Induction and Regdation of Dissolved Inorganic Carbon Transport in Green Algae

Gale Giancarlo Bozzo

A Thesis Submitted to the Faculty of Graduate Studies

in Partial Fulfillment of the Degree of Master of Science

Graduate Programme in Biology York University

Toronto, Ontario, Canada

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INDUCTION AM) REGULATION OF DISSOLVED INORGANIC CARBON TRANSPORT IN GREEN ALGAE

by Gale Giancarlo Bozzo

a thesis submitted to the Faculty of Graduate Studies of York University in partial fuifillment of the requirements for the degree of

Mas ter of Science

2000 O

Permission has been granted to the LIBRARY OF YORK UNI- VERSITY to lend or seIl copies of this thesis. to the NATIONAL LIBRARY OF CANADA to microfilm this thesis and to lend or seIl copies of the film, and ;O UNIVERSITY MICROFILMS to publish an abstract of this thesis. The author reserves other publication rights. and neither the thesis nor extensive extracts from it rnay be printed or other- wise reproduced without the author's written permission.

Abstract

Induction of the carbon concentrating rnechanism (CCM) was characterized

during the acclimation of 5 % CO2-grown Chlamydornonas reinhardtii (2137 mt+)

and Chlorella kessleri (UTEX 1808) ceiIs to well-defined dissolved inorganic carbon

@IC) lirnited conditions. The CCM components investigated in both ceIl types were

active HC03- and CO2 uptake. The maximum rate of photosynthesis (Pm) was similar

for high- and low-CO2 grown cells of Chlorella kessleri, but the apparent whoIe ceII

affinity for DIC and CO2 (&) of high COz-grown cells was about 30-fold greater than

that in air-grown cells, which indicates a lower affinity for DIC and CO2. Bicarbonate

and CO2 transport were induced after 5.5 h in Chlorella kessleri cells acclirnating to

CO3-fee air and air, in the presence and absence of 21 % 0 2 . This indicates that a

change in the C02/02 ratio in the acclimating medium does not trigger induction of DIC

transport. No active DIC transport was detected in high CO2-grown cells maintained on

high CO2 in the presence of 5 mM aminooxyacetate, an aminotramferase inhibitor; which

indicates no involvement of photorespiration in the induction mechanism. For

Chlarnydornonas reinhardtii cells, active CO2 and HC03- uptake were induced within 2 h

of acclimation to air, but active CO2 transport was induced prior to active HC03-

transport, and this was aIso evident in Chlorella kessleri. Sirnilar results were obtained

for both algae during acclimation to low CO2 in darkness. Active DIC transport

induction was inhibited in celIs treated with cyclohexirnide but was unaffected by

chloramphenicol treatrnent, indicating that the induction process requires de novo

cytoplasmic protein synthesis. Changes in extraceilular carbonic anhydrase (C4.r)

activity were measured only in Chlamydomonas reinhardtii. C L , activity increased 10-

fold within 6 h of acclimation to 360 ppm CO2 and there was a slight increase over the

next 18 h. C b t activity also increased substantiaily after an 8 h lag penod dunng

acclimation to air in darkness. This indicates that the induction of C&, and active DIC

transport are not correlated temporalIy in Chlamydomonas reinhardtii cells. The

concentrations of extemal CO2 required for maximum induction and repression of DIC

transport in Chlorella kesslet-i was O and 120 pM, respectively, and was independent of

the pH of the acclimation medium. In Chlamydomonas reinhardtii, the concentrations of

extemal CO2 eliciting maximum induction and repression of DIC transport and C L t

activity were 10 and 100 pM, respectively. ProIonged exposure to a critical external CO?

concentration elicits the induction of the CCM in Chlorella kessleri and Chlamydamonas

rein ha rdhi.

Ackmowledgements

A few years ago, Dr. Brian Colman presented me with the opportunity of doing a

B. Sc. Honours thesis project in his laboratory after months of not being able to find a

supervisor. 1 would like to express my sincere thanks to Dr. CoIman, who allowed me the

opportunity to explore and Iearn on my own, and has provided me with the tools

necessary to progress as a scientist. His generosity and assistance are very much

appreciated, and it was a great experience working with someone who is willing to

consider and discuss the viewpoints of a graduate student. Your efforts have made me a

better stsdent and scientist.

1 am also very appreciative of the time and effort of Dr. Yusuke Matsuda, who

carried out his post-doctoral research in the Colman lab. Yusuke brought an enormous

arnount of energy and insight into the research camed out in the lab, and for this I am

very grateful,

1 would like to express my sincere thanks to the present gang in the Colman lab. 1

would like to express rny gratitude to Dr. Emma Huertas (fiom the real Spain) whose

lighthearted and upbeat attitude always provided an enjoyable atmosphere in the lab, and

whose absence from the lab due to her research at Erindale, was always felt

tremendously. 1 would also like to include my other lab mates, Rich DeMarchi, Shabana

Bhatti, and Eva Szabo, whose generosity, assistance and friendship were extremely

valuable.

There are also some ex-Colmanites who need to be mentioned: these include

Jason Deveau, Steve Pollock, and Meryl John McKay. Al1 three have since left the lab,

vii

but our time shared will not be forgotten. 1 will remernber Jason (my pub crawling

partner in crime) for his witty jokes, zealous approach to life, and generosity will be

etched in my memory forever. Steve's unending assistance and willingness to discuss al1

things science is also weII noted and appreciated.

1 am also grateful to my farnily, whose support and encouragement have taught

me never to Iose sight of my goals and my dreams. 1 would like to thank my parents and

sisters, Eluana and Sabrina, whose selflessness and caring have given me strength. Your

love and caring have provided comfort, in both triumphant and difflcult points in my life

and my scholastic career.

1 can't forget about out my friends here at York, whose support and fnendship are

greatly appreciated, and who have helped to provide a good balance between work and

pIay. These include Mark Gaglairdi, Nancy Silva, Anthony Bruce, Natdie Rodrigues,

Maria Mazzurco, Sophia Stone, Lorainne Nunes Christie, Selena Kim, and others from

the Biology Department who have been kind and helpful over the past few years. 1 would

also like to thank "Danny" (3d Floor Custodian) who everyday provided an interesting

story or analogy, which kept me laughing for hours after

A730:

ABC:

AOA:

ATP:

MA:

BB:

C3:

C4:

OC:

Ci:

CA:

CA%,:

CAint:

CCM:

Chl:

DIC:

EZQ:

g :

h:

m:

optical absorbante at a wavelength of 730 nanometres

ATP binding cassette

aminooxyacetate

adenosine 5-triphosphate

acetazolamide

Bold's basai medium

three carbon

four carbon

degrees Celsius

inorganic carbon

carbonic anhydrase

extracellular carbonic anhydrase

intracellular carbonic anhydrase

carbon concentrating mechanism

chlorophyll

dissolved inorganic carbon

ethoxyzolarnide

gravitational force

hour

isonicotinic acid hydrazide

mg:

min:

rnL:

rnM:

nmol:

pCA:

P-gl ycolate:

3-PGA:

pH:

Pm=:

PPFD:

Modalton

Michaelis-Menten constant for half-saturation of enzyme velocity

concentration of CO2 required to yield half maximum photosynthetic

rate in intact cells

concentration of DIC required to yield haif maximum photosynthetic

rate in intact cells

litre

metre

miIligram

minute

millilitre

rnillimola.

rnessenger

mating type

nannomole

periplasrnic CA

phosphoglycolate

3-phosp hoglycerate

-log CHCl

maximum rate of photosynthesis

photosynthetic photon flux density

parts per million

Rubisco:

SD:

SE:

SG:

W-A:

ribulose- 1,s-bisphosphate carboxylase/oxygenase

ribulose- 1 ,S-bisphosphate

seconds

standard deviation

standard error

Sager-Granick

control time in carbonic anhydrase activity assays

sample time in carbonic anhydrase activity assays

microgram

micromole

University of Texas Culture Collection

Wilbur-Anderson

Table of Contents

Abstract

Acknowledgements

A bbreviations

List of Figures

List of Tables

Introduction

1.1 General Characteristics of Green Algae

1.2 Limitation in Dissolved Inorganic Carbon Availability

1.3 The Role of the CCM in Phototrophic Organisms

1.4 Characteristics of the Microalgal CCM

Page Number

iv

v1

vüi

xiii

xv

1

1

2

3

5

1.5 Carbonic Anhydrase and the Carbon Concentrating Mechanism

1.6 Regdation of the Carbon Concentrating Mechanism in Phototrophic Organisms

1.7 Purpose of the Study

Materials and Methods

2.1 Growth Conditions

2.2 Determination of the Apparent Whole Ce11 AfFinity for DIC

2.3 Time Course of Induction of Active Bicarbonate and Active CO:! Transport

2.4 Time Course of Induction of ExtraceIlular Carbonic Anhydrase Activiîy in Chlamydomonas reinhardtii

2.5 Determination of the Critical Ci Concentrations Required for the Induction of the CCM

2.6 Deterrnination of the Effect of Protein Synthesis and Metabolic Inhibitors on the Induction of the CCM in Chlorella kessleri

3.1 Affinity for DIC in Chlorella kessleri Under Various CO2 Regimes

3.2 Changes in Extracellular Carbonic Anhydrase Activity in Chlamydornonas reinhardtii During Acclimation to Low CO2

3.3 The Time Course of Acciimation in Chlorella kessleri and Chlamydornonas reinhardtii

3.4 Effect of Metabolic Inhibitors on the Acclimation of Chlorella kessleri to Low CO2

3.5 Critical Extemal DIC Concentration During Acclimation

4. Discussion

4.1 High Affinity for Inorganic Carbon is Induced in Chlorella kessleri Cells Under Low CO2 Conditions

4.2 Induction and Regulation of Active DIC Transport in Green Algae

4.3 Induction and Regulation of CL,, Activity in Chlamydomonas reinhardtii

4.4 Triggering the CCM in Green Algae

5 Literature Cited

List of Figures

Page Number

Figure 1 Schematic diagram of the green dgaI CCM.

Figure 2 Schematic diagram of the proposed light-dependent phosphoglycolate trigger in the induction of the CCM.

Figure 3 Schematic diagram of CCM induction triggered by the extracellular CO2 concentration.

Figure 4 Rate of photosynthesis at various DIC concentrations of cells of Chlorella kesslen' at pH 7.8 and 25OC.

Figure 5 Rate of photosynthesis at various DIC concentrations of

cells of Chlorella kessleri at pH 7.8 and 25OC in the presence of bovine CA.

Figure 6 Changes in CL& activity of high CO2-grown Chlamydomonas reinhardtii cells during acclimation to air.

Figure 7 Changes in C&,, activity of high COrgrown Chlamydomonas reinhardtii cells during acclimation to air in darkness.

Figure 8 The photosynthetic O2 evolution rate of high CO2-grown ceus of Chlorella kessleri during acclimation to air, and CO2-free air.

Figure 9 Response to extemal 0 2 concentration. The photosynthetic 0 2 evolution rate of high COTgrown cells of Chlorella kessleri during acclimation to Orfree air.

Figure 10 The photosynthetic 0 2 evolution rate of high CO2-grown cells of Chlamydomonas reinhardtii during accIimation to air.

Figure 11 The photosynthetic O2 evolution rate of high C02-grown ceils of Chlorella kesslen' during acclimation to CO2-free air in darkness,

Figure 12 The photosynthetic O2 evolution rate of high COTgrown cells of Chlorella kessleri during acclimation to air in darkness.

Figure 13 The photosynthetic O2 evolution rate of high CO2-grown cells of Chlamydomonas reinhardtii during acclimation to air in darkness-

xiv

36

Figure 14 The effect of inhibitors of protein synthesis and inhibition 40 of aminotramferases on the induction of active DIC transport in high COz-grown Chlorella kessleri cells acclimating for 5.5 h.

Figure 15 Acclimation of high COa-grown Chlamydomonas reinhardtii cells to various extemal CO2 concentrations at pH 5.5 for 2 h.

Figure 16 Acclirnation of high CO2-grown Chlamydornonas reinhardtii 43 cells to various externai CO2 concentrations at pH 7-5 for 2 h.

Figure 17 Determination of the critical COa concentration effecting the 45 induction of C4,, ac tivity in Chlamydomonas reinhardtii cells.

Figure 18 AccIimation of high CO2-grown Chlorella kessleri cells to 46 various externai concentrations of DIC and COz at pH 6.6 for 5.5 h.

Figure 19 Acclimation of high COrgrown Chlorella kessleri cells to 47 various external concentrations of DIC and CO-, at pH 7.5 for 5.5 h.

List of Tables

Table 1: Photosynthetic characteristics of Chlorella kessleri cells grown in high CO2 and low CO2 conditions.

Table 2: CO2 and DIC concentrations eliciting induction of active CO2 and HCO< transport in high COTgrown Chlorella kessleri ceils acclimating at two different pH values for 5.5 h.

Page Number

1 - Introduction 1.1 General Characteristics of Green MicroaIgae

Photosynthetic organisms inhabiting aquatic environments are major contributors

of O2 to the atmosphere. The contribution to the prirnary productivity of the hydrosphere

by marine phytoplankton is on the order of 45-50 1012 g carbon per annum (Falkowski

et al., 1998). Primary productivity is defined as the amount of organic carbon that is made

available to heterotrophic organisms. The aquatic phototrophs comprise prokaryotic

organisms, such as cyanobacteria, and eukaryotes, such as rnicroalgae and macroalgae.

Microalgae belonging to the division Chiorophyta exist as unicellular and rnulticellular

organisms and are distinct from other algae, because they contain the green plant

pigments, chlorophylls a and b, in their chloroplasts. The chloroplast is an organelle

found in organisms which are capable of autotrophic growth by the process of

photosynthesis. The photosynthetic reaction is dependent on light energy, and is as

follows:

6 C 0 2 + 6H20 C6H120:! i 6 02.

Absorption of light by chlorophyll molecules is converted into chemical energy, required

to h e l the carbon fixation process. The excitation of chlorophyll molecules by photons of

light initiates electron transfer to a nurnber of protein complexes in the electron transport

chain, and this is coupled to the oxidation of H20 to produce 02. There are two processes

invohed in photosynthesis: the light reactions, light dependent chemical energy

production, and the dark reactions, chemical energy dependent CO2 fixation. Green

rnicroalgae are phototrophic and CO2 available in the aquatic environment is assimilated

internaliy to form starch by means of reductive carbon flow through the Calvin cycle.

This process is dependent on energy derived from ATP and NADPH. Microalgae carry

out C3-type photosynthesis, which means ribulose- 1,s-bisphosphate carboxylase/

oxygenase (Rubisco) catdyses the carboxylation reaction between RuBP and CO2. The

products of the reaction are 2 molecules of 3-PGA. RuBP is regenerated by a series of

enzyrnatic reactions in the Calvin cycle. The process eventually culminates with the

accumulation of starch. Rubisco is localised in the chloroplast stroma of the green algal

cell (Fig. 1), and in certain organisms has also been found in varying arnounts within a

protein rich structure of the chloroplast known as the pyrenoid (Borkhsenious et al.

1998). Many green algae are biflagellate cells (e-g. Chlamydomonas reinhardtii) which

aids in rnotility, but this is not characteristic throughout the division (e-g. Chlorella

kessleri is a non-flagellate). In Iaboratory studies, green microalgae have also been

deterrnined to be capable of heterotrophic (e-g. acetate utilization) or mixotrophic growth

(e.g. acetate and CO2 utilization)-

1.2 Limitation in Dissolvecl Inorganic Carbon Availability

Aquatic plants are Iimited by the availabilie of CO2 in comparison to terrestrial

plants. The concentration of dissolved CO2 varies with the temperature of the aquatic

environment and the partial CO2 pressure in the air above water. An increase in

temperature is correlated with a decrease in CO2 solubility, but this is compensated for by

an increased rate of conversion of CO2 by the spontaneous dehydration of HC03' present

in the medium (Raven and Geider, 1988). The present day level of atmospheric CO2 is

approximately 360 ppm. Under these conditions of partial CO2 pressure and at 25OC, the

concentration of dissolved CO2 in a freshwater environment is approximately 10 p M

(Aizawa and Miyachi, 1986). CO2 diffusion in water is about 1OOOO fold slower than in

air (Badger, 1987). Dissolved COt is in equilibrium with HC03- and CO^" and in

freshwater environments, CO2 reacts with water to forrn the unstable intermediate

carbonic acid (H2C4), which is rapidly converted to HC03- and H? and further to ~ 0 ~ " .

At alkaiine pH (Le. pH 7-8), the eqiiilibrium between the DIC species will be driven

towards HC03; whereas at acidic pH (i.e. pH 5.5) the equilibrium will be driven to CO2.

Limitations in DIC may occur in natural populations when rapid growth in an algai

bloom occurs, since the algae are efficient at depleting the aquatic environment of the

available DIC. In this case, competing algal populations will be DIC-limited.

1.3 The Role of the CCM in Phototrophic Organisms

Under DIC-limiting conditions, a carbon concentrating mechanism is induced in

various cyanobacteria and green microalgae. The CCM functions to elevate the COz

concentration around Rubisco. The necessity for a CCM stems from the low affinity of

algal and cyanobacterial Rubisco for its substrate, CO2. In green algae, the Km (CO2) or

the COz concentration that half saturates the carboxylation reaction is on the order of 30

to 60 pM (Jordan and Ogren, 198 1). In contrat, the whole ce11 affinity (Kin) for DIC and

CO2 of DIC-Iirnited cyanobacteria and algae from which Rubisco had been isolated was

considerably lower than the Km (CO2) of Rubisco (Badger et al., 1998). Low Kin (CO2)

values are apparent in low COî-grown and acdimated green aigal ceils (Gehl et al., 1990;

Rotatore and Colman, 199 1 b; Matsuda and Colman, 1996a; 1996b), which suggests that

the cells express an efficient DIC uptake system. This is also indicated by low CO2

compensation points in DIC-limited cells of green algae (Rotatore and Colman, 1991a;

1991b; Matsuda and Colman, 1996a). The CO? compensation point is measured in the

Iight and is the COz concentration in the extemal medium at which the rate of

photosynthetic CO2 uptake is suni la to the rate of CO2 efflux by respiration. A low CO2

compensation point is indicative of low rates of photorespiration, and an efficient DIC

uptake mechanism, which is characteristic of algae with a functioning CCM. Two

different strategies have evolved to ded with a limitation of COz in growth: (1) the

evolution of an increased affinity for CO2 by Rubisco promoting increased efficiency of

catalysis (Badger et al. 1998), or (2) the presence of a CCM. The induction of the CCM

has been studied in cyanobacteria and green aigae, and to a Iesser extent in non-green

algae and dinoflagellates (Leggat et al. 1999) in laboratory expenments. Berman-Frank et

al. (1998) found that induction of CCM characteristics in Peridimium gatunense cells

inhabiting a freshwater lake occurred in response to a 40 % decrease in lake DIC

concentration resulting from a bloom in the dinoflagellate population.

CCMs are also present in terrestrial as well as aquatic phototrophs. Terrestrial

plant species that characteristically assimilate carbon by the Cq pathway express a CCM

that is biochemical as opposed to the biophysical CCM found in microalgae. In C4-

terrestrial plants, CCM requires two distinct structural components. CO2 enters the leaf

mesophyll ceii, is quickly converted to HC03-, and then is fxed by PEP carboxylase to

form oxaloacetate and subsequentiy malate or aspartate. One of these C4-acids is

transported into a separate entity, the bundle-sheath cell, where decarboxylation takes

place releasing CO2 into the bundIe sheath cell cytosol (Badger and Price, 1994). The

CO2 concentration around Rubisco is elevated and leakage is minirnized by the thick ceIl

walls of the bundle sheath ce11 (Moroney and Somanchi, 1999). The rnicroalgal CCM

contrasts with the Cq plant CCM in that a majority of algae exist as unicellular, rather

than multicellular organisms. The unicellular structure of green microalgai species does

not allow for a C4-type fixation but pennits a greater interaction with the extracellular

environment, which necessitates the requirement for physiological control of the CCM.

In green rnicroalgae, the CCM operates by accumulating HCOs' in the cytosol,

which rninimizes CO2 leakage to the external medium. HC03- accumuIates in the

chloroplast and is converted to CO2 by CA, and this results in an increase in the CO2

concentration around Rubisco localized within the chloroplast (Moroney and Somanchi,

1999). The cyanobacteriai CCM functions in a similar rnanner, where the accumulated

HC03- eventualIy leads to an increase in HC03- in the carboxysome, in which the

Rubisco is located. HC03- is converted to CO2 by a carboxysomal CA. The CCM

functions to improve the effkiency of carbon fixation in rnicrodgae when the CO2 in the

external environment is limi ting

1.4 Characteristics of the Microalgal CCM

A CCM possesses various hallmark characteristics. In almost d l algae where a

CCM is present, the internal accumulation of DIC occurs at concentrations in the mM

range which can be 10 to 1000-fold the external DIC concentration (Aizawa and Miyachi,

1986; Miller and Colman, 1980b). Intracellular accumulatioii of DIC is absent or very

much reduced in green algae grown under high CO2-conditions (Badger et al., 1980;

Palmqvist et al., 1988). The DIC species that is accumulated intemally is usually HC03-,

because of the alkaline pH of the cytosol and the chloroplast stroma Badger et al. (1980)

used the silicone oil centrifugation technique to demonstrate that Chlamydmonas

reinhardtii cells accumulate DIC intemally several fold higher than the extemal DIC

concentration. DIC is therefore accumulated against a concentration gradient. Coleman

and Colman (1981) were able to demonstrate an accumulation of internal DIC in

cyanobacteria during growth at alkaline pH. The active accumulation rnechanism is light-

dependent (Kaplan et al., 1980; Miller and Colman, 1980b). Miller et al. (1991)

dernonstrated that there was a several fold decrease in the internal DIC accumulation

during light to dark transitions in Synechococcus cells. It has also been shown that the

intemal accumulation of DIC is induced in Chlorella ellipsoidea during acclimation to

ambient CO2 conditions (Matsuda et al., 1995a). The DIC accumulation mechanism is

due to active uptake of HC03- and/or CO2 by the cells. Active DIC transport is usually

absent or reduced under high COz-growth (Shiraiwa and Miyachi, 1985; Sültemeyer et

al., 1989; Matsuda and Colman, 199%~).

Active HC03- transport across the whole-ce11 boundary was reported by Miller

and Colman (1980a) in a cyanobacterium CoccochloBs peniocystis using a kinetic

method of determination. Photosynthetic O2 evolution rates detennined under specific

DIC limiting conditions, pH and temperature, were compared with the caiculated

maximum rate of CO2 production in the medium from the spontaneous breakdown of

HC03-. Using this indirect approach, which assumes a 1 : 1 photosynthetic quotient

between the CO2 consumed and the Oz evolved, the O2 evolution rate was significantly

greater than the spontaneous dehydration rate, suggesting that photosynthesis is

supported by active HC03- uptake. Matsuda and Colman (1995a) were able to

demonstrate active uptake of HCOf in air-acclimated Chlorelia ellipsoidea using this

procedure.

A large amount of research has been conducted on the nature of active HCQ-

transport in cyanobacteria (Espie et al., 1988; Miller and Canvin, 1985). A Na+-

dependent HC03- uptake mechanism has been reported in Synechococcus cells growing

in standing culture; whereas Nac-independent HC03' transport activity has been reported

in air-grown cultures. The presence of a sodium chloride analogue, Lithium chloride, was

found to inhibit Nac-dependent HCOY uptake in Synechococcus leopoliensis (Miller and

Canvin, 1985). Na+-dependent transport activity has not been reported in green algae.

Inhibition of ~a+-dependent HC03- uptake by absence of sodium lead to the observation

that under these conditions, CO1 was taken up rapidly in the presence of bovine CA,

which suggested active CO2 transport (Espie et al., 1988).

Internal accumulation of DIC in cyanobacteria and green algae is also due to

active CO2 transport by the whole cells. Active CO2 transport has been studied in

cyanobacteria (Miller et al., 1989; Miller and Canvin, 1985) and to a lesser extent in

Chlamydomonas (Sültemeyer et al., 1989) and Chlorella species (Rotatore and Colman,

1991a, 1991~). Using mass spectrometric analysis, Badger and Andrews (1982) reported

a very rapid decline in the CO2 concentration in the external medium of Synechococcz~

cells to almost zero, which caused a marked disequilibnum between CO2 and HC03- in

the medium. This was correlated with an increase in the rate of intemal DIC

accumulation when a low concentration of 14coZ as opposed to ~'~~03' was supplied to

the illuminated Synechoccocus celIs. The rapid uptake of CO2 is characterised as an

active process, since it is against a concentration gradient (Miller et al., 1988), and must

involve an energy cost since CO2 and HC03- are pulied out of equilibrium in the

extracellu~ar medium. It is presumed that the extracellular DIC is out of equilibrium,

because the addition of CA results in a reestablishment of the DIC equilibrium. The

aforementioned experirnents were perfonned under alkaline conditions where the non-

enzyrnatic conversion of HC03- to CO2 is quite slow. Demonstration of active CO2

uptake by cyanobacteria using the mass spectromeûic technique is not complicated since

C k X t activity is not present. CA maintains the equilibrium between CO2 and HC03- in

the medium. Miller et al. (1990) proposed that active COz transport acts as a scavenger

for CO2 that has Ieaked out of the ce11 due to HC03- dehydration in the cyanobacterial

cytosol, and that active CO2 uptake is only present in cells expressing active HC03-

transport activity. However, active KC03- transport has been reported in a marine green

alga, Nannochloropsis gaditana, that does not have active CO2 transport (Huertas et al.,

2000), and the opposite case was found in a reIated species, Nannochloris maculata

(Huertas et ai., in press) and in the freshwater alga Erernosphaera viridis (Rotatore et al.,

1992). Sultemeyer et al. (1989) demonstrated active uptake of CO2 by air-grown cells and

isolated protoplasts of Chlamydornonus reinhardtii, where ceIls treated with

acetazolarnide or washed protoplasts had no C&,, activity. Active CO2 uptake also

explains the internal accumulation of DIC in ChloreZZa sp. grown at acid pH (Gehl and

Colman, 1985), in which a significant percentage of the DIC in the bulk medium is

present in the form of CO2.

A number of studies have been done to detect the molecular component

corresponding to active DIC transport in cyanobacteria and green algae. This is usuaiIy

approached by screening for mutants deficient in active DIC transport activity.

Impairment in HC03- uptake activity was evident in a high CO2-requiring mutant (IL-2)

of Synechococcus PCC7942: there was no saturation in the kinetic uptake of HC03-,

suggesting a possible lesion in the transport mechanism (Bonfil et al., 1998). The protein

encoded by the gene responsible for the lesion in IL-2 was homologous to a 42 kDa

polypeptide (CmpA), thought to represent a plasma membrane component of the

cmpABCD operon which encodes for an ABC-type transporter involved in HC03- uptake

in Synechococcw PCC7942 (Omata et al., 1999). The induction of high-affhity HC03-

uptake activity was shown to be correlated with the expression of crnpABCD (Omata et

ai., 7999). A moIecuIar component has not been found for active CO2 transport.

Active CO2 uptake occurs at the plasma membrane boundary in Chlorella

ellipsoidea cells, but is absent in isolated chloroplasts of air-grown cells (Rotatore and

Colman, 1991~). In contrat, Amoroso et al. (1998) have demonstrated that active CO2

and active HC03- transport occur at both the plasmalemma and chloroplast envelope of

air-acclimated Chlamydomonas reinhardtii cells. Chen et al. (1997) report the isolation of

a LIP-36 gene which encodes for a 36 kDa protein (Spalding and Jeffrey, 1989), and is

homologous to the rnitochondrial carrier protein superfamily. LIP-36 is induced under

low COTconditions, and is thought to be localized to the chloroplast envelope (Chen et

al., 1997). The role and precise regdation of the LP-36 protein are yet to be detennined.

1.5 Carbonic Anhydrase and the Carbon Concentrathg Mechanism

The induction of the microalgd CCM is correlated in some algae with an increase

in CA activity dunng acclimation to low COz conditions. CA is a zinc-containing enzyme

that reversibiy binds CO2 and HC03-, and maintains the equilibrium between these two

DIC species in solution. The role of CA in the CCM has been debated over the past few

decades. CA is localised to various cornpartrnents in the green algal cell, but the

physiological role of each CA in the CCM is yet to be fully determined. An increase in

extemal or ~eriplasrnic CA @CA or C&,J activity has been reported in Chlamydomonas

r e i n h a d i cells during acciimation to DIC-limiting conditions (Aizawa and Miyachi,

1986; Badger and Price, 1994; Coleman, 199 1).

The presence of C L t is not a characteristic of al1 green microdgae. An absence

of this activity has been reported in Chlorella ellipsoidea (Rotatore and Colman, 1 99 1 a;

1 9 9 1 ~ ) ~ which has a fully operational CCM under DIC-limiting conditions (Matsuda et

al., 1995a), and in Chlorella kessleri (Matsuda et al., 1999). The presence of C&x,

activity is thought to increase the apparent whole-ceIl photosynthetic affinity for CO2

(Aizawa and Miyachi, 1986).

The importance of C L t activity in the Chlumydornonas CCM has been debated

in past studies. Moroney et al. (1985) demonstrated that C&, is necessary for the

utilization of DlC at low external CO2 concentrations and alkaline pH. However,

Vdliams and Turpin (1987) demonstrated that C L , is not required under these

conditions, since a membrane-impermeant inhibitor of CA, acetazolamide, has no effect

on DIC utilization, In another green algal species, Chlorella ellipsoidea C-27, Shiraiwa

et ai. (1991) demonstrated that C L t activity was highest in cells acclimating to low CO2

in the pH range of 7.0 to 8.0, and lower in cells acclimating at pH 5.5. Gehl et al. (1990)

demonstrated that C&,, activity in Chlorella saccharophila is suppressed by growth at

acid pH. These results suggest that C&x, activity is induced under conditions where the

ratio of dissolved HC03- concentration to dissohed CO2 concentration is high. Williams

and Colman (1996) found that C b t activity increased with a concomitant decrease in

DIC suppiy. Kt appears as though C&,, is required to supply CO2 to high affinity DIC

transporters operating in the CCM response.

The necessity for CAint activity for CO2 fixation under DIC-limiting conditions

was suggested as a result of studies where CAi,, was absent from or inhibited in

Chlamydomonas reinhardtii cells (Spalding et al., 1983; Moroney et al., 1985). In cells of

a high CO2 requiring mutant of Chlamydomonas reinhardtii, ca- 1 - 12- lc, an increase in

the internai DIC accumulation and lower photosynthetic rates in cornparison to wild-type

cells were observed under low CO;! growth (Spalding et al., 1983). The decrease in

photosynthesis is the result of a slow conversion of the internal accumulated HC03- in

ca- 1- 12-lc cells lacking CAint activity, which results in a decreased CO2 pool available to

Rubisco. The lesion in the ca-1-12-lc mutant is in the gene, CAH3, encoding for an

intracelMar CA (Funke et al., 1997). C M 3 encodes for an insoluble carbonic anhydrase

localised to the chloropIast of Chlamydomonas reinhardtii cells (Karlsson et al., 1995).

Figure 1. Schematic diagram of the green algal CCM. In the extracelIular medium, DIC is present in the form of CO, and HC0,-. Periplasmic CA (pCA) maintains the extemal DIC equilibrium. High afinity transporters at the plasma membrane actively transport CO2 and HCO,- into the cytosoI raising the intracellular DIC concentration to a level several fold greater than the extemal DIC concentration. CO:, andor HC03- is actively or passively taken up at the chloroplast enveiope. Accumulated HC03- in the chloroplast is converted to COz by chloroplastic CAS, or is transiocated to the pyrenoid, Pyrenoidal CAS would convert HCO,- to CO,. The elevated CO,

concentration in the pyrenoid and chloroplast stroma is in close proximity to Rubisco molecules.

This diagram has been modified fiom Badger and Price (1 994).

Similar efr'ects on the CCM in Chlamydomonas reinhardtii were observed using the

membrane-permeable sulfonamide, EZA, which is a potent inhibitor of CA activity

(Spalding et al., 1983; Moroney et aI., 1985).

Various CAS have been shown to be induced under low CO2 conditions (Fig. 1).

These include a thylakoid bound CA (Karlsson et al., 1998), and a mitochondrial CA

(Eriksson et al., 1998) in Chlamydomonas reinhardtii cells. The induction of a pyrenoid-

based C A in Chlorella vulgaris cells occurs during acclimation to low CO2 (Villerago et

al., 1998). The importance of these three intemal CAS in the green algal CCM is yet to be

elucidated.

1.6 Regdation of the Carbon Concentrating Mechanism in Phototrophic

Or ganisms

De novo protein synthesis of cytopiasrnic proteins encoded by the phototroph's

nuclear genome is thought to arise during the induction of the CCM (Shiraiwa and

Miyachi, 1985; Palmqvist et al., 1988; Matsuda and Colman, 1995a; Matsuda et al.,

1998). The regulation of de novo protein synthesis in the CCM is important in

understanding the acclimation response to low CO2. In the past few decades, much debate

has centred on what is the physiological trigger for induction of the CCM. AIthough the

signdling pathway which initiates induction in green algae and cyanobacteria is not

known, it has been proposed that the induction of high afXnity photosynthesis may be in

response to the accumulation of photorespiratory pathway intermediates within the ce11

(Marcus et al., 1983). The photorespiratory signal mode1 @ig. 2) had been proposed

because there is an intracellular accumulation and release of glycolate into the externa1

Figure 2. Schematic diagram of the proposed light-dependent phosphoglycolate trigger in the induction of the CCM. Extracellular O, is proposed to diffuse readily into green algal celis, and CO2 is able to be taken up actively and passively. In the presence of light, Rubisco has the ability to bind to O, or CO,, which is catalyzed by the oxygenase or carboxylase activity of Rubisco. Under low CO, acclimation, where the C 0 2 / 0 2 ratio is small in comparison to high CO,-cells, the oxygenation reaction catalyzed by Rubico is favoured, and P-glycolate is formed. The accumulation of photorespiratory intermediates is thought to serve as a trigger to induce the CCM response (Mode1 is as represented by Matsuda et al., 1998).

medium during the acclimation of high COTgrown Chlamydomonas reinhardtii cells to

ambient CO2 Ievels (Neison and Tolbert, 1969). This is thought to be the result of a

decrease in the C02:02 ratio in the growth medium which would stimulate the oxygenase

activity of Rubisco, and cause an increase in photorespiratory pathway intermediates.

Glycolate release ceases and photorespiration is suppressed with the induction of the

CCM- The trigger for induction is therefore light and Oz-dependent. A requirement for

light has also been reported in regulating the activity of C&,, (Spalding and Ogren,

1982). For example, Dionisio-Sese et al. (1990) found that an increase in the levels of CA

mRNA occurred in Chlamydomonas reinhardtii cells within 2 h of acclimation to low

CO2 in the light; but remained unchanged when cells were acclimated in darkness.

However, Rawat and Moroney (1995) demonstrated that periplasmic CA transcript was

made in the dark after a lag period.

There is increasing evidence to suggest that cells do not respond to an interna1

metabolic signal but to a critical concentration of dissolved CO2 in the external growth

medium (Matsuda and Colman, 1995b; Matsuda et al., 1998). In the unicellular green

alga, Chlorella ellipsoidea, the induction of the CCM occurs when cells are acclimated to

low COz in darkness (Matsuda and Colman, 199%). Similarly, Umino et al. (1% 1)

reported a decrease in the Kir, (CO2) for Chlorella regulan's during acclimation to low

CO2, which was independent of photosynthesis. These results can not be explained by the

light-activated photorespiratory metabolite trigger model. The induction of active DIC

transport in Chlorella ellipsoidea is in response to a critical concentration of COz in the

CO* CO, CO, HC0,-

HC0,- CO2

CO, CO, CO, HC03-

Figure 3. Schematic diagram of CCM induction triggered by the extracellular CO2 concentration. Matsuda et al. (1998) have proposed that the CCM in green algae may be regulated by the external CO, concentration through a CO2 sensor at the algal ce11 suface. Interaction of the sensing mechanism with CO2 would in triggering the repression of active HCO,- andor active CO, transport in high CO2-grown cells. In low CO2-acclimated cells, derepression of DZC transport would occur as a result of the absenc5 of an interaction between the sensor and CO,, leading to de novo transporter synthesis. The diagram is modified fiom Matsuda et al. (1998).

external bulk medium (Matsuda and Colman, 1995b). In Chlorella eElipsoidea, active

COz transport is induced during acclimation at a CO2 level lower than 0.37 %, regardless

of the pH of the extracellular medium (Matsuda and Colman, 1995b), whereas active

HCQ' transport was induced at a CO2 level of 0.21 5% or below. A continuum of active

DIC transport activities was induced in response to an increasing concentration of

dissolved CO2 in the extracellular medium. Mayo et al. (1986) report intermediate Kin

(DIC) values for Synechococcus leopoliensis ceus acclimated to CO2 and DIC

concentrations between hi& and low CO2. Matsuda and Colman (1996a) isolated CO2-

insensitive mutants of Chlorella ellipsoidea, which had sirnilar affinities for DIC and

CO-, when grown under high or low COî conditions. High-affrnity photosynthesis is

constitutively expressed in Chlorella saccharophila cells grown under various external

CO2 regirnes (Matsuda and Colman, 1996b), whereas high-affinity photosynthesis is

induced in wild-type Chlorella ellipsoidea cells during acclirnation to air. During

acclimation of wild-type Chlorella ellipsoidea celis to Iow CO2, the induction of active

DIC transport is dependent upon de novo protein synthesis (Matsuda and Colman,

1995a). Matsuda et al. (1998) proposed that a CO2 sensor at the plasmalemma surface in

Chlorella cells, plays a pivota1 role in triggering the CCM response. Under high CO2

conditions, the CCM in Chlorella ellipsoidea is repressed when the sensor is bound with

CO1 molecules. Under low CO2 growth, when the sensor wouId be depleted of CO2, the

CCM would be derepressed. The derepression is correlated with de novo protein

synthesis involved in the expression of active DIC transport by intact celis (Figure 3).

The signal transduction pathway in the CCM response has not been detennined. In the

case of cells which constitutively express a CCM, such as Chlorella saccharophila

(Matsuda and Colman, 1996b) and CO2-insensitive mutants of Chlorella ellipsoidea

(Matsuda and Colman, 1996a), the sensing mechanism or signalling pathway may be

absent.

1.7 Purpose of Study

PhysiologicaI characteristics of the CCM in unicellular green microalgae,

Chlorella kesderi and Chlamydornonas reinhardtii, were studied in response to a

limitation in the extemal DIC supply during growth. The CCM response has been

investigated in Chlamydornonas reinhardtii to a great extent in the past, but not the

regulation of its CCM. C&,, activity is induced in Chlamydomonas reinhardtii cells

under Iow CO2 conditions, whereas Chlorella kessleri contains no detectabIe C&,,

activity (Matsuda et al., 1999). Matsuda et al. (1999) report high whole-ce11 rate constants

for HC03- and COa and a high photosynthetic afinity for DIC in Chlorella kessleri cells

grown by aeration with air at a rate less than 10 mL min-', as compared to high CO2-

grown celis which do not express similar high affinity photosynthetic charactenstics.

These results suggested that active DIC transport and high affinity photosynthesis may be

induced in response to CO2-limited growth. In confiming the presence of a CCM in

Chlorella kessleri, it is important to deterxnine the trigger for induction of the CCM in

both organisms. Matsuda and Coiman (1995a, 1995b, 1996a) have determined that an

inducible CCM, in particular active DIC transport, is regulated in response to a critical

dissolved CO2 concentration in the bulk medium during growth, The object of this study

was to determine how the CCM in Chlorella kessleri and Chlamydomonas reinhardtii is

regulated in order to provide evidence whether the trigger for induction is response to

extemal CO2 concentration and therefore a cornrnon phenornenon in green algae.

2 - Materials and Methods 2.1 Growth Conditions

Axenic cultures of Chlorella kesslen' ( 1 8 08) and Chlamydomonas reinhardtii

(2137 mt+) were originally obtained from the University of Texas Culture Collection and

the Chlarnydomonas Genetics Center at the University of Duke Culture Collection,

respectively. Cells were transferred axenically to batch culture, as described previously

(Gehl et al., 1990). Chlorella kessleri cells were grown in BB medium (Nichols and

Bold, 1965); Chlamydomonas reinhardtii cells were grown in a modified SG medium

(Sager and Granick, 1953). Modifications to SG medium were the replacement of 0.38

rnM NH4N03 with 0.42 rnM NWCI, and the exdusion of organic components such as

citrate or acetate. Cultures were illuminated under a PPFD of 100 p o l s-'. Ce11

cultures were grown under a variety of CO2 regimes, which included aeration at a rate of

3.6 L min-' with air containing either 0.036 % CO2 (low COz), 5 % COz (high COz), or

COTfree air. CO2-free grown cells could also be obtained by aeration with air at a rate of

10 mL min-', which ensured a DIC concentration in the medium of O pM.

2.2 Determination of the Apparent m o l e Ce11 Affinity for DIC

The physiological characteristics of cells grown under the various CO2 regimes

were assessed. Cells were harvested at the rnid logarithmic stage of growth (A730 0.4-0.5)

by centrifugation at 4500 g (Sorvall R3-B Superspeed Centrifuge) for 3 min at room

temperature. Ceils were washed twice with N2-equilibrated, 50 mM sodium/potassium

phosphate bufTer (pH 7.8)- containing less than 5 pM DIC, and resuspended in the same

buffer. Rates of photosynthetic oxygen evolution at various DIC concentrations were

rneasured in a Clarke-type O2 electrode (Hansatech Instruments Ltd.) as described

previously (Gehl and Colman, 1985) with a PPFD of 400 p o l m-Z s-'. The apparent

whole cell affinity (Kin) for DIC and CO2 with and without the addition of bovine CA

was determined according to the procedure of Rotatore and Colman (1991b). The CO2-

compensation point was measured by a gas chromatographie procedure (Birmingham and

Colman, 1979).

2.3 Time Course of Induction of Active Bicarbonate and Active CO2 Transport

Physiological changes in high COz-grown cells acclimating to air were exarnined.

An aliquot of high CO2-grown Chlorella kessleri ce11 culture and/or high CO2-grown

Chlamydomonas reinhardtii cell culture was harvested at the mid logarithrnic stage of

growth by centrifugation as described above. The pellet of cells was resuspended in BB

medium (pH 6.6) for Chlorellu kessleri, or in SG medium (pH 6.5) for Chlamydomonas

reinhardtii. The two different ce11 suspensions were cultured axenically under low CO2

aeration. Cells were acclimated for 24 h and the DIC concentration of the medium was

monitored using gas chromatography. During the acclimation process, cells were

harvested periodically as described above. The chlorophyll concentration of the ce11

suspension was approxirnately 40 pg Ch1 mL? Cells were incubated in the 0 2 electrode

apparatus under a PPFD of 400 p o l m" s-L and ailowed to reach the CO2 compensation

point. The capacity of whole cells to actively take up HC03- was assessed by comparing

the photosynthetic O2 evolution rate at defined conditions of DIC concentration, pH of

the assay buffer and temperature, with the theoretical O2 evolution rate that can be

supported by the maximum rate of the uncatalyzed breakdown of HC03- in the medium

to form CO2 under the same conditions, which was calculated according to the method of

Miller and Colman (1980a). The O2 evolution rate of Chlorella kessleri cells was

measured at 50 ph4 DIC, pH 7.8 and 25'C, for Chlamydomonas reinhardtii cells, it was

measured at LOO pM DIC, pH 8.0 and 25"C, in the presence of 5 pA4 AZA. Stimulation

of the Os evolution rate upon the addition of bovine CA was used as a measure of active

CO2 uptake by the whole cells in suspension. The effect of O2 concentration in the

medium of the acclimating culture was examined in ChZorella kessleri cells by

transferring high COrgrown cells to BB media, and culturing thern axenicaily by

aeration with 02-free Nt enriched with 0.036 % COa. The photosynthetic O3 evolution

rates were measured periodically in acclimating cells with and without CA, as described

above. The effect of darkness on the induction of active DIC transport during acclimation

to low CO2 was also examined.

2.4 Time Course of Induction of Extracellular Carbonic Anhydrase Activity in

Chlamydornonas reinhardtii

The tirne course of induction of extracelluiar carbonic anhydrase (C&,J activity

during the acclimation of high CO~grown Chlamydornonas reinhardtii celIs to air was

assessed using a potentiornetric assay (Williams and Colman, 1996). High CO2-grown

cells were harvested at the mid-logarithrnic stage of growth (AT3() 0.4) by centrifugation at

5000 g for 3 min at room temperature. Cells were resuspended in SG medium at pH 6.6,

and allowed to acclimate to a i . level CO2. During the acclimation process, ceils were

harvested penodically, and the C&, activity was measured. CeUs were harvested,

washed in 20 mM Na-barbital buffer (pH 8.3), resuspended in 1.5 rnL of the same buffer

and placed in a water-jacketed chamber (2.0 to 4.0°C) containing a pH electrode. COz-

saturated water (0.5 mL) was added to the ce11 suspension after a one-minute incubation,

and the time for a drop in pH from 8.3 to 8.0 was measured. C L , activity is measured in

W-A units mg CM' and was calculated by the following formula:

CA& = (t& - l)/ [CH].

In this calculation, t, represents the time for the pH to change from 8.3 to 8.0 upon the

addition of CO2-saturated distilled Hfi; t, represents the time taken for the pH change

when cells are present. In the Iatter case, a shorter time period indicates an increase in the

rate of acidification of the medium (the conversion to CO2 to HC033. The activity was

normaiized to chlorophyll concentration of the ce11 suspension, which was determined as

descnbed previously (Williams and Colman, 1993).

2.5 Determination of the Critical Ci Concentrations Required for the Induction of

the CCM

The critical Ci conditions causing the induction of active CO2 and HC03-

transport were assayed according to the procedure of Matsuda and Colman (1995b).

Briefly, high CO2-grown cells of Chlorella kessleri and Chlamydomonas reinhardtii were

harvested at the rnid-logarithmic stage of growth. (A730 0.4)- Chlorella kessleri cells were

resuspended in BB medium (phosphate buffered at pH 6.6 or 7.5). Chlamydomonas

reinhardtii cells were resuspended in SG medium (phosphate buffered at pH 5.5 or 7.5).

The cells suspensions were axenically transferred to 0.5-L cylindrical culture vesseIs

equipped with a sarnpling port plugged with a rubber serurn stopper. Cells were aerated

with defined inflow CO2 concentrations, in the range of O to 0.42 % and 0.036 to 0.84 %

for Chlorella kessleri and Chlamydornonas reinhardtii, respectively. The dissolved CO2

concentration in the medium was monitored by adjusting the pH to 2 0.1 units, by

injections with 2.0 M HCI or 2.0 M NaOH; and by maintaining a constant inflow CO2

concentration. M o w CO2 concentrations and the DIC concentration of the medium were

measured by the gas chrornatographic technique. DIC equiIibrium conditions were best

maintained when the A730 of the acclimating culture was 0.2. Equilibrium conditions

between HC03- and CO2 in the culture medium were verified by comparing the

calculated concentrations of DIC at each pH and inflow CO2 concentration (Buch, 1960;

Sturnrn and Morgan, 1981) with the measured concentration in the medium. Chlorella

kessleri and Chlamydornonas reinhardtii cells acclimating to defined CO2 concentrations

were harvested after 5.5 h and 2 h, respectively. At this point, the O2 evolution rates

were measured as described above. CkXt activity was measured in Chlamydomonas

reinhardtii cells acclimating for 6 h to defined CO2 concentrations.

2.6 Determination of the Effect of Protein Synthesis and Metabolic Inhibitors on

the Induction of the CCM in Chlorella kessleri

The effect of protein synthesis inhibitors on the acclimation of high CO2-grown

cells to low CO2 was assayed according to the method of Matsuda and Colman (1995a).

High CO2-grown cells were harvested by cenwgation and resuspended in BB medium

containing 5 pg rd-' cycloheximide or 400 pg de' chloramphenicol. Ce11 cultures

were aerated with air containing 0.036 % CO? for 5.5 h. Cells were assayed for the

capacity to transport HC03- and CO2 imrnediately following the acciimation penod

according to the procedure described above. The effect of 5 rnM AOA, an

aminotransferase inhibitor, was examined during the acclirnation of high COz-grown

cells to high CO2 for 5.5 h.

3 - Resdts 3.1 Affirnity for DIC in Chlorelia kessleri Under Various COz Regirnes

Chlorella kessleri ceUs were grown under various CO2 regimes comprising

growth under air supplemented with either 5 % CO2, or 0.036 % CO2 and CO2-free air.

Photosynthetic oxygen evolution rates of cells grown under the various COî conditions

were measured over a range of DIC concentrations at pH 7.8 in a closed system, once the

CO2 compensation point of the ce11 suspension had been reached. Chlorella kessleri celIs

grown under DIC-limited conditions demonstrated a higher photosynthetic affinity

for DIC and CO2 (Table 1) in cornparison to 5 % C02-grown cells. The (DIC) was

Iowest in cells grown in CO2-free medium (Table 1). Intermediate K 1 ~ ( DIC) values

were obtained for Chlorella kessleri cells grown under air (Table 1, Fig. 4). When bovine

CA was added to algal ce11 suspensions during the O2 evolution assay there was a further

decrease in the K112 (DIC) and Kin (CO2) values under al1 growth conditions (Table 1,

Fig. 5). The Pm, was similar for cells grown in CO2-enriched and COrlimited media

(Table 1, Fig. 4, Fig. 5). The CO2 compensation point was also found to decrease when

the CO2 level in the growth medium was reduced (Table 1).

3.2 Changes in Extracellular Carbonic Anhydrase Activity in Chlàmydomonas

reinhardtii During Acclimation to Low CO2

ChZamydornonas reinhardtii cells grown under high CO2 conditions were

acclimated to ambient CO2 conditions. During the acclimation process, CL&,, activity

was measured periodicaliy. C&,, activity increased markedly within the first 5 h of

acclimation to 0.036 % CO2 (Fig. 6) . Within 6 h of acclimation to low CO2, there was a

10-fold increase in C k X t activity, when cornpared to the basal level of activity measured

in high CO2-grown cells (Fig. 6). There was a slight increase in C L t activity between 6

h and 24 h of acclimation. Changes in C&,, activity were also measured with cells

acclimating to air in darkness (Fig. 7). A slight lag in the induction of C&, activity was

apparent during acclimation to low CO2 in darkness. A 3-fold increase in C&,, activity in

comparison to high CO-grown cells was evident within 8 h of acclimation. At 10 h of

acclimation under low CO2 and darkness, C&,, activity was approximately 2-fold greater

than cells acclimated for 8 h. There was no significant increase in C k X t activity between

10 h and 24 h of acclimation (Fig. 7).

In order to detect the induction of active HC03- transport induction in

Chlarnydomonas reinhardtii cells during acclimation to low CO2, it was necessary to

block any C L t activity. Under alkaline pH conditions, the presence of C&,, activity

maintains the CO2-HC03- equilibrium, and therefore comparison of the spontaneous

dehydration rate with the measured photosynthetic OZ evolution rates is not an

Time of Acclimation (h)

Figure 6. Changes in CA,,, activity of high CO,-grown Chlamydomonas reinhardtii cells during acclimation to air. Values represent the mean c SE of four separate experiments.

O 5 10 15 20 25

Time of Acclimation (h)

Figure 7. Changes in CA,, activity of high CO,-grown Chlamydomonas reinhardtii cells during acclirnation to air in darkness. Values represent the mean + SE of four separate experiments.

appropriate assessrnent of active HCOY uptake. AZA (5 p.M) was found to completely

inhibit CAd, activity in air -grown cells harvested at rnidLlog phase (data not shown).

3.3 The T h e Course of Acciimation in Chlorella kessleri and Chluntydomonus

rein hardtii

Suspensions of high CO2-grown cells were transferred to COTlimiting conditions.

Chlorella kessleri cells were allowed to acclimate separately to air, CO2-fiee air, and Oz-

free nitrogen supplemented with 0.036 % CO2 for 24 h, whereas Chlamydomonas

reinhardtii cells were allowed to acclimate to air for 24 h. The DIC concentration in the

acclimation medium of Chlorella kessleri and Chlamydornonas reinhardtii cells was

initially about 5.0 mM at pH 6.6, and thïs decreased to approximately 30 pM at pH 6.6

after 2 h of acclimation. Niquots of the ce11 suspensions were taken at intervals over the

24 h period in order to determine photosynthetic rates. Photosynthetic 0 2 evolution rates

for Chlorella kessleri cells acclimating to 0.036 % CO2 in the presence and absence of 21

% 02, and to CO?-free conditions, were measured at 50 pM DIC, pH 7.8 and 25OC. O2

evolution rates for Chlamydomonas reinhardîii cells acclimating to air were determined

at 100 p M DIC, pH 8.0 and 25OC. Under these defined conditions, the theoretical O2

evolution rate at which the production of CO2 from the available HC03- is maximum is

5.39 nmol O2 mL-' min-' and 7.30 nmol 0 2 d-' min-' for Chlorella kesslen and

Chlamydomonas reinhardtii cells, respectively- In the case of Chlorella kessleri cells,

within 2 h of acclimation to air in the presence and absence of 0 2 (Fig. 9), O2 evolution

rates measured in the absence of CA were significantly greater than the caicuIated

maximum rate of CO2 supply, and assurning a photosynthetic quotient of unity, this

O 5 10 15 20 25 30

Tirne of Acclimation (h)

Figure 8. Photosynthetic O evolution rate of high CO,-grown cells of Chlorella kessleri dunng acclimation to air and CO,-fiee air. 0, evolution rates were

measured at 50 p M DIC, pH 7.8 and 2S°C, and approximately 40 pg Ch1 rnL-'. O, evolution rates in cells: acclirnating to air with (l ) and without CA (a ); and acclirnating to CO,-fiee air with (v) and without added CA (A ).The dashed line represents the calculated rate O, evolution rate at which the spontaneous CO, - supply from HCO; is maximum. Values are the means + SE of three separate experiments.

Time of Acclimation (h)

Figure 9. The photosynthetic O evolution rate of high COrgrown cells

of Chlorella kesslen' dunng acclimation to air containing 21 % 0, and 0,-

free air. O, - evolution rates were measured at 50 pM DIC, pH 7.8 and Z°C, at approximately 40 pg Ch1 mL-l. O evolution rates in cells: acclimating to air assayed with (0 ) and without ( O ) added CA; and acclimating to 02-free N, supplemented with 350 ppm CO, assayed with ( ) and without ( A ) added CA. The dashed line represents the rate at which the spontaneous formation of CO, is maximum at 50 pM DIC, pH 7.8 and 2S°C. Values are the means + SE of three separate experiments.

indicates that active bicarbonate transport was induced in cells within 2 h. The same

phenornenon of induction was apparent in Chlorella kessleri cells acclimating to CO2-

free air, but there was a 2.5-fold increase in the O2 evolution in comparison to air

acclimated ceI1s (Fig. 8). The Oz evolution rates were greater in 6 h acciimated Chlorella

kessleri cells regardess of the low COrregime. HC03- transport was fully induced in

Chlorelia kessleri cells within 5.5 h during acclimation to low CO2 (Fig. 8, Fig. 9). In the

case of Chlamydomonas reinhardtii cells, O2 evolution rates were measured at the

aforementioned conditions in the presence of 5 p M AZA. In high COrgrown

Chlamydomonas reinhardtii cells, 0 2 evolution was significantly lower than the

spontaneous dehydration rate (Fig. IO), which suggests there is no active HC03- transport

present. Within 2 h of accIimation to air, there was a marked increase in O2 evolution in

the presence of AZA, which was 1.5 foId greater than the spontaneous dehydration rate.

This suggests that active HC03- transport is induced in Chlamydomonas reinhardiii cells

within 2 h of acclimation to low CO2 (Fig. 10).

During the sarne acclimation processes for both cells, 0 2 evolution was measured

in the presence of bovine CA. In 2 h air-acclimated Chlorella kessleri and

Chlamydomonas reinhardtii cells, the addition of CA stimulated the O2 evoIution rate

1.5-fold (Fig. 8) and 3-fold (Fig. IO), respectively, in cornparison to 0 2 evolution

without CA . This suggests that active CO2 transport is induced, since the addition of

excess CA maintains the CO2 supply available to the cells. Active CO2 transport was

hl ly induced within 2 h in Chlamydomonas reinhardtii and within 6 h in Chlorella

kessleri (Fig. 8 , Fig. 10). In Chlorella kesslen, the rate of induction of active CO2

O 2 4 6 8 10 12

Tirne of Acclimation (h)

Figure 10. The photosynthetic O2 evolution rate of high CO2-grown cells cells of Chlamydomonas reinhardîii during acclimation to air. O, evolution rates were measured at 100 p h i DIC, p H 8.0, and 2S°C, at approximately 40 pg Ch1 mL-l, with AZA (a ) and with added CA(. ). Values represent the mean + SE of four experiments. Dashed line represents the calculated rate of spontaneous CO2 formation from 100 pM HCO,- at pH 8.0.

O 2 4 6 8 10 12 14

T h e of Acclimation (h)

Figure 11 The photosynthetic O, evolution rate of high CO,-grown ceiis of Chlorella kessleri during acclimation to COrfree air in darkness. 0, evolution rates

were rneasured at 50 p M DIC, pH 7.8 and 25OC, at approximately 40 pg Chl mLL, with (m ) and without added bovine CA ( ). As a control, cells were transferred to high CO, in the dark, and the 0, evolution rates were determined with ( V ) and without CA ( A ). The dashed line represents the rate at which the spontaneous formation of CO, is maximum at 50 pM DIC, pH 7.8 and 25OC. Values represent the means + SE of three separate experiments.

O 2 4 6 8 10 12 14

Time of Acclimation (h)

Figure 12. The photosynthetic O2 evolution rate of high CO,-grown cells of Chlorella kessleri during acclimation to air in darkness. 0, evolution rates were

measured at 50 p M DIC, pH 7.8 and 2S°C, at approximately 40 pg Ch1 rd,-', with (a ) and without added bovine CA ( 0 ). As a control, cells were tranferred to high CO, in the dark, and the 0, evolution rates were determined with and without CA (Data not shown). The dashed line represents the rate at which the spontaneous

formation of CO, is maximum at 50 pM DIC, pH 7.8 and 25OC. Values represent the

means t SE of three separate experiments.

Time of Acclimation (h)

Figure 13. Changes in the photosynthetic O, evolution rate in high COigrown cells of Chlamydomonas reinizurdtii during acclimation to air in darkness. 0, evolution rates were measured at 100 p M DIC, pH 8.0 and 25OC, at approximately 40 pg Ch1 mL-l, with AZA ( ) and with added CA (. ). Values represent the mean + SE of four separate experiments, Dashed line represents the calculated rate of - spontaneous CO, formation fiorn 100 p M HC03- at pH 8.0.

transport was similar in air-acciimated cells, in the presence and absence of 21 % 02, and

in CO2-free acclimated cells. O2 evolution rates were higher in the latter case.

The same parameters of induction were assessed during the acclimation processes

of Chlorella kessleri and Chlamydornonas reinhardtii cells to low CO2 in darkness. The

DIC concentrations in the medium of Chlorella kessleri cells acclimating to air and C O -

free air, and Chlamydornonas reinhardtii cells acclimating to air were 50,4, and 45 pM,

respectively. The time course of acclimation of Chlorella kessleri cells (Fig. 12) and

Chlamydornonas reinhardtii cells (Fig. 13) to air was similar to that of cells in the light,

except that the maximum rate of photosynthetic O2 evolution corresponding to fully

induced dark-acclimated cells was lower than that of cells acclimated in the light (Fig. 8,

Fig. 10). In dark-acclimated Chlorelia kessleri cells, O2 evolution rates were higher in

cells acclimating to CO2-free aeration (Fig. 11) rather than aeration with 0.036 % COz

(Fig. 12), which was also apparent in the acclimation of Chlorella kessleri cells to

various low CO2-regirnes in the light (Fig. 8). Maximum induction of active CO2

transport was slightly slower in Chlorella kessleri cells acclimated to air (Fig. 12) than in

CO-free conditions (Fig. II). The results indicate that active HC03- and COz transport

are induced in Chlorella kesslen and Chlamydornonas reinhardtii cells dunng

acclimation to low CO2 in darkness.

3.4 Effect of MetaboIic Inhibitors on the Acclimation of Chlorella kessleri to Low

coz.

Hïgh CO2-grown cells were allowed to acclimate to 0.036 % CO2 at pH 6.6 for

5.5 h in the presence of protein synthesis inhibitors. Treatment with a cytoplasmic protein

High CO, + Aminooxyacetate (5 mM) p

High CO, (Control) i I

Air (Control) CLni Air + Chloramphenicol

Air + Cycloheximide F'

Oxygen evolution rate (mol O, mL-l min-')

Figure 14. The effect of inhibitors of protein synthesis and inhibition of arninotransferases on the induction of active DIC transport in high CO,-grown Chlorella kesslet-i cells

acclimating for 5.5 h. O, evolution rates were determined at 50 p M DIC, pH 7.8, and 2S°C with (m) and without (O) added CA. Values represent the mean + SE of three to five separate experiments. High CO,-grown cells were also - acclimated to air and to high CO, as control experiments. The dashed line

represents the calculated maximum rate of CO, formation from 50 p M HCO,- at pH 7.8 and 25°C.

synthesis inhibitor, cycloheximide (5 w mL-l), resulted in measured 9 evolution rates in

the presence and absence of CA which remained comparable to those of cells maintained

on 5 % CO2 for 5.5 h (Fig. 14). Cycloheximide inhibited the induction of active HC03-

and CO2 transport- Treatment with the chloroplastic protein synthesis inhibitor,

chlorarnphenicol (400 mg d - 1 ) , did not inhibit the induction of active CO2 and HCOs-

transport in Chlorella kessleri cells acclimating to low CO2, and the 0 2 evolution rates

measured in the presence and absence of CA were similar to ceUs acclimating to low CO2

with no inhibitor (Fig. 14). It has been suggested that the accumulation of intermediates

of the photorespiratory pathway, possibly phosphoglycolate (Marcus et al,, 1983; Suzuki

et al., 1990) or glycolate could act as triggers for the induction of the CCM in algae. In

order to test this hypothesis, high CO2-grown cells, maintained on high CO2, were treated

with the photorespiratory pathway inhibitors, 5 rnM AOA and IO mM INH for 5.5 h.

Neither AOA (Fig. 14) nor INH (data not shown) had a stimulatory effect on the

induction of active D K transport of Chlorella kessleri cells.

3.5 Critical External DIC Concentration During Acclimation

Ce11 suspensions of high CO2-grown Chlorella kessleri and Chlamydomonas

reinhardtii were aerated with various inflow COa concentrations, in the range of O to 0.42

% and 0.036 to 0.84 % CO2, respectively. Chlorella kesslen' cells were allowed to

acclimate for 5.5 h at pH 6.6 or pH 7.5, whereas Chlamydomonas reinhardtii cells were

allowed to acclimate for 6 h at pH 5.5 or pH 7.5. In Chlamydomonas reinhardtii, after 2

h of acclimation, a small aliquot of ce11 suspension was harvested in order to measure

photosynthetic O2 evolution, and the rate of HC03- and CO2 transport at 100 pM DIC, pH

External [DIC] during Acclimation (PM)

O 50 100 150 200 250 300

O 50 100 150 200 250 300

External [CO,] during Acclimation (PM)

Figure 15. AccIimation of high CO,-grown Chlamydornonas reînhardtii cells to various concentrations of DIC (top mis) and CO, (lower mis) at pH 5.5 for 2 h. O, - evolution rates were deterrnined at 100 p M DIC, pH 8.0, and 2S°C with AZA (O ) and with added CA (. ). The dashed line represents the calculated maximum rate of CO, formation from 100 p.M HCO,- at pH 8.0 and 2S°C.

External [DIC] during Acclimation (PM)

O 1000 2000 3000 4000

Extemal [CO,] during Acclimation (FM)

Figure 16. Acclimation of high CO,-grown Chlamydornonas reinhardtii cells to various concentrations of DIC (top axis) and CO, (iower axis) at pH 7.5 for 2 h. O,

evolution rates were detennined at 100 pM DIC, pH 8.0, and 25°C with AZA (0 ) and added CA (. ). The dashed Iine represents the calculated maximum rate of CO, formation fiom 100 ph4 HCOy at pH 8.0 and 25OC.

8.0 and 25OC was assayed. With an increase in the external CO2 concentration during

acclirnation, there was a concomitant decrease in O2 evolution measured in the presence

of AZA (Figs. 15 and 16). In cells acclimating at pH 5.5, HC03- transport was fully

induced at approximately 11 pM DIC, whereas at pH 7.5 HC03- transport was fully

induced at approximately 160 pM DIC. Regardless of the pH of the cultlare medium to

which the cells were acclimating, HC03- transport was firlly induced at approximately 10

@M dissolved CO2 in the extemal medium. HC03- transport was fully rzpressed at

approximately 1600 pM DIC and 100 ph4 DIC, during acclimation at pH 7.5 and 5.5

respectively. At both pHs of acclimation, HC03- uptake was fully repressed at 98 plkl

dissolved CO2. The same phenornenon was apparent with 0 2 evolution measurements in

the presence of bovine CA. Active CO? transport was fully induced in ceiis at

approximately 13 /AM and 192 p M DIC during acclimation at pH 5.5 and 7.5,

respectively (Figs. 15 and 16). At both pHs, active CO2 transport was fully induced at 12

p M dissolved COa in the external medium. Transport of this inorganic carbon species

was fully repressed when cells were acclimated to 100 p.M dissolved CO2, at both pH

values. C&,, activity was measured in Chlamydornonas reinhardtii cells acclimating to

various external CO2 concentrations at pH 5-5 or 7.5. Induction of C L , activity

increased with a concomitant decrease in the external CO2 concentration during the 6 h

acclimation (Fig. 17). The highest level of C&, activity was approximately 68 WA units

mg CM-'. Regardless of the pH at which the cells were acclimating, C L , was fully

induced during acclimation to 10 ph4 dissolved CO2. Basal CkXt activity (40 WA units

Extemal [CO,] during Acclimation (PM)

Figure 17. Determination of the critical CO, concentration effecting the induction of CAa, activity in Chlamydornonas reinhardtii cells. High CO,-grown

Chlamydornonas reinhardtii cells were acdimated to various

concentrations of CO, at pH 5.5 (1) and pH 7.5 (+ ) for 6 h.

O 20 40 60 80 10012014 Extemal [COJ during

Acclimation (pM)

O 50 100 150 200 250 300 350

External [ DIC] durhg Acclirnation (PM)

Figure 18. Acclimation of high CO,-grown Chlorella kessleri cells to various external concentrations of DIC and CO, (inset) at pH 6.6 for 5.5 h. O, evolution rates were determined at 50 p M DIC, pH 7.8, and 2S°C with (O ) and without (a ) added CA. The dashed Iine represents the calculated maximum rate of CO, formation from 50 p M HCO,- at pH 7.8 and 25°C.

O 20t 40 60 80 10012014 External [CO,] during

Acclimation (pM)

External [DICI during Acclimation (PM)

Figure 19. Acciimation of high CO-grown Chlorella kessleri cells to various external concentrations of DIC and CO, - (inset) at pH 7.5 for 5.5 h. O, evolution rates were determined at 5r0 pM DIC, pH 7.8, and 25OC with ( ) and without (0 ) added CA- The dashed line represents the cdculated maximum rate of CO, formation fkom 50 p M HC0,- at pH 7.8 and 2S°C.

mg CH-') was apparent after acclimation to dissolved extemal CO2 concentrations of 100

p M CO2 and higher (Fig. 17).

In Chlorellu kessleri, the rates of HC03- and CO2 transport at 50 pM, pH 7.80 and

25OC were assayed at the end of each acclimation period. At pH 6.6, O2 evolution rates

measured in the absence of CA, indicated that HC03- transport was repressed at

approximately 240 ph4 DIC (Fig. 18), when the 0 2 evolution rate was compared to

themaximum rate of uncatalyzed CO2 supply. At pH 7.5, the DIC concentration in the

external medium that repressed active HC03- transport was 1300 pM (Fig. 19). At both

pH 6.6 and 7.5, the dissolved CO2 concentration eliciting the fully repressed responses

were sirnilar, being 86.4 and 86 pM, respectively. ChLorella kessleri cells acclimating to

CO2 concentrations in the externd medium lower than 86 pM had 0 2 evolution rates

measured without CA that increased in a non-linear relationship with a decrease in the

DIC and CO2 in the acclimation medium. Maximum 0 2 evolution rates were obtained

with and without CA in cells that had acclimated to extemal COz concentrations

approaching O (Figs. 18 andl9). These results indicate that the full induction of active

CO2 and HC03- transport occurs during acclimation to CO2-free conditions.

The DIC concentration corresponding to the 0 2 evolution rate at which 75 % of

the maximum DIC transport is induced was greater in ceils acclimating at pH 7.5 than in

cells acclimating at pH 6.6 (Table 2). The dissolved extemal CO2 concentration

corresponding to the induction of the half maximum DIC transport was approximately 15

p.M at both pH values (Table 2). Induction of HC03- transport was 50 % of the maximum

in Chlorella kessleri cells during acclimation at an external dissolved CO2 concentration

Table 2. CO2 and DIC concentrations eliciting induction of active CO2 and HC03-

transport in high CO2-grown Chlordla kessleB cells acclimating at two different pH

values for 5.5 h.

DIC Concentration (uM) at Acclimation pH of

6.6 7.5

Concentration at which 75 % of maximum DIC transport is induced

Total [PIC]

Total [CO21

Concentration at which half of maximum HC03- transport is induced

Total PIC] Total [Cod

Concentration at which bicarbonate transport is first repressed

Total DIC] Total [CO2]

of 15 p M (Table 2). O2 evolution measured in the presence of excess CA indicated that

the total DIC concentration in the external medium corresponding to repression of active

CO2 transport in Chlorella kessleri cells was approximately 5.5-fold greater in cells

acclimating at pH 7.5, in comparison to ceiis acclimating at pH 6.6 (Figs. 18 and 19).

The dissolved CO2 concentration corresponding to the full repression of active CO2

transport in Chlorellu kessleri was 120 pM, regardless of pH during acclimation. High

COrgrown Chlorella kessleri cells acclimating to extemal CO2 concentrations greater

than 120 pM, showed no significant difference in 0 2 evolution rates measured at 50

DIC, pH 7.80 and 25OC with and without added CA (data not shown), indicating that cells

had a greatly reduced capacity to transport CO2 when acclirnated to free CO2

concentrations of 120 pM or greater. Active DIC transport is fully repressed at a slightly

higher extemal CO2 concentration in the acclimation medium in Chlorella kessleri than

in Chlumydornonas reinhurdtii.

4 - Discussion 4.1 Hi&-Mity for Inorganic Carbon is Induced in Chlorella kessleri Ceils Under

Low CO2 Conditions

A CCM is induced in Chlorella kessleri and Chlarnydomonas reinhardtii during

acclimation to a critical dissolved CO2 concentration in the external medium. In both

species, cells grown under a 5 % CO2 aeration, exhibit a low affinity for DIC and CO2 in

comparison to Iow CO2-grown cells, although there is no difference in the Pm of cells

grown under the two growth regimes (Table 1, Fig. 4, Fig. 5; Sültemeyer et d., 1991).

High affinity photosynthesis in low COrgrown Chlorella kessleri cells was similar to

that in other green algae (Badger et al., 1980; Matsuda and Colman 1995a; Mayo et al.,

1986; Sültemeyer et al., 199 l), and to cases where the CCM is constitutively expressed

under al1 CO2 concentrations in the external medium as in Chlorella saccharophila and

CO2-insensitive Chlorella ellipsoidea mutants (Matsuda and Colman, 1996% 1996b).

Air-grown Chlorella kessleri cells have a high affinity for dissolved CO2 (Table 1) and

there was a marked increase in affinity in CO2-free grown celIs, indicating a Mly

inducible CCM may be responding to an external CO2 concentration between arnbient

and CO2-free conditions. The sarne affinity comparison was not made for

Chlamydomonas reinhardtii grown under COî-free conditions.

Air-grown Chlorella kessleri cells had a high affinity for CO2 as indicated by the

low (CO2) of 1.2 pM in the presence of bovine CA which was used to maintain a

constant CO2 concentration in the medium (Table 1, Fig. 5). The CO2 affinity of air-

grown and CO2-free grown cells is high, but the increase in CO2 affinity of high COz-

grown cells upon the addition of CA was the first indication of the possibility of some

CO2 transport activity in these celIs (Table 1). Matsuda et al. (1999) reported that air-

grown Chlorella ellipsoidea and ChIorella saccharophila had high rate constants for CO2

in cornparison to HCO). Whole-ce11 rate constants are a quantitative determination of the

contribution of active CO2 and HC03' transport to photosynthesis when the absolute rate

of O2 evolution is measured at a lirniting DIC concentration in the absence of C&,

(Matsuda et al., 1999). In the same study, Chlorella kesslerr' showed the same

phenornenon but had rate constants that were significantly higher for both DIC species.

It appears that Chlorella kessleri utilizes available inorganic carbon better than other

Chlorella spp (Matsuda et al., 1999). High affinity photosynthesis is also apparent in air-

grown Chlamydomonas reinhardtii cells. The low Kin (CO?) of approximately 2 p M

(Aizawa and Miyachi, 1986) is comparable to Kin (COa) of Chlorella kessleri and other

green algae containing a CCM. The high affinity photosynthesis present in Chlorella

kessleri under DIC-Limiting conditions suggests that a functional CCM is present.

High ainity photosynthesis in DIC-lirnited cultures is often associated with an

increase or induction of intemal DIC accumulation (Badger et al., 1980; Palmqvist et ai.,

1988; Matsuda and Colman, 1995a; Matsuda et al., 1998). The internal DIC accumulation

in various green dgal species is often on the order of 40 to 75 fold, when the internal

DIC concentration is compared to the extemal DIC concentration (Badger et al., 1998).

AIthough the intemal DIC accumuiation is a recognized component of green algal and

cyanobacterial CCMs, some researchers argue the presence or absence of this factor as

the defining cornponent of a CCM. Badger et al. (1998) present evidence on nongreen

algal species that maintain a low internal accumulation of DIC, but display high affinity

photosynthesis; they proposed that the reduction in internal DIC accumuIation levels may

be due to thylakoid CA activity using protons from the thylakoid lumen to convert HC03'

to CO2, thereby causing a decrease in the stromal HC03- pool. This mechanism would

rely on a high thylakoid CA activity, but more evidence is needed to establish the validity

of this mechanism, since thylakoid CAS have been reported only in Chlamydomonas

(KarIsson et al., 1998). Intemal DIC accumulation seen in CCM-containing green algae is

not due to difisive entry (Badger et al., 1980), but rather an active accumulation process

involving uptake of CO2 and HC03' across the plasmalemma boundary (Marcus et al.,

1984; Rotatore and Colman, 199 lc).

4.2 Induction and Regulation of Active DIC Transport in Green Algae

Active transport of HC03- across the whole ce11 boundary was assayed by the

kinetic method of Miller and Colman (1980a) during acclimation of Chlamydomonas

reinhardrii and Chlorek kessleri to 0.036 % CO2. This qualitative analysis is used as

evidence to suggest that photosynthesis is supported by active HC03- uptake. A definitive

quantitative approach of the contribution of a particular extemal DIC species to

photosynthesis of a unicellular rnicroalga is provided by the method of Matsuda et al.

(2999). HC03- transport is induced in Chlorella kessleri and Chlurnydornonirs reinhardtii

cells during acclirnation to low CO2 (Figs. 8 and IO), and the presence of light is not a

deterrnining factor in this response (Figs. 1 1-13). Induction of active DIC transport was

slower in cells acclimated to low CO2 in darkness, than in the light. Matsuda et al. (1998)

suggest that the lower photosynthetic 0 2 evolution rates that are evident in dark-

acclimated cells may be explained by an increase in the rate of respiration, which would

contribute to an increase in the CO2 concentration in the bulk medium. This phenornenon

of induction was ails0 evident in Chlorella ellipsoidea cells acclimated to low CO2 in

darkness (Matsuda and Colman, f 995b). Under similar acclimation conditions, full

induction of HC03' transport occurs in a shorter time penod in Chlamydornonas

reinhardtii as com-pred to Chlorella kessleri. In both systems, the induction of active

HC03- transport is preceded by the induction of active CO2 transport. The characteristics

of induction of active DI% transport were sirnilar in Chlorella ellipsoidea (Matsuda and

Colman, 1995a), except that was a Iag in the induction of HC03' uptake (Matsuda and

Colman, 1995a). Bicarbonate transport was first observed in Chlorella kessleri and

Chlamydornonas reinhardtii after 2 h (Fig. 8) and after I h (Fig. 10) acclirnation to air,

respectively. It appears that the time of induction of active HC03- transport varies

between green a l g d species.

Under high CO2-growth conditions, HC03- transport is repressed in green algae

(Figs.15, 16, 18 amd 19; Matsuda and Colman, 1995b; Matsuda et al., 1998). HC03-

transport is induced during acclimation to a continuous low dissolved COz concentration

in the external medium, which is pH-independent. The higher level of HC03- transport

activity in Chlorellk kessleri cells acclimated to COrfiee aeration as compared to air-

acclimation sugges-ted the possibility of a continuum of HC03- transport activities in

response to acclimation to a continuum of CO2 regirnes (Fig. 8). This was evident in

Chlorella kessleri cells acclimated to various external CO2 concentrations (Figs. 18 and

19). Mayo et al. (1986) report a similar response in the cyanobacterium, Synechococcus

leopoliensis, where intermediate KIR PIC) values were obtained in cells grown under

intermediate DIC levels. In Chlorella kessleri and Chlarnydomonus reinhardtii, active

HC03- transport activity is repressed during acclimation to 60 pli4 CO2 or greater, and

induced dunng acclimation to dissolved CO2 concentrations lower than 60 @A (Table 2).

Repression of active HC03- transport in Chlorella ellipsoidea cells occurs at a sirnilar

external CO-, concentration (Matsuda and Colman, 1995a). Full induction of active

HC03' transport in Chlorella ellipsoidea (Matsuda and Colman, 1995b), Chlamydomonas

reinhardtii and Chlorella kessleri occur in response to acclimation at 35, 10, and O pM,

respectively. The inflow CO2 concentrations that effect the full induction responses in

Chlorella ellipsoidea, Chlamydomonas reinhardtii and Chlorella kessleri are 0.1 %,

0.036 % and O % CO2, respectively.

There is a marked difference i