carbon acquisition by diatoms
TRANSCRIPT
REVIEW
Carbon acquisition by diatoms
Karen Roberts Æ Espen Granum Æ Richard C. Leegood ÆJohn A. Raven
Received: 31 August 2006 / Accepted: 11 April 2007 / Published online: 12 May 2007
� Springer Science+Business Media B.V. 2007
Abstract Diatoms are responsible for up to 40% of pri-
mary productivity in the ocean, and complete genome
sequences are available for two species. However, there are
very significant gaps in our understanding of how diatoms
take up and assimilate inorganic C. Diatom plastids origi-
nate from secondary endosymbiosis with a red alga and
their Form ID Rubisco (ribulose-1,5-bisphosphate carbox-
ylase-oxygenase) from horizontal gene transfer, which
means that embryophyte paradigms can only give general
guidance as to their C acquisition mechanisms. Although
diatom Rubiscos have relatively high CO2 affinity and
CO2/O2 selectivity, the low diffusion coefficient for CO2 in
water has the potential to restrict the rate of photosynthesis.
Diatoms growing in their natural aquatic habitats operate
inorganic C concentrating mechanisms (CCMs), which
provide a steady-state CO2 concentration around Rubisco
higher than that in the medium. How these CCMs work is
still a matter of debate. However, it is known that both CO2
and HCO3– are taken up, and an obvious but as yet unproven
possibility is that active transport of these species across
the plasmalemma and/or the four-membrane plastid enve-
lope is the basis of the CCM. In one marine diatom there is
evidence of C4-like biochemistry which could act as, or be
part of, a CCM. Alternative mechanisms which have not
been eliminated include the production of CO2 from HCO3–
at low pH maintained by a H+ pump, in a compartment
close to that containing Rubisco.
Keywords Active transport � Bacillariophyceae �Carbon dioxide � CO2-concentrating mechanism �Carbonic anhydrase � C4 photosynthesis � Form ID
Rubisco � Pyrenoid
Introduction
There are at least 10,000 extant species of diatoms, con-
stituting the class Bacillariophyceae (phylum Hetero-
kontophyta, kingdom Chromista: Falkowski et al. 2004).
Diatoms occur in the benthos and plankton of the ocean
and inland waters, and account for up to 40% of the total
marine primary productivity of approximately 50 Pg C per
year (Field et al. 1998; Granum et al. 2005). Complete
genome sequences for the marine diatoms Thalassiosira
pseudonana and Phaeodactylum tricornutum have revealed
a unique mixture of characteristics, as befits organisms
derived from secondary endosymbiosis, in a clade separate
from plants and from opisthokonts (animals and fungi)
(Baldauf 2003; Armbrust et al. 2004; Montsant et al. 2005;
Allen et al. 2006). A unique characteristic of diatoms is the
bipartite, silicified cell wall which greatly influences their
ecology (Raven and Waite 2004).
In chromists, the endosymbiotic origin of plastids from
red algal cells (Falkowski et al. 2004) accounts for their
two additional membranes (‘plastid endoplasmic reticu-
lum’). These additional membranes require different,
incompletely characterised plastid targeting sequences
from those found in green and red algae and embryophytes
with just the two ‘normal’ plastid envelope membranes,
making it difficult to predict if a given nuclear-encoded
protein is targeted to the plastids (Apt et al. 2002). The
additional membranes also have implications for the
K. Roberts � J. A. Raven (&)
Plant Research Unit, University of Dundee at SCRI, Scottish
Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
e-mail: [email protected]
E. Granum � R. C. Leegood
Department of Animal and Plant Sciences, University of
Sheffield, Sheffield S10 2TN, UK
123
Photosynth Res (2007) 93:79–88
DOI 10.1007/s11120-007-9172-2
transfer of inorganic C species, and other low Mr solutes,
into plastids. Replacement of the Form IB Rubisco inher-
ited from the red algal plastid ancestor by a Form ID Ru-
bisco in chromists probably occurred by horizontal gene
transfer not directly related to secondary endosymbiosis.
With this background we consider the implications of
the available data for inorganic C acquisition by diatoms
and early stages of organic C metabolism (Raven et al.
2007).
Diatom Rubisco and the need for a CCM
Form ID Rubiscos have relatively high affinities for CO2,
high CO2/O2 selectivities, and a relatively low CO2-satu-
rated specific reaction rate (Badger et al. 1998; Tcherkez
et al. 2006). While diatom selectivity and affinity values
are not the highest measured for Form ID Rubiscos, they
are higher than those in coccolithophores (Badger et al.
1998; Whitney et al. 2001; Shiraiwa et al. 2004). Form ID
Rubiscos generally have higher CO2/O2 selectivities than
the other Rubiscos found in algal cells.
It has long been known that diatom cells have a very
high affinity for inorganic C (Barker 1935). While Barker
(1935) used Warburg buffers with very high inorganic C
concentrations and high, and varying, pH values to obtain
the required CO2 concentrations, such conditions could be
experienced in nature by diatoms in saline, high-carbonate,
inland waters. Badger et al. (1998) (see also Burkhardt
et al. 2001; Rost et al. 2003) showed that the affinity for
inorganic C expressed as CO2 in photosynthesis by diatom
cells can be forty times that of isolated diatom Rubisco. To
explain this with diffusive CO2 entry there would have to
be at least a 40-fold excess of inorganic C-saturated Ru-
bisco activity over the inorganic C-saturated rate of pho-
tosynthesis in vivo, both expressed on a cell basis (Raven
1984; Badger et al. 1998). In this case inorganic C-satu-
rated photosynthesis in the cells would be limited by
reactions such as reduction of NADP+ and phosphorylation
of ADP (Raven 1984). ‘At least’ refers to the 10,000-fold
lower diffusion coefficient of CO2 in water compared to
that in air, and in even the smallest diatoms the restriction
imposed by aqueous phase diffusion exceeds that in C3
land plants (Raven 1984). Such a 40-fold, or more, excess
of Rubisco protein and activity is practically impossible.
Assuming only a two-fold excess of Rubisco activity over
that required for the observed inorganic C-saturated rate of
photosynthesis in vivo, to half saturate Rubisco would re-
quire a two-fold accumulation of CO2 at the Rubisco active
site relative to that in normal seawater (~2 mol m–3 inor-
ganic C) at pH 8.2. A 20-fold CO2 accumulation would be
needed with 1/10 the normal inorganic C concentration in
seawater, a value in the range used in many experiments on
CO2 accumulation.
The observed ratio of internal to external inorganic C
concentration in marine diatom cells during photosynthesis
is up to 3.5 in a medium with more than twice the seawater
inorganic C concentration at pH 7.5 (Colman and Rotatore
1995), and is higher than 3.5 at lower external concentra-
tions at pH 7.5–8.0 (Burns and Beardall 1987; Rotatore
et al. 1995; Johnston and Raven 1996; Mitchell and
Beardall 1976; Reinfelder et al. 2004). Even higher ratios
occur in freshwater diatoms (Rotatore and Colman 1992).
Relating these findings to the CO2 concentration at the
active site of Rubisco involves assumptions about the
intracellular location of the accumulated inorganic C pool
and the pH at this site. Table 1 shows calculations
assuming that there is an equilibration of CO2 across the
plasmalemma, but not across the plastid envelope, and vice
versa. The calculations are for two external inorganic
carbon concentrations and for a steady-state CO2 concen-
tration in the plastid stroma that is twice the concentration
in normal seawater. For any of the assumptions made the
predicted inorganic C concentration averaged over the
whole cell volume (Table 1) is consistent with measured
mean intracellular accumulations. Even a requirement for a
CO2
concentration in the plastid stroma which is two times
that found in normal seawater would still yield predicted
inorganic C concentrations averaged over the whole cell
that are within the range of measured values. Hence, sig-
nificant inorganic C accumulation inside the chloroplast,
and even the cytoplasm, can be obtained without a large
increase in mean intracellular concentration due to the high
volume fraction of vacuole with little inorganic C. How-
ever, some of the diatoms that are widely used in experi-
ments (e.g., P. tricornutum) probably have a lower fraction
of the protoplast occupied by vacuole than the organisms
considered in Table 1, and so would have higher predicted
mean intracellular inorganic C concentrations. The range
of cells considered in Table 1 was restricted by the avail-
ability of morphometric data. We now consider the pro-
posed diatom CCMs which could deliver the required
increase in CO2 concentration around Rubisco.
Proposals for diatom CCMs
Table 2 and Fig.1 give an overview of possible diatom
CCMs, based on the CCMs already known, or proposed,
for O2-producing photosynthetic organisms. Mechanisms
Ia-c in Table 2 are based on active transport of CO2, HCO3–
and H+ across membranes, i.e., vector energized reactions.
Mechanisms IIa-c in Table 2 are based on energized
reactions in aqueous solution, i.e., scalar energized reac-
80 Photosynth Res (2007) 93:79–88
123
tions, although IIb also requires active transport for the
night-time accumulation of malic acid in vacuoles.
Active influx of CO2 (Table 2, Ia) has only been found
in eukaryotes, where it occurs at the plasmalemma and/or
the plastid envelope (Giordano et al. 2005). Diatoms can
take up CO2 from the medium (reviewed by Giordano et al.
2005) with CO2 accumulation in the cells (Rotatore and
Colman 1992). However, it is not known where the active
transport of CO2 occurs, i.e., across the plasmalemma or
the plastid envelope (see Raven 1997, and Table 2, Icii).
Wittpoth et al. (1998) showed that isolated plastids from
two species of diatom had over a third (on a chlorophyll
basis) of the intact cell rates of inorganic C-dependent
photosynthetic O2 evolution, but did not determine the
inorganic C species taken up, or whether inorganic C was
accumulated by the plastids. Since almost all the isolated
plastids had lost their chloroplast endoplasmic reticulum
membranes, their in vivo relevance is unclear. Uptake of
HCO3– rather than CO2 into the chloroplasts would facilitate
the operation of the mechanism of pyrenoid function de-
scribed below involving conversion of HCO3– from the
stroma to CO2 in the pyrenoid.
Active influx of HCO3– (Table 2, Ib) occurs, using sev-
eral mechanisms, at the plasmalemma of cyanobacteria.
HCO3– transport also occurs at the plasmalemma and plastid
envelope of eukaryotes, and leads to accumulation at both
the plastid and the whole cell level. HCO3– is taken up by
almost (see Cassar et al. 2002) all diatoms (Giordano et al.
2005), but it is not known whether it is transported across
the plasmalemma or plastid envelope. Despite the inside-
negative electrical potential (Boyd and Gradmann 1999)
across the plasmalemma of diatoms, 1:1 symport with H+
or 1:1 antiport with OH– would permit HCO3– entry without
energy input (see Raven 1997, and Table 2, Icii). Operation
of the mechanism of pyrenoid function discussed below
would be facilitated by HCO3– rather than CO2 influx from
the cytosol into the plasid stroma.
The required CO2 concentration around Rubisco is
more readily accommodated if the observed inorganic C
accumulation averaged over the whole cell volume is
recalculated for accumulation only in the plastids. Hence,
studies of inorganic C transport across the plastid enve-
lope of diatoms, and further investigation of mechanisms
based on active H+ transport (Raven 1997) are urgently
needed.
If there is accumulation of HCO3– in the plastid stroma
then a mechanism of CO2 supply to Rubisco of the kind
believed to occur in cyanobacteria could be envisaged,
with the pyrenoids found in the great majority of diatoms
(Schmid 2001) taking the place of carboxysomes. A diatom
version may have active HCO3– influx across the plastid
envelope and/or the plasmalemma, yielding a higher con-
centration of HCO3– in the chloroplast stroma than is found
in the medium, with Rubisco and carbonic anhydrase (CA)
mainly localized in the pyrenoid. In this model, HCO3–
enters the pyrenoid with ribulose bisphosphate (RuBP),
CO2 generated by CA is assimilated by Rubisco, and the
resulting phosphoglycerate (PGA) diffuses into the stroma
where it is further metabolised by the photosynthetic C
reduction cycle (PCRC). Diatom pyrenoids are typically
surrounded by a membrane of unknown composition
(Schmid 2001); is this membrane analogous to the pro-
teinaceous shell of carboxysomes (Kerfeld et al. 2005),
with anion-selective pores permitting fluxes of the sub-
strates HCO3– and RuBP and the product PGA? Kerfeld
et al. (2005) suggests that these channels restrict loss of the
CO2 generated by CA and entry of O2 that competes with
CO2 at the active site of Rubisco.
Table 1 Model calculations of total inorganic C accumulation in four
diatoms based on non-energized (= passive) CO2 equilibration across
the plasmalemma and tonoplast with active transport at the chloro-
plast envelope, or CO2 equilibration across the chloroplast envelope
and tonoplast with active transport at the plasmalemma
Organism Diatomatenue
Cyclotellameneghiniana
Fragilariacapucina
Stephanodiscusbinderanus
Protoplast volume 230 lm3 250 lm3 320 lm3 760 lm3
Vacuole volume as fraction of protoplast volume 0.45 0.40 0.45 0.62
Cytoplasm volume as fraction of protoplast volume 0.35 0.37 0.32 0.24
Chloroplast volume as fraction of protoplast volume 0.20 0.23 0.23 0.14
Mean [inorganic C] in protoplast for active transport at the chloroplast envelope and
external [inorganic C] of (i) 0.2 mol m–3 or (ii) 2 mol m–3(i) 0.82 (i) 0.94 (i) 0.94 (i) 0.57
(ii) 0.97 (ii) 1.10 (ii) 1.07 (ii) 0.68
Mean [inorganic C] in protoplast for active transport at the plasmalemma and external
[inorganic C] of 0.2 or 2 mol m–31.13 1.27 1.23 0.80
Other assumptions made were chloroplast stromal [inorganic C] of 4 mol m–3 and external medium [inorganic C] of either 0.2 or 2 mol m–3.
Quantitative morphometry yielding compartmental volume fractions was obtained from Sicko-Goad et al. (1977, 1988) and Sicko-Goad and
Stoermer (1979). Compartmental pH values (seawater medium 8.2; vacuole 5.0; chloroplast stroma 8.2; cytoplasm 7.6) were obtained from
Raven (1984), Johnson and Raven (1996) and Nimer et al. (1999, for a marine dinoflagellate). At equilibrium the ratios of [inorganic C] between
compartments of pH 8.2, 7.6 and 5.0 are 1:0.23:0.0065
Photosynth Res (2007) 93:79–88 81
123
It must be admitted that there is no evidence either for
the occurrence of CA in diatom pyrenoids, or of the pro-
posed properties of the pyrenoid membrane. However,
there is good evidence for the occurrence of a b-CA in
aggregations in a ring at the periphery (near the girdle
lamellae) of the stroma in a strain of P. tricornutum which
lacks pyrenoids (Tanaka et al. 2005). These b-CA aggre-
gates seem to be involved in the CCM (Satoh et al. 2001);
do they contain Rubisco and function as membrane-less
pyrenoids (Tanaka et al. 2005)? Less is known about the
Table 2 Summary of proposed CCMs, with their relevance to diatoms
Mechanism Location in cell Phylogenetic distribution Occurrence in
diatoms?
References
Ia Active influx of CO2 Plasmalemma and/or plastid
envelope; HCO3– rather than
CO2 influx from the cytosol
into the plastid stroma
would be more compatible
with present views of
pyrenoid function
Eukaryotic algae (but see Icii).
Hornworts? Some aquatic
flowering plants?
Yes, provided
mechanisms Icii
and Iciii are not
operative, but not
clear where active
transport occurs
Colman et al. (2002),
Burkhardt et al. (2001),
Cassar et al. (2002), Rost
et al. (2003)
Ib Active influx of
HCO3–
Plasmalemma and/or plastid
envelope; HCO3– rather than
CO2 influx from the cytosol
into the plastid stroma
would be more compatible
with present views of
pyrenoid function
Cyanobacteria. Eukaryotic
algae (but see Icii).
Hornworts? Some aquatic
flowering plants?
Yes, provided
mechanisms Icii
and Iciii are not
operative, but not
clear where active
transport occurs
Badger et al. (2006), Colman
et al. (2002), Burkhardt
et al. (2001), Rost et al.
(2003) cf. Laws and Cassar
(2002)
Ici Active efflux of H+
to cell wall,
generation of CO2
from HCO3–
Plasmalemma; CO2 generated
in cell wall, diffuses into
plastid stroma
Some characeans and some
freshwater aquatic plants;
perhaps some marine
macrophytes
Unlikely; only
possible in larger
cells
Walker et al. (1980)
Icii Active influx of H+
to thylakoid lumen,
generation of CO2
from HCO3–
Thylakoid membrane; HCO3–
from stroma enters
thylakoid lumen where CO2
is generated, and then
diffuses into the stroma and,
specifically, the pyrenoid
Chlamydomonas reinhardtii? Possibly; not readily
compatible with
mechanisms Ia or
Ib
Pronina and Semenenko
(1992), Raven (1997),
Hanson et al. (2003), Mitra
et al. (2005)
Iciii Active efflux of H+
from cytosol or
stroma to plastid
inter-membrane
space, generation of
CO2 from HCO3–
Membranes of chloroplast
endoplasmic reticulum; CO2
generated in inter-
membrane space, diffuses
into stroma
Some eukaryotic algae with
plastids obtained by
secondary endo-symbiosis?
Possibly; not readily
compatible with
mechanisms Ia or
Ib
Lee and Kugrens (1998,
2000)
IIa Single-cell C4
biochemistry
(C3 + C1) carboxylation of a
C3 carboxylic acid occurs in
the cytosol and (C4 – C1)
decarboxylation of a C4 acid
occurs in the plastids, with
refixation of CO2 by
Rubisco
Single-cell C4 in some
terrestrial and aquatic
flowering plants, and
marine algae such as.
Udotea flabellum and
(perhaps) Emiliania huxleyi
Thalassiosiraweissflogiisuggested to be C4;
more likely C3 –
C4 intermediate
Reinfelder et al. (2000, 2004),
Johnston et al. (2001),
Morel et al. (2002),
Edwards et al. (2004),
Granum et al. (2005), Tsuji
et al. (2006)
IIb Crassulacean acid
metabolism
(C3 + C1) carboxylation occurs
in the cytosol at night, malic
acid is stored in the vacuole,
and (C4 – C1)
decarboxylation occurs in
the cytosol during day, with
refixation of CO2 by
Rubisco
Terrestrial and aquatic ferns,
lycopods and flowering
plants
Possibly, but only in
larger cells with a
high vacuole:
cytoplasm ratio
Keeley and Rundel (2003),
Holtum et al. (2005)
IIc Passive CO2 entry,
active conversion to
HCO3– on thylakoid
membrane
Active generation of HCO3– in
cytosol, transported into the
carboxysomes
Cyanobacteria Possibly (on
chloroplast
endoplasmic
reticulum)
Tchernov et al. (2001),
Badger et al. (2006)
See also Giordano et al. (2005) and Raven et al. (2007)
82 Photosynth Res (2007) 93:79–88
123
role of the other CA found in diatoms, including two other
novel CAs, one able to use either Zn or Co as cofactor, the
other specific for Cd (Roberts et al. 1997; Cox et al. 2000;
Lane et al. 2005; Park et al. 2007; Szabo and Colman
2007).
The final category of CCMs based on active transport
(Table 2, Ic) relies on acidification of a compartment
accessible to HCO3– from the medium; formation of the
equilibrium (high) CO2: HCO3– ratio, usually requiring CA
activity, is followed by diffusion of CO2 to the compart-
ment containing Rubisco. A mechanism (Table 2, Ici)
where such acidification occurs in zones at the surface of
diatom cells is unlikely in view of their relatively small
size (Raven 1984), and the limited expression of extra-
cellular CA in diatoms in nature (Martin and Tortell 2006).
Intracellular variants on this acidification theme include
CO2 generation in the thylakoid lumen (Table 2, Icii). Here
the suggestion is that HCO3–, ultimately from the medium,
enters the thylakoid lumen of the plastid through anion
channels, and a CA then generates CO2 which diffuses to
Rubisco in the stroma, or, more specifically, the pyrenoid
(Pronina and Semenenko 1992; Raven 1997). In agreement
with this hypothesis the freshwater green microalga Chla-
mydomonas reinhardtii has a luminal a-CA (Cah3) essen-
tial for the CCM located in thylakoid tubules in the
pyrenoid (Hanson et al. 2003; Mitra et al. 2005). However,
the lack of any detectable HCO3– channels in the thylakoids
of three strains of green algae with CCMs, including a
wall-less strain of C. reinhardtii (van Hunnik and Sulte-
meyer 2002), is clearly a problem. Could this mechanism
occur in diatoms? No Cah3 analogue has been found in
diatoms, but the ubiquitous luminal photosystem II protein
PsbO has CA activity (Enami et al. 2005; Lu et al. 2005;
Hillier et al. 2006; cf. Stemler and Govindjee 1973).
Nevertheless, the CCM of C. reinhardtii is absolutely
dependent upon the expression of Cah3 (Hanson et al.
2003). Furthermore, a few diatoms have no pyrenoids, and,
when pyrenoids do occur, they do not always have thy-
lakoids running through them. However, the ‘mem-
brane’(when present) around the pyrenoids could represent
the ends of thylakoids (Drum 1963; Drum and Pancratz
1964; Schmid 2001; Mayama et al. 2004).
The hypothesis of Lee and Kugrens (1998, 2000) (Ta-
ble 2, Iciii), with the acidified compartment that generates
CO2 identified as the chloroplast endoplasmic reticulum,
has not been explicitly tested on diatoms, or any of the
other algae to which it may apply. The hypothesis could be
tested when photosynthetically active diatom plastids can
be isolated with their chloroplast endoplasmic reticulum
intact (see Wittpoth et al. 1998).
The possibility that diatoms have C4-like photosynthesis
(Table 2, IIa) was first suggested in the 1970s when short-
term (2s) inorganic 14C supply experiments (Beardall et al.
1976) showed labelling of malate, typical of C4 photo-
synthesis, rather than PGA and sugar phosphates, typical of
C3 biochemistry. Furthermore, assays of the in vitro
activity of carboxylases showed a relatively high ratio of
phosphoenolpyruvate carboxylase (PEPC) to Rubisco.
Other, usually rather longer-term, labelling experiments
showed a predominance of PGA and sugar phosphates
(Coombs and Volcani 1968; Holdsworth and Colbeck
1976; Kremer and Berks 1978; Mortain-Bertrand et al.
1987). The implicit or explicit assumption that diatoms
have only C3 photosynthetic biochemistry went unchal-
lenged until Reinfelder and colleagues (Reinfelder et al.
2000, 2004; Morel et al. 2002; cf. Johnston et al. 2001;
Granum et al. 2005) found significant evidence of C4
photosynthesis in the marine diatom T. weissflogii.
tsalporolhcmsalpotycmsalpirep
AC AC
CPEP
OC 2OC 2
OCH 3-OCH 3
-
uR ocsib
aIOC 2
OCH 3-
eloucav
C4 dicadiokalyht
cimsalpodneuluciter m
CRCP
AC AC
C3 dica
C4 dica
aI aI
bI bI bI
cI icII
baII aII
bII
cI iii cI ii
H+
bII
AC
C4 dica
C3 dica
OC 2
OCH 3-
OC 2
OCH 3-
uR PB AGP×2
EM/KCPEP
HC[ 2 ]O
H+ H+H+
dioneryp
OCH 3-
OC 2
Fig. 1 Schematic presentation
of the possible CCMs in
diatoms (Table 2, and text). The
simultaneous occurrence of all
of these mechanisms would lead
to futile cycles; only restricted
subsets of these processes can
function together as parts of
CCMs
Photosynth Res (2007) 93:79–88 83
123
The inorganic 14C labelling results for T. weissflogii
were confirmed and extended by Roberts et al. (unpub-
lished). Their 2s and 5s labelling experiments showed a
rather lower and variable ratio of malate to PGA and sugar
phosphates than that found by Reinfelder et al. (2000), but
still with a predominance of malate. The labelling pattern
was not influenced by the growth inorganic C concentra-
tion. Overall, the labelling pattern resembled that of certain
C3 – C4 intermediate flowering plants such as Flaveria
linearis, with PGA and malate in parallel as the initial
fixation products, and subsequent label transfer from C4
acids to PGA and sugar phosphates (Monson et al. 1986).
By contrast, T. pseudonana strictly showed a C3 labelling
pattern, again regardless of the inorganic C concentration
used for growth (Roberts et al. unpublished), and little or
no up-regulation of C4-related genes by low inorganic C
concentration (Granum et al. 2005; Granum et al. unpub-
lished). Armbrust et al. (2004), in analysing the complete
genome sequence of T. pseudonana, suggested that this
diatom (like T. weissflogii) had C4 photosynthetic metab-
olism. This suggestion was based on the presence of genes
for PEPC, phosphoenolpyruvate carboxykinase (PEPCK)
and pyruvate-orthophosphate dikinase which, with plastid
envelope transporters, could allow single-celled C4 photo-
synthetic metabolism; these genes also occur in P. tricor-
nutum (Montsant et al. 2005). The metabolic labelling
experiments show that the presence of these genes, as in C3
flowering plants, is inadequate to demonstrate C4 photo-
synthetic metabolism; this is also the case for a similar
suggestion made for the picoplanktonic prasinophyte Ost-
reococcus tauri (Derelle et al. 2006). More work is clearly
needed (see Granum et al. 2005) to determine more pre-
cisely the pathways of inorganic C assimilation, and their
relative contributions to overall photosynthesis, in T.
weissflogii.
Is it unexpected to find both C3 and C3 – C4 interme-
diate photosynthetic pathways in a single morphologically
defined genus of diatoms? It is instructive to look at the
distribution of C3, C4 and C3 – C4 intermediate photo-
synthetic pathways in flowering plants. Molecular phylo-
genetic analyses show that there have been at least 31
independent origins of C4 photosynthesis among flower-
ing plants (Kellog 1999), yielding some 8,000 C4 species
(Sage et al. 1999). Sage et al. (1999) list five morpho-
logically defined genera of the Poaceae with both C3 and
C4 species, and Kellog (1999) points out that two of these
genera also have C3 – C4 intermediate species. Kellog
(1999) cautions that there is no adequate phylogeny of the
subfamily Panicoideae and that, while the genus Panicum
has C3 and C3 – C4 intermediate species as well as spe-
cies with all three subtypes of C4, Panicum is probably
paraphyletic. Interestingly, molecular phylogenetic studies
of the diatom order Thalassiosirales show that T.
pseudonana and T. weissflogii are not closely related
within the paraphyletic genus Thalassiosira (Kaczmarska
et al. 2006). Returning to the question at the beginning of
the paragraph, it seems no more unexpected to find a
diversity of photosynthetic pathways of inorganic C
assimilation in a genus of diatoms than in a genus of
flowering plants. Additional evidence of photosynthetic
variation within the genus Thalassiosira comes from work
on T. oceanica from a low-iron area in the North Pacific.
T. oceanica, unlike T. pseudonana, T. weissflogii and all
other chromists so far examined, uses plastocyanin rather
than cytochrome c6 in electron transport from the cyto-
chrome b6-f complex to P700 (Strzepek and Harrison
2004; Peers and Price 2006).
A temporally rather than spatially separated variant of
C4 photosynthesis is Crassulacean acid metabolism (CAM;
Table 2, IIb). The volume fraction of vacuoles in larger
diatom cells is similar to that of CAM vascular plants, so
there is adequate malic acid storage capacity (Raven 1987,
1995), as shown by the following calculation based on data
for the giant diatom Ethmodiscus (Villareal et al. 1999).
The organic C content is 0.48 · 10–6 mol cell–1, and the
cell volume is 2.2 · 10–9 m3. Assuming that the vacuole
contributes 90% of the cell volume (Raven 1995), and that
CAM involves a relatively modest (Raven and Spicer
1996) diel variation of vacuolar malic acid of 200 mol m–3,
the CO2 stored in malic acid overnight for refixation in the
day is 200 · 0.9 · 2.2 · 10–9 or 0.40 · 10–6 mol CO2 per
diel cycle. This is not quite adequate for one cell doubling
per day based on CAM alone, especially if CO2 losses in
leakage and respiration and dissolved organic C losses are
taken into account. However, the estimated doubling time
of Ethmodiscus cells in the North Pacific Gyre is not less
than 45 h, so CAM could be a possible means of supplying
all of the inorganic C used for growth. Nevertheless, there
is the important question of what could be the selective
advantage of CAM in the oligotrophic ocean. For the
smaller-celled diatoms that have been examined the mea-
sured rates of dark inorganic 14C fixation (e.g., Mortain-
Bertrand et al. 1998) match the anaplerotic requirement for
inorganic N assimilation (Granum and Myklestad 1999),
thus not leaving significant surplus for CAM. Furthermore,
small-celled diatoms do not have a large enough fraction of
their cell volume occupied by vacuoles (Raven 1987, 1995)
to permit CAM to support their high growth rates (Banse
1982).
The final possible CCM in diatoms involves passive
CO2 entry and energized conversion to HCO3–, a mecha-
nism only found so far in cyanobacteria (Table 2, IIc).
Plastid NADH dehydrogenases are not known in diatoms
(Peltier and Cournac 2002; Armbrust et al. 2004), so the
mechanism suggested for cyanobacteria with energized
CO2 conversion to HCO3– in the cytosol, involving a H+
84 Photosynth Res (2007) 93:79–88
123
gradient produced by a NADH dehydrogenase, cannot
occur in diatoms (Tchernov et al. 2001; Badger et al.
2006).
In conclusion, there are many missing links in the story
of diatom CCMs. None of the proposed mechanisms are
completely ‘joined up’ and, while some of them could be
complementary, e.g., active influx of CO2 (Ia) with a
cytosol CA producing HCO3– that then enters plastids (IIc),
or active influx of HCO3– (Ib) with HCO3
– using C4
metabolism (or, more likely, C3 – C4 intermediate metab-
olism) (IIa), some other combinations could result in futile
cycles. An example of such a combination is that of a
mechanism that delivers CO2 to Rubisco in the pyrenoid by
C4 photosynthesis involving PEPCK (IIa), or by CO2 dif-
fusion across the thylakoid membrane after CA-catalysed
conversion of HCO3– to CO2 in the acidic thylakoid lumen
(Icii), with mechanism Ib involving CA-catalysed conver-
sion of HCO3– to CO2 in the pyrenoid. A more general
aspect of futile cycling is that of leakage of accumulated
inorganic C, especially CO2, during the operation of
CCMs. Leakage of inorganic C from diatom cells (or
chloroplasts) is probably small as suggested by the high
photon yields (on an absorbed photon basis) of photosyn-
thesis by T. pseudonana (Myers 1980) and P. tricornutum
(Geider et al. 1985, 1986), at least in the latter case for cells
grown in air-equilibrated artificial seawater medium.
Leakage is further discussed by Granum et al. (2005) and
Raven et al. (2007)
Regulation of diatom CCMs
The affinity of diatom photosynthesis for inorganic C in-
creases with decreasing inorganic C supply (Johnston and
Raven 1996; Fielding et al. 1998; Burkhardt et al. 2001;
Rost et al. 2003). Matsuda et al. (2001, 2002) showed that
CO2 rather than HCO3– is the external inorganic C species
involved in this acclimation in P. tricornutum, as in
freshwater green microalgae, but different from that in
cyanobacteria (Badger et al. 2006). Cyclic AMP is a
mediator in the response of the CCM of P. tricornutum to
external CO2 (Harada et al. 2006).
Photorespiration in diatoms
The CCMs of diatoms do not completely suppress the
oxygenase activity of Rubisco, especially when there is
significant CO2 depletion and/or O2 accumulation relative
to air-equilibrium conditions, and/or high irradiances and
temperatures (Birmingham and Colman 1979; Colman and
Hosein 1980; Birmingham et al. 1982; Parker et al. 2004;
Parker and Armbrust 2005). Roberts et al. (unpublished)
found significant labelling of phosphoglycolate, glycolate
and glycine among the 2–5s products of inorganic 14C
assimilation by T. pseudonana and T. weissflogii under
near-ambient conditions.
Parker et al. (2004) and Parker and Armbrust (2005)
have shown increased transcription of glycine decarbox-
ylase, an enzyme of the photorespiratory C oxidation cycle
(PCOC) of embryophytes and many green algae, under
conditions which permit more Rubisco oxygenase activity
in T. pseudonana and T. weissflogii, and Granum et al.
(unpublished) have confirmed this for T. pseudonana under
low CO2 conditions. Armbrust et al. (2004) suggested that
the PCOC is the pathway of phosphoglycolate metabolism
in T. pseudonana; while there can still be doubts as to the
occurrence of all of the genes required for this cycle in the
genome, it is salutory that a bacterial like glycerate kinase
was only recently recognised as a cycle enzyme in Ara-
bidopsis thaliana (Boldt et al. 2005). It has also not proved
possible to establish at the genetic level the occurrence of
the alternative tartronic semialdehyde pathway (Eisenhut
et al. 2006) of phosphoglycolate metabolism in diatoms,
although genes required the glyoxylate cycle are present.
Enzymes in diatom peroxisomes include a glycolate
dehydrogenase that also oxidises L-lactate and enzymes of
the glyoxylate cycle (Winkler and Stabenau 1995).
Mitochondria have a glycolate dehydrogenase that also
oxidises D-lactate, and can convert the resulting glyoxy-
late to serine and CO2. Malate synthase in peroxisomes
can convert glyoxylate, with acetyl CoA which comes
ultimately from photosynthetically produced triose phos-
phate with CO2 release, into malate. Provided the rate of
phosphoglycolate synthesis is not too great, the products
of the PCOC up to serine hydroxymethyl transferase, and
of malate synthase, can all be used in biosynthesis of the
amino acids glycine and serine and those of the aspartate
and glutamate families, and as tetrapyrols, replacing
PEPC activity. The least energetically costly means of
metabolising any excess malate is conversion to triose
phosphate with further CO2 release, although complete
oxidation of malate to CO2 allows much of the energy put
in as reductant and ATP to be recouped as ATP (Raven
et al. 2000). Further work is needed on the relationship
between glycolate metabolism and N assimilation in
diatoms (Allen et al. 2006).
Conclusions
Despite recent advances, much work is required before we
have a clear understanding of the pathways of inorganic C
assimilation and related reactions in diatoms, and of how
Photosynth Res (2007) 93:79–88 85
123
very substantial inorganic C leakage is avoided (Granum
et al. 2005; Raven et al. 2007).
Acknowledgements Unpublished work reported here was per-
formed under grant NER/A/S/2001/01130 from the Natural Envi-
ronment Research Council, UK. We are grateful to Professor D G
Mann for pointing out the work of Schmid (2001), and to the com-
ments of two anonymous reviewers which have significantly im-
proved the paper.
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