Photosynth Res DOI 10.1007/s11120-014-0016-6

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CO2 acquisition in Chlamydomonas acidophila is influenced mainly by CO2, not phosphorus, availability Elly Spijkerman • Slobodanka Stojkovic John Beardall



Received: 24 September 2013 / Accepted: 19 May 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The extremophilic green microalga Chlamydomonas acidophila grows in very acidic waters (pH 2.3–3.4), where CO2 is the sole inorganic carbon source. Previous work has revealed that the species can accumulate inorganic carbon (Ci) and exhibits high affinity CO2 utilization under low-CO2 (air-equilibrium) conditions, similar to organisms with an active CO2 concentrating mechanism (CCM), whereas both processes are down-regulated under high CO2 (4.5 % CO2) conditions. Responses of this species to phosphorus (Pi)-limited conditions suggested a contrasting regulation of the CCM characteristics. Therefore, we measured external carbonic anhydrase (CAext) activities and protein expression (CAH1), the internal pH, Ci accumulation, and CO2-utilization in cells adapted to high or low CO2 under Pi-replete and Pi-limited conditions. Results reveal that C. acidophila expressed CAext activity and expressed a protein cross-reacting with CAH1 (the CAext from Chlamydomonas reinhardtii). Although the function of this CA remains unclear, CAext activity and high affinity CO2 utilization were the highest under low CO2 conditions. C. acidophila accumulated Ci and expressed the CAH1 protein under all conditions tested,

E. Spijkerman (&) Universita¨t Potsdam, Am Neuen Palais 10, 14469 Potsdam, Germany e-mail: [email protected] S. Stojkovic  J. Beardall School of Biological Sciences, Monash University, Clayton, VIC 3800, Australia S. Stojkovic Marine and Atmospheric Research, CSIRO, Hobart, TAS, Australia

and C. reinhardtii also contained substantial amounts of CAH1 protein under Pi-limitation. In conclusion, Ci utilization is optimized in C. acidophila under ecologically relevant conditions, which may enable optimal survival in its extreme Ci- and Pi-limited habitat. The exact physiological and biochemical acclimation remains to be further studied. Keywords CO2 concentrating mechanism  Inorganic phosphorus limitation  Varying CO2 condition  Extremophilic green alga  Co-limitation  Internal pH  Inorganic carbon accumulation  Affinity for CO2 uptake

Introduction The extremophilic green microalga Chlamydomonas acidophila inhabits very acidic environments (pH 2.3–3.4) where inorganic carbon (Ci) and phosphorus (Pi) concentrations limit its growth (Spijkerman 2008a; Tittel et al. 2005). The water has a low pH that is buffered by iron and sulfate and contains a negligible concentration of bicarbonate, resulting in an environment where CO2 is the sole inorganic carbon source (Stumm and Morgan 1970). Earlier experiments under Pi-replete conditions revealed that C. acidophila accumulated Ci up to 35-fold under low CO2 conditions whereas it did not do so under high CO2 (Spijkerman 2005). In the same study, high affinity CO2 utilization, as measured by O2-evolution, was found under both low and high CO2 conditions, whereas when this experiment was repeated in dilute semi-continuous cultures, the affinity for CO2 utilization was threefold higher at low than at high CO2 conditions (Spijkerman 2008b). The same difference in affinity was obtained in similar CO2 but Pi-limiting conditions (Spijkerman et al. 2011). Thus,

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C. acidophila has a high affinity CO2 utilization under both Pi-replete and Pi-limited conditions when grown under low CO2, but are CO2 or Pi conditions major players affecting this species? Therefore, we posed the following research questions: (1) (2)

Is the Ci accumulation of C. acidophila influenced by the cellular P quota? Is an external carbonic anhydrase (CAext) involved in the high affinity CO2 utilization of C. acidophila?

CCM characteristics in neutrophilic algae were first described in Chlamydomonas reinhardtii (e.g., Badger et al. 1980), while CCM acclimation in relation to ecological features has been described for C. reinhardtii and other microalgae (Giordano et al. 2005). One important feature of the CCM in C. reinhardtii is a fourfold enhanced CO2 concentrating factor (CCF) under low CO2 compared to high CO2 conditions (Badger et al. 1980). Another acclimation to low CO2 is an enhanced affinity for Ci utilization as shown by a 20-fold lower affinity constant (K0.5) compared to high CO2 (Su¨ltemeyer et al. 1991; Villarejo et al. 1996). A third important acclimation is the expression of carbonic anhydrases (Moroney et al. 2011), among which the periplasmic CAext is highly expressed under low CO2, enabling the use of bicarbonate by its conversion to CO2 at the cell surface, although its essential role is questionable (Spalding et al. 2002; Van and Spalding 1999). Unfortunately, less is known about the regulation of the CCM under Pi-limiting conditions. As far as we know only two studies to date have reported on the interaction between a CCM and Pi-limiting conditions and these studies, both performed on Chlorella species, showed contrasting changes in their CCM acclimation in response to Pi-limitation. KozlowskaSzerenos et al. (2004) showed stimulation of photosynthetic rates and affinity for Ci under high light and CO2 when Pi-limitation was applied, whereas Beardall et al. (2005) reported that Pi-limitation caused a down-regulation of CCM activity. From these limited studies of the ecological relevant interaction of Pi and Ci acquisition, these contradictory results cannot be resolved. From an ecological viewpoint Pi-limitation is of great importance as Pi is often a limiting factor for freshwater algal growth, especially in acidic lakes (Spijkerman 2008a). Therefore, we decided to study Ci acquisition in C. acidophila under variable Pi conditions in a direct Ci uptake assay and examined molecular/biochemical aspects of this acclimation. We measured the Ci concentrating factor (CCF), CAext activity, the expression of CAH1 protein, the photosynthetic response to the CA inhibitor acetazolamide and the K0.5(CO2) by O2 evolution in cultures adapted to high or low CO2, and Pi-replete and Pi-limited conditions.

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Materials and methods Cultures Chlamydomonas acidophila Negoro (CCAP 11/137) was cultured in modified WH medium at pH 2.6 (Gerloff-Elias et al. 2005). Pi-limiting conditions were brought about by lowering the medium Pi concentration from 50 to 1.5 lM, and either culturing semi-continuously at a growth rate of 0.2 day-1 or, for some experiments, by Pi-depleting for 10 days in batch growth. Pi-replete cultures were either cultured semi-continuously at a growth rate of 0.4 day-1 or grown in batch mode. Cultures were non-aerated during Piacclimation and were subsequently measured under these low CO2 conditions or after acclimation to high CO2 for 2 days by aerating with a 4.5 % CO2/air mixture (v/v). This short treatment did not fully acclimate all cells to high CO2 conditions, but was necessary to maintain a similar cellular P quota (Qp) at both high and low CO2. Earlier experiments revealed that long-term acclimation to a certain medium P-concentration resulted in a 5.5-fold lower Qp under high CO2 than under low CO2 conditions (Spijkerman 2011). All cultures were kept at 20 °C under continuous light and were monitored for external pH, cell density, and Qp determined by measuring cellular P and C on appropriate filters (Spijkerman et al. 2012). Cell densities were determined on a CASY1 cell counter (Scha¨rfe Systems GmbH). CO2 concentrations in the cultures were measured by direct injection into a carbon analyzer (HighTOC ? N, Elementar). Statistical analyses were performed in SigmaPlot (version 11.0). Ci accumulation, cell volume and internal pH To measure Ci accumulation, internal pH and cell volume, cultures were harvested by centrifugation (1,5009g, 5 min) and concentrated to an optical density of 1.5 at 750 nm. After 30–60 min acclimation in the light (200 lmol PAR m-2 s-1) to empty the internal Ci pool, the culture suspension was loaded on a silicon oil layer on top of a killing fluid (1 M glycine pH 10; Spijkerman 2005). The oil consisted of a mixture of 1:3 (v/v of silicon oil ‘‘type 3’’:‘‘550’’ for gas chromatography, Merck, Darmstadt, Germany). About 50–70 lM NaH14CO3 (specific activity 1,739 GBq mmol-1; Perkin Elmer) was provided to the algae in the light (200 lmol PAR m-2 s-1), and uptake was terminated after 10 s by quick centrifugation (12,0009g, 15 s). Algae from all different treatments (i.e., high and low CO2, Pi-replete and Pi-limited) were exposed to approximately the same external Ci and consequently the same CO2 concentration, although Ci concentrations turned out to be higher in the experiments with high CO2 cultures (73 ± 1 lM) than with the low CO2 cultures

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(57 ± 4 lM).The centrifuge tube was quickly frozen in liquid nitrogen, and the algal pellet was removed by cutting the end off the tube. Cells were resuspended in 400 ll, 0.1 M NaOH, of which 150 ll was transferred to 150 ll of 0.1 M NaOH and 150 ll into 150 ll of 0.5 M HCl in methanol. Acid-labile carbon was allowed to evaporate overnight under a fume head after which all samples received 2.5-ml Hionic Fluor (Perkin Elmer, RodgauJu¨gesheim, Germany), and radioactivity was determined in a liquid scintillation analyzer (Tri-Carb 2810 Perkin Elmer). The accumulation of Ci was determined from the difference between acid-stable and acid-labile fractions. Similarly, in the same algal suspension, internal pH was measured by equilibration with 0.5 lCi 14C-DMO (5,5dimethyl-2-[14C]oxazolidine-2,4-dione; specific activity 55 mCi mmol-1, Hartmann-analytic, Germany) and cell volume by incubation with 1 lCi 3H–H2O (specific activity 1 mCi ml-1, Hartmann-analytic, Germany) following Beardall (1981). In short, DMO was equilibrated in the algal suspension for 20 min after which cells were centrifuged through the oil. Supernatant and cell pellet were counted, and internal pH was calculated using the Henderson–Hasselbalch equation using a pKa value of 6.14. The Ci accumulation factor (CCF) was expressed as accumulated Ci over 10 s based on cell volume, related to the added Ci concentration at time zero. CAext activity CAext was measured using a potentiometric method following methods described in Ratti and others (Ratti et al. 2007; Stojkovic et al. 2013). Cultures were harvested by centrifugation (1,5009g, 5 min), and pellets were resuspended to a final volume of 5 ml in 20 mM phosphate buffer (pH 8.6). We used a Metrohm pH meter and recorded the pH change from 8.2 to 7.2 after addition of CO2-saturated water at 4 °C. Activity is calculated from Eq. 1: Relative enzyme activity (REA) = 10  ½ðTblank = Tcells Þ  1  ½ml culture1

ð1Þ

in which Tblank and Tcells are the times (s) for 1 unit pH change for blank and samples, respectively. Equal volumes of buffer and substrate (2 ml) were used with 0.5 ml sample, or 0.5 ml buffer as a blank. Activity was expressed on a cell basis by dividing REA by cells ml-1. No cell lysis or proton extrusion was observed during the assay. Expression of CAH1 For this assay, cultures of Chlamydomonas reinhardtii Dangeard (SAG 11–32b) were grown in Pi-replete (50 lM

P) and Pi-limited (1.5 lM P) WH medium at pH 7, buffered with 10 mM HEPES without aeration (low CO2). After 10 days of batch growth under Pi-limited conditions, acclimation to high CO2 was established by aerating with a 4.5 % CO2/air mixture (v/v) for 2 days. For both C. reinhardtii and C. acidophila, dilute Pi-replete cultures were grown at high CO2 for 2 days to suppress CAH1 expression and to diminish the effect of CAH1 possibly produced under standard low CO2 conditions. Cultures were harvested by centrifugation (2,5009g, 5 min), and pellets were washed with distilled water. After extraction of protein and denaturation (90 °C, 5 min.), protein concentrations were determined according to Ghosh et al. (1988), and 20 lg of protein was loaded per lane on an SDS gel as described in the BioRad manual (Gerloff-Elias et al. 2006). Protein expression of the CAH1 protein was determined via Western blotting using a specific polyclonal antibody against the CAext (a kind gift of J. Moroney; Ynalvez et al. 2008). An antibody against the nuclear H3 protein served as a loading control (AS10 710, Agrisera). The specificity of the reaction obtained with the CAH1 antibody was tested by affinity chromatography on a paminomethylbenzene sulfone amide (p-AMBS) agarose column (Sigma) following Satoh et al. (2001). After loading the sample on the gel bed, six-eluted aliquots of 100 ll were collected by an elution buffer (elution buffer 1) with 25 mM Tris-H2SO4 containing 0.1 M Na2SO4 followed by elution with the same buffer containing 0.3 M NaClO4 (elution buffer 2; Satoh et al. 2001). The column will initially bind many proteins, most of which are eluted with the first buffer. All proteins containing sulfonamidebinding sites (e.g., CA) will only be eluted by the second buffer. Samples rich in CA (i.e., C. reinhardtii -CO2 ?P) were run over the affinity chromatography gel bed, and the most protein-rich eluted fractions were then loaded on a gel together with total cell extracts, and CAH1 protein determined via Western blotting as described above. Affinity for CO2 uptake by photosynthesis and chlorophyll determination The affinity for CO2 uptake was determined via measuring the response in O2 evolution after addition of eight different Ci concentrations in a light dispensation system (PLD 2, Topgallant LLC, USA) equipped with a Clark-type O2 electrode (Microelectrodes Inc, USA; Spijkerman 2005). Cultures were harvested by centrifugation (1,5009g, 5 min) and concentrated to an optical density of 0.2 or 0.6, for Pireplete and Pi-limited cells, respectively, at 750 nm in Cidepleted growth medium. Measurements were performed at 500 lmol PAR m-2 s-1 and 20 °C. After correcting rates for chlorophyll a (Chl a) concentration, Michaelis–Menten kinetics were linearized according to Hofstee (1952) and the

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maximum uptake rate (Vmax) and K0.5(CO2) estimated. From these two parameters, the affinity for CO2 uptake as defined by Vmax/K0.5(CO2) was calculated. From the concentrates, samples were extracted with 90 % acetone and measured in a spectrophotometer (UV2401PC, Shimadzu, Japan) at 750, 664, and 647 nm for calculation of the Chl a concentration according to Jeffrey and Humphrey (1975). Photosynthetic response to the CA inhibitor acetazolamide Cultures were harvested by centrifugation (1,5009g, 5 min), and pellets were resuspended to an optical density of 0.2 at 750 nm in a Ci-depleted medium, buffered at pH 5.0 with 5 mM MES buffer or at pH 7.0 with 5 mM HEPES buffer. O2 evolution was recorded in the light dispensation system at 500 lmol photons m-2 s-1 and 20 °C for one minute after addition of 10 lM Ci. Then, 35 lM acetazolamide (final concentration; AZ) was added and the response to this CAext inhibitor was followed. The effect is expressed as Effect of AZ addition ¼O2 evolution after AZ addition= O2 evolution before AZ addition  100 % Results CCF After 10 s of Ci incorporation in the light there was no difference in CCF between the different growth conditions (ANOVA, F3,8 = 1.47, P = 0.30; Fig. 1). The CCF was higher in Pi-limited, low CO2 grown cells than in the other conditions (T test, t10 = -2.25, P \ 0.05; Fig. 1). All CCF values were higher than the theoretical value for a CCF totally dependent on diffusive CO2 incorporation (i.e., 1.7). Thus, the CCF in C. acidophila was possibly saturated under all conditions tested and not under regulation of CO2 or cellular P quota, although both medium CO2 concentrations and Qp varied considerably (Table 1). In general, CCF values ranged between 11.3 and 22.6 based on external Ci concentrations measured and the cellular Ci pool based on the cell volume estimated with 3H–H2O. The external pH values were slightly influenced by culture density and CO2 aeration and ranged between 2.75 in Pilimited, low CO2 cultures and 2.95 in Pi-replete, high CO2 cultures (Table 1). The internal pH values as measured by 14 C-DMO were similar under all culture conditions and ranged between 5.83 in Pi-replete, low CO2 cultures and 6.11 in Pi-limited, high CO2 cultures. Accumulated Ci

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Fig. 1 CCF in C. acidophila after 10 s. Mean ± SD of three independent replicates grown in Pi-limited (-P), Pi-replete (?P), low CO2 (-CO2), and high CO2 (?CO2) conditions. Cell densities (cells ml-1 in mean ± SD) used in the assays were 2.0 ± 0.2 9 107 (-P -CO2), 1.22 ± 0.3 9 107 (-P ?CO2), 2.0 ± 0.4 9 107 (?P -CO2), and 1.9 ± 0.2 9 107 (?P ?CO2)

pools ranged between 0.6 and 1.2 mM without any clear pattern between growth conditions (Table 1). External carbonic anhydrase Measured CAext activity was low (but present) in high CO2-grown C. acidophila (Fig. 2), whereas significantly higher activity was measured in cells growing at low CO2 conditions (Mann–Whitney-U, T = 16 (n1,2 = 6), P \ 0.01). Although the Pi status of the cells varied considerably (Table 1), it did not influence CAext activity in this species under low CO2 conditions as there was no significant difference between the Pi-replete and Pi-limited cells (Mann–Whitney-U, T = 7 (n1,2 = 3), P = 0.33). Western blots using a polyclonal antibody against CAH1 showed the typical CAH1 protein expression in C. reinhardtii at about 37 kDa in cells grown at low CO2 and hardly any expression in cells grown at high CO2, Pireplete conditions (Fig. 3a). C. reinhardtii also expressed CAH1 under Pi-limitation. In C. acidophila a protein at approx. 40 kDa was detected under all conditions tested, and expression was the highest under Pi-replete, high CO2 conditions and the lowest under both Pi-limiting conditions (Fig. 3a). The proteins reacting with the CAH1 antibody (from C. reinhardtii and C. acidophila) eluted only with elution buffer 1 (Fig. 3b). If results on a b-CA in Phaeodactylum tricornutum hold true for the method applied in the present paper, the fraction from elution buffer 1 consists of bulk proteins, but not CA (Satoh et al. 2001). CA and other proteins which have a sulfonamide-binding site elute with elution buffer 2 (Fig. 3b). The loading control, H3, eluted with elution buffer 1 only. Results thus suggest that the C. acidophila protein at 40 kDa was a CA, the

Photosynth Res Table 1 Cellular and medium concentrations, from the Ci accumulation experiment, of the cultures of C. acidophila grown under Pi-limited and Pi-replete, low CO2 or high CO2 conditions

Cellular

Pi-limited

Pi-replete

Low CO2

High CO2

Qn (mol N:mol C)

0.15 ± 0.01

0.13 ± 0.01

0.16 ± 0.01

0.17 ± 0.01

Qp (mol P:mol C)

1.3 ± 0.2

0.8 ± 0.1

15.5 ± 0.7

13.9 ± 1.4

Chl a:C (g:g)

48.8 ± 8.1

50.0 ± 6.2

21.3 ± 3.8

30.3 ± 1.2

pg C cell-1

28.1 ± 2.6

44.8 ± 9.7

25.4 ± 1.1

28.8 ± 2.3

pHi (in cells) Ci pool (mM, after 10 s)

Low CO2

High CO2

5.9 ± 0.2

6.1 ± 0.1

5.8 ± 0.3

6.0 ± 0.3

0.87 ± 0.09

0.93 ± 0.23

0.73 ± 0.20

0.88 ± 0.14

Medium CO2 (lM in medium) Values are mean ± SD from three independent replicates

pH (in medium)

4±2

792 ± 140

14 ± 4

856 ± 22

2.75 ± 0.01

2.74 ± 0.01

2.85 ± 0.02

2.95 ± 0.01

(Fig. 2), we cannot exclude the possibility that AZ inhibited internal CA. Affinity for CO2 utilization

Fig. 2 CAext activity in C. acidophila as expressed on a cell basis. Mean ± SE of three independent replicates grown in Pi-limited (-P), Pi-replete (?P), low CO2 (-CO2), and high CO2 (?CO2) conditions. Cell densities (cells ml-1; mean ± SD) used in the assays were 4.4 ± 0.2 9 106 (-P -CO2), 2.7 ± 0.4 9 107 (-P ?CO2), 5.7 ± 1.2 9 106 (?P -CO2), and 3.0 ± 0.3 9 107 (?P ?CO2)

The affinity constant (K0.5(CO2)) for CO2-dependent O2 evolution was lower in low CO2 acclimated cultures of C. acidophila independent of their cellular P quota (2-way ANOVA, F1,19 = 15.1, P \ 0.005 for CO2 and F1,19 = 2.1, P = 0.17 for cellular P quota; Fig. 5a). Under low CO2 conditions, the K0.5(CO2) was lower (higher affinity) under Pi-limited than under Pi-replete conditions (t test, t10 = -3.59, P \ 0.05). The pattern in K0.5(CO2) directly translated into a higher affinity (measured as Vmax * K0.5(CO2)-1, i.e., the slope of the uptake kinetics curve) for CO2 utilization in low CO2 grown cells than in high CO2 grown ones (Fig. 5b). The difference between low and high CO2 acclimated cells in both K0.5(CO2) and affinity for CO2 utilization was approximately 1.5-twofold.

expression of which did not coincide with the CA activity presented in Fig. 2. Discussion Effect of AZ on O2 evolution The presence of CAext was additionally shown by partial inhibition of O2 evolution by addition of AZ at pH 7.0 and about 10 lM Ci (Fig. 4a). Cells grown at pH 2.6 and resuspended at pH 5.0 were not susceptible to AZ (Fig. 4b), but when resuspended at pH 7.0, the addition of AZ resulted in an inhibition of O2 evolution by about 20 % (2-way ANOVA, F1,16 = 15.9, P \ 0.005; Fig. 4b). This experiment was only conducted on cells grown at low CO2 concentrations, as normally these should be the conditions under which CAext is maximally expressed. Cellular P quota did not influence the response to AZ (2-way ANOVA, F1,16 = 0.02, P = 0.89; Fig. 4b). Although these observations contrast with the CAext activities that might be higher under Pi-limiting than Pi-replete conditions

In this study, we focussed on three important CCM characteristics of Chlamydomonas acidophila in response to changing CO2 and Pi concentrations. These features were the CCF, the affinity for CO2 uptake, and the expression and activity of CAext. We show that most CCM characteristics in C. acidophila were under regulation of CO2 and not Pi. Based on former experiments, we expected a CCF of 35 under Pi-replete, low CO2 conditions (Spijkerman 2005), but we were not able to reproduce this result under our standardized conditions. Our results were not influenced by the incubation time; similar to observations in Chlorella vulgaris at acidic pH, the maximum CCF pool in C. acidophila was filled within a 10 s period (Tsuzuki et al. 1985), and we obtained similar Ci pools after 30 s as with

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Fig. 3 a Compiled Western blots result of total protein extracts (20 lg) of C. reinhardtii (Cr) and C. acidophila (Ca) grown under Pireplete (?P) or Pi-limited (-P) conditions either without CO2 aeration (-CO2) or aerated with CO2-enriched air (?CO2). Dilute Pireplete, high CO2 cultures are marked with an asterisk (see ‘‘Materials and methods’’ section for more details). b Compiled Western blots result of total protein extracts (20 lg) of C. reinhardtii (Cr) and C. acidophila (Ca) grown under Pi-replete, low CO2 (-CO2) or high CO2 (?CO2). Dilute Pi-replete, high CO2 cultures are marked with an asterisk. Protein eluents from elution buffer 1 (el1) and elution buffer 2 (el2) after affinity chromatography on a p-AMBS column were prepared from Pi-replete, low CO2 cultures of both species. The blot from the highest protein concentrations in the eluent is shown. The upper part of the blot shows binding of protein to the CAH1 antibody and the lower part to the H3 antibody. Molecular weight of the marker (M) protein is indicated on the right side of the figure

10 s (values ranging between 0.6 and 1.6 mM Ci). The CCF is still higher than that expected on the basis of diffusive uptake as that would account for a factor of only 1.7 over a 10 s period and with a 50 lM initial external Ci concentration. Because K0.5(CO2) was not much regulated by CO2 or Qp in C. acidophila (Spijkerman 2011), our results indicate that a CCF between 12 and 16 (as based on

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Fig. 4 The effect of acetazolamide (AZ) addition (as indicated by the arrow) on O2 evolution in C. acidophila cultured at pH 2.6 and measured at pH 7.0 in Pi-limited and Pi-replete, low CO2 cultures (a). O2 evolution following AZ addition as a % of activity before AZ addition for cultures resuspended in pH 5.0 and 7.0 medium (b). Mean ± SE of three independent replicates are shown from Pi-replete and Pi-limited, low CO2 cultures

total Ci) is the maximum under a wide range of Qp (0.8 and 15.5 mmol P (mol C)-1) when using an initial Ci concentration of *50–70 lM. The Ci pool was rather constant ranging between 0.6 and 1.2 mM Ci, which is in the lower range of that reported before (compare with 1.3–2.5 mM in Spijkerman, 2011), but is still high enough to support the presence of active CO2 acquisition. The CCF was independent of P status, although the Qp varied between 0.8 and 15.5 mmol P (mol C)-1. The variation in Qp covers values formerly reported for C. acidophila under Pi-limiting conditions: the minimal Qp ranging between 0.6 and 1.1 mmol P (mol C)-1 under high and low CO2 conditions, respectively (Spijkerman 2010), and values reaching 20 mmol P (mol C)-1 under low CO2, Pireplete conditions (Spijkerman 2011). This contrasts with the positive relationship found between the CCF based on

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Fig. 5 Affinity constants (K0.5(CO2); a) and affinity (Vmax:K0.5(CO2); b) for CO2 uptake by O2 evolution in C. acidophila. Mean ± SD of at least three independent replicates. All measurements performed at pH 2.6

the accumulation of Ci and Qp in C. acidophila as observed within one CO2 growth condition (Spijkerman 2011) and a similar observation of a negative correlation between Pi-limitation, as realized by steady state growth rate, and CCF in Chlorella emersonii (Beardall et al. 2005). As the latter studies were both conducted in (semi-)continuous cultures, possibly prolonged exposure to Pi-limiting conditions might result in a regulation of CCF with cellular P quota for which our 10 day acclimation was too short. Ci accumulation, therefore, might only be downregulated by Qp after prolonged acclimation to low Pi stress, this despite the fact that Qp in the Pi-limiting cells was close to the minimum observed for this species (Spijkerman 2010). Interestingly, CCF values showed a roughly similar pattern to those for K0.5(CO2) and affinity values, as the highest CCF corresponds to the highest affinity for CO2 uptake under Pi-limited, low CO2 conditions. An intermediate position was observed in the Pi-replete, low CO2 conditions, whereas cells cultured at high CO2 had similar affinities. The higher affinity for CO2 uptake under Pilimitation than with Pi-replete conditions under low CO2 conditions might be a result of the lower growth rate being adjusted to obtain a Pi-limitation in contrast to a twofold higher growth rate to retain Pi-replete conditions. In C.

acidophila, this results in a regulation in the number of, rather than in a change for high affinity uptake, transporters (Spijkerman et al. 2011) grown under such potentially Pi/ CO2 co-limiting conditions. It is likely that CCF and whole cell CO2 uptake were positively correlated: the CCF was increased when affinity for CO2 utilization was increased. Interestingly, under these Pi/CO2 co-limiting conditions, physiological acclimation in C. acidophila was stronger toward CO2- than toward Pi-limitation. High affinity utilization of CO2 in C. acidophila was characterized by a low affinity constant in photosynthesis (K0.5(CO2) values varying between 2 and 3 lM), which are in range of values reported for other species measured under low pH conditions; K0.5(CO2) values varying between 1 and 3 lM; (Tsuzuki et al. 1985; Gehl et al. 1990; Zenvirth et al. 1985). For Chlorella saccharophila grown at low external pH of 2.0 and 4.0, a relatively high K0.5(CO2), ranging between 4 and 12 lM, was reported (Beardall 1981; Gehl et al. 1987); possibly as a result of higher cell densities during the measurement (Tsuzuki et al. 1985), similar to the very high K0.5(CO2) of 37 lM at pH 2.5 measured in an acid-tolerant Chlamydomonas (formerly designated as C. acidophila; Balkos and Colman 2007). The high affinity CO2 utilization and its significant Ci accumulation factor after 10 s in C. acidophila suggest that this species relies on active CO2 uptake independent of cellular P status, which could be mainly driven by the proton gradient. The internal pH measured here in C. acidophila (on average 6.0) might be flawed by the low external pH, this being nearly 3 pH units below the pKa of DMO (Addanki et al. 1968; Boron and Roos 1976). Therefore, we only compare pHi between treatments and not absolute values as these were rather low compared with earlier studies on the same species (i.e., 6.6 in Messerli et al. 2005 and 7.6 in Gerloff-Elias et al. 2006). There were no differences in pHi between treatments. The expression of CAext under low CO2 conditions has been often observed in many green microalgae, for example in Scenedesmus obliquus (Findenegg 1985), thus enabling the use of bicarbonate as well as CO2 (Spalding et al. 2002). Because bicarbonate is not present in the very acidic habitat of C. acidophila, we did not expect to find any CAext activity. In contrast to this expectation, CAext activity was always present and quite high (up to 1.5 REA 10-6 cells) under low CO2 conditions, especially when compared to Dunaliella tertiolecta (0.6 and 0.8 REA 10-6 cells (Young et al. 2001) and Emiliania huxleyi (0.16 and 0.32 REA 10-6 cells (Stojkovic et al. 2013). The same holds true if we base CAext activity on Chl a, which resulted in activities ranging between 10 and 1,400 REA (mg Chl a)-1 in high and low CO2 cultured C. acidophila, respectively. These activities lie well within the values

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reported for the cell-wall-less mutant of C. reinhardtii at high CO2 (50 REA (mg Chl a)-1), and low CO2 (1,000 REA (mg Chl a)-1) conditions (Coleman et al. 1985). Therefore, the enzyme is possibly involved in CO2-acquisition in C. acidophila, especially under low CO2 conditions. In neutrophiles, such as C. reinhardtii, culturing at acidic pH resulted in low or non-detectable CAext activity (Patel and Merrett 1986), and similar patterns in activities were described in the acid-tolerant Chlamydomonas sp. and Chlorella saccharophila with low or undetectable CAext when grown at acid pH 5, but with activities increasing at alkaline culture pH (Balkos and Colman 2007; Williams and Colman 1995). In contrast to those observations, but similar to observations in the green microalga Dunaliella acidophila grown at pH 1.0 (Geib et al. 1996), we detected significant CAext in C. acidophila at pH 2.6, although the physiological function for this enzyme in a ‘‘HCO3--free’’ environment is still unclear. Geib et al. (1996) suggested for Dunaliella acidophila that CAext facilitates a HCO3uptake system, but this option seems unlikely as pH drift experiments did not indicate that C. acidophila can use HCO3- (unpubl. results). More likely, CAext is a relic from ancient evolutionary processes occurring before the phylogenetic differentiation to an acidophilic species similar to the expression of alkaline phosphatase enzymes in C. acidophila in response to a Pi-limitation (Spijkerman et al. 2007). Recent experiments with several phytoplankton species suggested that evolutionary traits connected with CO2 adaptation do not evolve in relatively short evolutionary time scales of 1,000 generation times (Collins and Bell 2006; Low-Decarie et al. 2013). In contrast to the lack of a cross-reaction with the CAH1 antibody in Dunaliella acidophila (Geib et al. 1996), we detected a clear band in C. acidophila. This band appears to be a CA, though there is some uncertainty in this identification as we used whole cell extracts and the antibody has been shown to cross-react with the chloroplastic CA (CAH3) in Chlorella vulgaris (Villarejo et al. 1998). CAH3 is also an alpha-CA of about the same size (Moroney et al. 2011) and, similar to the results in our Western blot, constitutive or moderately expressed under different CO2 conditions and, as for CAH1, also glycosylated (Buren et al. 2011). Future sequence work will hopefully resolve this uncertainty. Interestingly, this CA was less expressed under Pi-limiting conditions and was most abundant under Pi-replete, high CO2 conditions in C. acidophila. In C. reinhardtii cultured under low CO2 conditions, CAH1 can constitute up to 1 % of total protein in C. reinhardtii (Moroney et al. 2011), and this expression was also realized or maintained under Pi-limitation. The CAH1 signature in high CO2, Pi-limited cells in C. reinhardtii likely resulted from the low CO2 acclimation during the

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first acclimation period, as Pi-limited cultures did not grow much after acclimation to high CO2 conditions. Ecology In summary, the CCF, affinity for CO2 acquisition and CAext activity were all maximally expressed under low CO2, Pi-limited conditions in C. acidophila showing that this species is well adapted to its low CO2, Pi-(co-)limited habitat (Spijkerman 2008a; Tittel et al. 2005), under which conditions it realizes pronounced photosynthetic rates and primary productivity. How this acclimation of C. acidophila to low Pi and low CO2 conditions is realized remains to be studied as our studies of the physiological and biochemical adaptations involved did not allow for a clear conclusion and is one of the future goals of our research. Acknowledgments ES acknowledges support from the German Science Foundation (SP695/4-2 and SP 695/5) and thanks Francesco Memmola, Anna Leetz, and Barbara Schmitz for practical assistance. Work in JB’s laboratory on inorganic carbon uptake, and its interactions with nutrient availability, was supported by the Australian Research Council. We are very grateful for the reviewers and editor’s comments on earlier versions of the manuscript.

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CO2 acquisition in Chlamydomonas acidophila is influenced mainly by CO2, not phosphorus, availability.

The extremophilic green microalga Chlamydomonas acidophila grows in very acidic waters (pH 2.3-3.4), where CO2 is the sole inorganic carbon source. Pr...
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