in CO2-grown Chlamydomonas reinhardtii cells. Temperature = 28°C, pH = 7.0, Chl-content = 2.54#gml ~, and P F R = 700#mol photons m -2 s -I • 02 concentration was 244/~M (22.3%) and 368/~M (33.6%) at the beginning and at the end of the light period, respectively. Dark respiration five minutes after darkening was 40 #tool 02 mgChl ~h -l. One typical response curve out of four replicates with similar trends is shown.

28

/

6001

,,, ./---*

,._,400t ~ ,,T=

| ,/

~T*

~/

E° NET _.-/---4--

(°°t/'.-y-" c,40

~

o~

-'/,Z - - ~

-

o

/"

OI/"

10

5

15

45

"

CO' !ONEENTRATION [#M-] 20

I+

250

DIE[,uM]

Fig. 2. Short-term effect of increasing DIC-concentrations on gross 02 evolution (E0), 02 uptake (U0), and net 02 exchange (NET) in air-adapted Chlamydomonas reinhardtii cells after 6 h adaptation. Temperature = 28°C, pH = 7.02, Chl content = 1.01#gml -~, and PFR = 700#tool photons m-as 1.02 concentration was 206pM (19%) and 340/#M (31%) at the beginning and at the end of the light period, respectively. Dark respiration five minutes after darkening was 160 #tool 02 mgChl-~ h ~. One typical response curve out of four replicates with similar trends is shown.

w~

/+00

Eo NET

-'r-

x E 200 u~

OE

o

io '

2b

CO 2

CONCENTRATION [laM ]

'

~0

'

6'0

DIE [pM ]

'

2'0

8'0

Fig. 3. Short-term effect of increasing DIC-concentrations on gross O z evolution (E0), 02 uptake (U0) and net O2 exchange (NET) in air-grown Chlamydomonas reinhardtii cells. Temperature = 28°C, pH = 7.0, Chl-content = 1.55#gml -~, and PFR = 700/~mol photons m 2s- 1. 02 concentration was 241 pM (22.0%) and 378 pM (34.5%) at the beginning and at the end of the light period, respectively, Dark respiration five minutes after darkening was 59 #mol 02 mgChl -~ h-L One typical response curve out of four replicates with similar trends is shown.

29 At DIC compensation, E 0 and U 0 were approximately 40 (CO2-grown), 100 (air-adapted), and 60 #mol 02 mg Chl-~ h-J (air-grown). With increasing DIC, E0 of CO2-grown cells increased to 360 #tool 02 mg Chl-I h-1 at 610 #M DIC, whereas E0 of the air-adapted and air-grown cells reached 590 and 420#molOzmgChl-~h 1 at 100#M DIC, respectively. E0 became rate saturated at 480 #M DIC in the CO2-grown algae and at 60 to 80 #M DIC in both air-adapted and air-grown cells (Figs 1-3). U0, as a function of DIC, rose to 78 #tool O 2 mg Chl-1 h - 1 at 610 #M DIC in CO2-grown cells. This enhanced rate of 02 uptake in relation to increasing DIC was more pronounced in air-adapted and air-grown cultures where U0 reached 265 and 120 #tool 02 mg Chl- l h 1at 100 #M DIC, respectively. U0 seemed to be saturated at 260 #M DIC in CO2-grown and at 60-80/~M DIC in air-adapted and air-grown algae. Five minutes after darkening the mitochondrial respiratory O2 consumption was highest in air-adapted cells, namely 164/~mol O2mg Chl ~h-~, while airgrown and CO2-grown cells showed 59 and 38.5 #tool 02 mg Chl-1 h -~, respectively. These dark respiratory rates were always considerably lower than U0 just before darkening in all cultures (Figs 1-3). The ability of air-adapted and air-grown cells to consume more O2 than CO2-grown cells (Figs 1-3) was further analysed by comparing the U0/E0 ratios of these cultures at different DIC concentrations (Table 1). At 700 #mol photons m-Zs -1 the U0/E0 ratios were higher in air-adapted and air-grown than in CO2-grown algae, except at DIC compensation where the ratios reached unity (Figs 1-3; Table 1). With increasing DIC the ratio decreased to 0.28 (CO2grown), 0.45 (air-adapted) and 0.37 (air-grown) at saturating DIC concentrations (Table 1).

Discussion

The responses of net 02 exchange in relation to different DIC concentrations presented in Figs 1-3 show that air-grown algae exhibit a higher affinity for DIC than cells grown in 5% CO2. The apparent Ku (DIC) was 5-8 times higher in CO2-grown than air-grown C. reinhardtii (64 versus 8-13 #M DIC). As with earlier results [6, 12], the DIC compensation point of air-grown algae was Table 1. U 0/E0-ratios of CO 2-grown, air-adapted and air-grown Chlamydomonas reinhardtii cells at three DIC concentrations: CP = DIC-compensation point, K M (DIC), DICsAT = DIC saturating for photosynthesis. For both experiments these were: 10/~M, 64/~M, 480pM (CO2-grown) and 0/zM, 10/~M, 100/IM (air-adapted and air-grown). The values represent the means of 3 to 5 different experiments (+ SD). DIC Concentration

CO2-grown

Air-adapted

Air-grown

CP K M (DIC) DICsAT

1.00 + 0.00 0.38 + 0.08 0.28 _+ 0.07

1.00 + 0.00 0.56 + 0.03 0.45 + 0.01

1.00 + 0.00 0.42 + 0.06 0.37 _+ 0.02

30 approximately zero and photosynthetic DIC saturation was achieved at much lower DIC concentrations than in CO2-grown algae. Air-adapted cultures showed similar responses to external DIC as did air-grown algae (Fig. 2). These air-grown cells transferred to 5% CO2 for at least 12 h responded similarly to those which were CO2-grown (data not shown). This indicates that a 6h exposure to ambient air is sufficient to induce the high DIC affinity of air-grown cells. For all algae, however, maximal NET was similar at about 300 #tool Oz mg Chl-~ h -1 at saturating DIC levels. This is in accordance with other results [6] and correlates well with the same capacity for CO2 fixation in all cells [5].

Oxygen evolution in the light (Eo) At a PFR level which saturated net photosynthesis and at low concentrations of DIC, the algae were limited for CO2 the primary acceptor of photochemically generated electrons (Figs 1-3). Consequently, E0, as a direct measure of total electron flow from water via PS II, P S I to ferredoxin, was inhibited and finally equalled U0 at the DIC compensation point. In contrast to Radmer and Kok's view [17], under our experimental conditions at light saturation, 02 had only a limited capacity to replace CO2 as an electron acceptor in all algal cultures. Both, air-grown as well as air-adapted cultures have higher E0 values than CO2-grown algae above DIC compensation and up to DIC saturation. In contrast, air-grown cultures transferred to 5% CO2 for at least 12h lost their high E0 values (data not shown). This indicates that the enhanced electron flow of air-adapted and air-grown cells is induced during adaptation to ambient air and that it is correlated with the induction of the mechanism(s) responsible for the high DIC affinity of air-grown cells [2, 6]. Because air-adapted and air-grown algae have the same capacity to reduce CO2 at levels of DIC which saturate photosynthesis [5], this enhanced electron flow must be associated with an increased reduction of some other electron acceptor in these cells.

Oxygen uptake in the light (Uo) PCO cycle activity is one of the possible contributors to U0 in the light [7, 8]. U 0 by all algae increased with increasing DIC concentrations (Figs 1-3). The increase to U0 was proportionally less in CO2-grown algae (about 60%; Fig. 1) compared to air-adapted (about 160%; Fig. 2) and air-grown cells (about 110%; Fig. 3). This rise of U0 with increasing DIC cannot be explained by 02 uptake in the PCO cycle, although this cycle is active under limiting DIC conditions in both CO2- and air-grown C. reinhardtii [12, 15, 24]. From the results obtained at 28 °C (Figs 1-3) it is suggested that increasing the DIC in the algal suspension every third minute may induce extra O 2 consuming reactions [24]. Especially at higher DIC concentrations, there is very little if any 02 consumption by the PCO

31 cycle of these cells [12, 15]. Thus, 02 uptake under these conditions probably consists of both mitochondrial 02 uptake and photoreduction of 02. Mitochondrial O2 consumption measured five minutes after illumination was lower for CO2-grown cultures than for air-adapted and air-grown ceils (Figs 1-3). The question that now arises is whether the enhanced U0/E0 ratio of these latter cells (Table 1) is caused only by higher mitochondrial 02 consumption in the light. Although this possibility cannot be fully excluded, it is not very likely because increased mitochondrial 02 uptake in the light cannot explain the enhanced E0 of air-adapted and air-grown cells (Figs 2 and 3). Therefore we conclude that especially at DIC saturation, the enhanced U 0 of air-adapted and air-grown cells is caused by 02 reduction (Mehler reaction) which could explain the higher electron flow of air-grown algae. This conclusion is supported by reports from other laboratories which suggest that dark respiration is either inhibited or only a minor part of U0 in the light [8, 9, 20, 25]. This, however, contradicts Peltier and Thibault's view [16] that U0 is mainly composed of mitochondrial respiration in air-grown algae. Presumably this discrepancy could be attributed to the different experimental conditions (for more details see [25]). The ratio of U0 to E0 was always higher in air-grown cells compared to CO2-grown cells, except at the DIC compensation point of net photosynthesis (Figs 1 and 3; Table 1). Similar ratios for CO2- and air-grown algae have been reported in light response experiments [25] as well as by Fock et al. [9]. This pattern, together with the enhanced U0 of air-grown compared to CO2-grown cells, is probably induced during adaptation to ambient air, because air-adapted algae showed even higher values (Table 1). On the other hand, when air-grown cells were transferred to 5% CO2 for at least 12 h, this higher rate of 02 uptake disappeared. This induced higher O2 uptake seems to be correlated to the induction of the CO2/HCO3 concentrating mechanism known to be present in air-adapted and air-grown cells [2, 21, 25]. Although the energy requirement for this process is not known, ATP is probably the primary energy source for this mechanism in eukaryotic organisms [4, 23]. As in C4 plants [11], part of this extra ATP could be synthesized by 02 consumption in the Mehler reaction [13] via pseudocyclic phosphorylation [18]. Pseudocyclic phosphorylation has been suggested in the case of active phosphate transport in Hydrodictyon [19]. However, under certain conditions [14, 22] cyclic photophosphorylation also appears to drive the CO2/HCO3 concentrating mechanism which indicates a role of both cyclic and pseudocyclic ATP generation in supplying energy for the accumulating process.

Acknowledgement We are grateful to Professor R. Germerdonk (Fachbereich Maschinenwesen, Universit/it Kaiserslautern) for providing the GD 150 mass spectrometer used during the course of these experiments. We also wish to thank Prof. G. Bate

32 ( U n i v e r s i t y o f P o r t E l i z a b e t h , S o u t h A f r i c a ) f o r critical r e v i s i o n o f the m a n u script. T h i s w o r k w a s s u p p o r t e d by the D F G g r a n t F O 72/13-3 to H . P . F .

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