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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
M3J 1P3
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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