J. Phycol. 41, 1073–1076 (2005)
r 2005 Phycological Society of America
DOI: 10.1111/j.1529-8817.2005.00156.x
DEFINING THE MOLECULAR BASIS FOR ENERGY BALANCE IN MARINE DIATOMS
UNDER FLUCTUATING ENVIRONMENTAL CONDITIONS
The effectiveness of photorespiration as a protective
mechanism against photooxidative stress and as a necessary pathway for the dissipation of excess photochemical energy in vascular C3 plants has been
established (Kozaki and Takeba 1996, Wingler et al.
2000). Evidence for and against a functional photorespiratory pathway in photosynthetic chromoalveolates
such as diatoms has been extensively debated along
physiological and theoretical grounds. The extent of
photorespiratory activity in marine diatoms is a critical
physiological parameter with major implications for
phytoplankton growth efficiency and pelagic carbon
(C) and nitrogen (N) biogeochemistry. In vascular
plants, photorespiratory CO2 release is estimated to
be approximately 25% of the rate of net CO2 assimilation and NH3 loss (in the absence of recycling mechanisms) far exceeds primary NH3 assimilation from
NO3! reduction (Sharkey 1988, Keys et al. 1978).
Photorespiration is the light-dependent release of
CO2 that results from glycolate metabolism. Light-dependent glycolate production occurs as a result of substrate competition between O2 and CO2 for the active
site of RUBISCO, the enzyme responsible for photosynthetic CO2 fixation. Photorespiratory glycolate is
subsequently converted to serine in the mitochondria
through a pathway that involves the decarboxylation of
glycine and release of CO2 and NH3. In C3 vascular
plants, the rate of photorespiration increases at elevated O2/CO2 ratios and at higher temperatures because
the specificity of RUBISCO for CO2 is diminished under these conditions (Brooks and Farquhar 1985, Tolbert et al. 1995). Other studies have documented a
decrease in CO2 affinity and an overall reduction in
Calvin cycle activity at colder temperatures (Yokota
et al. 1989, Hutchison et al. 2000). It is likely that deviation above and below the optimal temperature for
CO2 incorporation causes inefficiency in CO2 uptake
mechanisms that lead to increased fluxes of carbon and
nitrogen through photorespiratory pathways. Elevated
levels of photorespiration are known to lead to a de-
pletion of carbohydrates and accelerated senescence
(Wingler et al. 2000).
Numerous studies on diatoms and a variety of other
alga taxa in the 1970s utilized in vivo isotopic labeling
(18O2 and 14CO2) and biochemical approaches to investigate metabolite fluxes and enzymatic activities for
pathways related to photorespiratory and glycolate
metabolism (Beardall 1989). Key findings include the
detection of glycolate dehydrogenase activity in the
mitochondria of the marine pennate diatoms Cylindrotheca fusiformis and Nitzscia alba (Paul et al. 1975). In
vascular plants, green algae, and non-vascular cryptograms, glycolate is oxidized to glyoxylate in the peroxisome by glycolate oxidase (Somerville 2001). Other
early findings concerning photorespiration in diatoms
include the relative insensitivity of photosynthesis in
the pennate diatom Phaeodactylum tricornutum, compared with C3 plants, to elevated O2 levels. In this aspect, P. tricornutum behaves more like the C- and Nefficient C4 plants, which have a variety of mechanisms
for alleviating competition between O2 and CO2 by
concentrating CO2 at the active site of RUBISCO,
thereby reducing C and N losses associated with photorespiration (Beardall and Morris 1975). Also, it was
shown that Thalassiosira pseudonana cells grown under
different O2 concentrations had nearly constant rates
of glycine and serine 14C labeling (Burris 1977).
Therefore, although the existence of a photorespiratory pathway in diatoms was confirmed, it is evident
that it differs substantially from that of C3 green lineage organisms. Recent evidence for the centric diatom
Thalassiosira weissflogii does in fact support the idea that
a significant fraction of the CO2 flux through RUBISCO is derived from C4 organic acids (Reinfelder
et al. 2004). The existence of a CO2 concentrating
mechanism (CCM) such as this one, and a variety of
others involving different types of carbonic anhydrases
(Lane and Morel 2000, Tanaka et al. 2005), appears to
be antithetical to photorespiration and would in all
likelihood greatly diminish the flux of C and N
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ANDREW E. ALLEN
through photorespiratory pathways in diatoms. It
should be noted, however, that C4 vascular plants do
harbor photorespiratory pathways that operate at
much reduced rates compared with C3 plants (Wingler
et al. 2000). Therefore, a major challenge for diatom
research is to conceptually integrate photorespiratory
metabolism into the framework of overall cellular
energy balance, C and N status and turnover.
The first direct molecular evidence for a photorespiratory pathway in diatoms was provided by Parker
et al. (2004) in series of experiments where gene expression levels of the T protein of glycine decarboxylase (GDCT) were monitored concomitantly with
glycolate release in cells that were pre-acclimated to
varying light intensities between 15 and 400 mmol "
photons " m ! 2 " s ! 1 before being placed in the dark for
24-h and subsequently transferred to high light
(400 mmol " photons " m ! 2 " s ! 1). Cells were assayed
for GDCT mRNA and glycolate release over the 24-h
period following transfer to high light. Significant glycolate release as well as a 3-fold increase in GDCT
transcript abundance was observed in the treatment
that received the most extreme shift in light intensity
(cells acclimated to 15 mmol " photons " m ! 2 " s ! 1 before
transfer to 400 mmol " photons " m ! 2 " s ! 1). Such a shift
in light regime is highly reminiscent of the fluctuating
conditions that are typical for marine phytoplankton
cells that are mixed from the nutricline at the bottom
of the euphotic zone into high-light surface waters. For
such cells, the protective benefits of photorespiration
to photooxidative stress probably outweigh potential C
and N losses. Indeed, there are many reports of dissolved organic nitrogen (DON) and NH4þ release by
marine phytoplankton communities in response to
rapid increases in light intensity (Lomas et al. 2000).
Such DON fluxes are critical components of the overall
N budget in stratified temperate marine waters (Bronk
et al. 1994)
Interestingly, in other experiments where cells were
acclimated to constant light intensities GDCT mRNA
transcript abundance was highly correlated with
steady-state light levels with the highest amount of
GDCT mRNA detected at light intensities well above
saturation. Also, cells that were acclimated to constant
light intensities, compared with cells that experienced
a shift in light regime, released much less glycolate at
higher irradiances. This series of experiments provided several extremely valuable insights regarding photorespiration in marine centric diatoms. First, these
results ostensibly indicate that under high-light conditions, regardless of O2 and CO2 dynamics, photorespiratory pathways are up-regulated in order to
facilitate alternative electron cycling scenarios. Second,
the concurrent release of the photorespiratory compound glycolate and up-regulation of GDCT following
‘‘light shock’’ provides good evidence for a correlation
between GDCT mRNA levels and photorespiration in
diatoms. Third, the observation that cells acclimated to
constant levels of high light also had relatively higher
levels of GDCT mRNA but less glycolate release indi-
cates that these cells were recycling much of the photorespiratory glycolate. Presumably, in such cells, with
an up-regulated photorespiratory pathway but little
glycolate release, photorespiration is facilitating the
conversion of fixed C and N to CO2 and NH3.
In addition to light, temperature is a factor that can
cause an imbalance between light assimilation and
growth. In high-light and cool-water environments,
where carbon uptake and metabolism are limited by
temperature, phytoplankton are likely to resort to alternative electron cycling strategies in order to buffer
electrons derived from the light reactions that are in
excess of what is required for balanced growth and
metabolism. Lomas and Glibert (1999) presented a
compelling case for a novel role for nitrate reductase
(NR) in such situations. Data presented in their study
indicate that under high-light and cold-water conditions, when NO3! is available as an N source, NO3!
reduction can become uncoupled from overall cellular
N demand. In a series of in situ field experiments involving diatom-dominated natural phytoplankton assemblages Lomas and Glibert (1999) do indeed
document an inverse relationship between NO3! uptake rate and temperature with higher rates of NO3!
uptake occurring at lower temperatures. The trend is
the opposite for NH4þ and urea, where, as expected,
higher uptake rates occur in accordance with higher N
demand and growth rates at higher temperatures.
This important study demonstrated that under very
specific conditions, NO3! can serve as a terminal electron sink in order to preserve overall cellular energy
balance. Operationally, this form of NO3! reduction is
very similar to photorespiration in that it facilitates a
type of alternative electron cycling.
In a new study that appears in this issue, Parker and
Armbrust (2005) generate gene expression data for the
centric diatom T. pseudonana for a variety of genes central to carbon and nitrogen metabolism and photorespiration to infer the relative importance of different
alternative electron cycling and metabolic pathways in
response to differences in light level, temperature, and
inorganic N source (NH4þ compared with NO3! ). Numerous novel relationships related to the effect of light
level, temperature, and N source on cellular energy
management and C and N metabolism are apparent
from this work. Transcriptional changes in mRNA
abundance in response to the differing experimental
light intensities and temperatures were monitored for
NR, glutamine synthetase II (GSII), phosphoglycolate
phosphatase (PGP), glycine decarboxylase T protein
(GDCT), and sedoheptulose 1,7-bisphosphatase (SBP).
NR and GSII are molecular markers for NO3! assimilation. PGP and GDCT are photorespiratory genes
and SBP is a genetic marker for changes associated
with Calvin cycle activity.
Perhaps the most fundamental result of this study is
the finding that both of the photorespiratory genes assayed, PGP and GDCT, are significantly up-regulated
under conditions of high light at 121 C when NH4þ is
the N source. In the case of GDCT, mRNA transcripts
ENERGY BALANCE IN MARINE DIATOMS
are significantly more abundant in all of the high-light
treatments regardless of temperature and N source.
However, cultures which were grown on NH4þ at 121 C
under high-light levels were significantly higher for
GDCT transcripts than all of the other high-light treatments. These data suggest that, as expected, photorespiratory activity is higher in high light compared
with low light but that temperature and N source are
also important variables. Parker and Armbrust also detected an effect of N source on measured levels of glycolate release in the various treatments. Cells cultured
under high-light intensities on NO3! had the greatest
net release of glycolate per cell, and across all treatments cells cultured on NH4þ released significantly less
glycolate into the media.
In the case of NR, cells grown on NH4þ , not surprisingly, had very low levels of NR mRNA. All of the
high-light-grown cells had significantly more NR
mRNA than cells cultured under low light and the
positive effect of light was significantly increased at
121 C compared with 221 C. This result nicely supports
the hypothesis of Lomas and Glibert (1999) that nitrate
reduction is enhanced at lower temperatures as a result of its role as an electron sink. Parker and Armbrust
note that the pattern of expression for the Calvin cycle
gene SBP, relative to the photorespiratory gene PGP
and NR, indicates an up-regulation of photorespiration and nitrate reduction relative to carbon fixation
under conditions of high light and cool temperature.
In such conditions, Calvin cycle activity is probably
constrained by lower temperatures and light is harvested in excess of what is required for balanced
growth. This scenario promotes the demand for
alternative electron cycling pathways. It is well
known that diatoms employ a variety of strategies
for energy dissipation including non-photochemical
quenching (NPQ) via the xanthophyll cycle and cyclic
electron flow around PSII (Lavaud et al. 2002,
Ruban et al. 2004). The data of Parker and Armbrust
strongly suggests that T. pseudonana cells will also utilize
nitrate reduction as a means of diverting excess
electrons from CO2 fixation. However, when NO3! is
not available photorespiratory metabolism is enhanced
significantly.
In temperate continental shelf waters, NO3! is supplied periodically via upwelling processes and atmospheric deposition, but the majority of the time
phytoplankton assemblages are fueled by low levels
of recycled NH4þ . Also, it is known that during phytoplankton bloom situations gross O2 production can
lead to supersaturating O2 levels (Luz et al. 2002),
providing an additional variable that favors photorespiration. It is likely, therefore, that depending on
localized light, temperature, and O2/CO2 regimes, diatom photorespiratory pathways are frequently upregulated in situ. The dynamics of glycolate metabolism in diatoms, including whether or not it is converted completely to NH3 and CO2, partially metabolized
and converted to the important antioxidant glutathione, or released from the cell, remains an important
1075
research topic. The fate of C and N that is cycled
through photochemically based alternative electron
pathways such as nitrate reduction and photorespiration remains poorly understood and is of great importance to overall diatom biology.
In a seminal study on nitrogen metabolism in vascular C3 plants, Keys et al. (1978) defined the photorespiratory nitrogen cycle. This study demonstrated
that mechanisms for recycling photorespiratory N are
critical for the overall N status of plants. Defining these
pathways in diatoms is an important research challenge. Recently it was reported that, compared with
the green alga Chlorella vulgaris, the diatom P. tricornutum is capable of a much higher conversion efficiency
of photosynthetic energy to biomass in a fluctuating
light regime (Wagner et al. 2005). One explanation for
this observation could be that diatoms possess particularly efficient means of diverting and then recovering
energy that is cycled through alternative electron pathways. Diatom genome sequencing efforts are revealing
numerous genes and pathways that are known not to
exist in green-lineage organisms (Armbrust et al.
2004). Many of these genes are related to the incorporation of fixed N, and in at least one case CO2, into
biomass. For example, diatoms apparently contain a
carbamoyl phosphate synthetase III gene that is predicted to encode a mitochondrial protein that is responsible for incorporating NH4þ or glutamine and
CO2 into carbamoyl phosphate, which is then available
for conversion into arginine, the important polyamine
precursor ornithine, or the high-energy molecule creatine phosphate. Diatoms also contain genes that encode the putatively cytosolic proteins NAD(P)H nitrite
reductase and prismane. Both of these are genes
known to exist primarily in bacteria (generally on the
same operon) and are also not present in green-lineage organisms. In bacteria, prismane functions as a
hydroxylamine assimilatory protein (Cabello et al.
2004). Together these bacterial-like copies of nitrite
and hydroxylamine reductase could serve as additional
electron sinks for redox balancing as well as aid in the
downstream metabolism of any NO2! that results from
alternative electron cycling nitrate reduction.
Parker and Armbrust have taken a very key step in
defining the conditions under which particular types
of alternative electron cycling are likely to occur, especially photorespiration. They also identified important
trade-offs, at the molecular level, between different
metabolic routes and energy dissipation pathways as a
function of temperature, light, and N source. The
challenge for the future is to integrate these basic physiological and molecular insights into the context of diatom genome biology and evolution. An important
research priority is also to calibrate the flux of carbon
and nitrogen through photorespiratory pathways under various light, temperature, and nutrition regimes.
Genetic tools for diatom research, such as the stable
transformation and over-expression of particular
genes, used in combination with physiological tracer
studies and gas chromatgraphy/mass spectrometry
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ANDREW E. ALLEN
metabolomics approaches will help to elucidate the
role of particular proteins in governing the overall flux
of C and N through photorespiratory pathways and
metabolites. There are many indications that the general photobiology of chromoalveolates is fundamentally different from that of well-studied green-lineage
organisms. The study of the interaction between the
novel C4-like mechanisms recently described in diatoms (Reinfelder et al. 2004) and the photorespiratory
pathway investigated by Parker and Armbrust (2005)
and the overall conversion efficiency of light energy
into biomass is likely to yield novel information concerning the mechanisms behind the extraordinary
ecological flexibility and dominance of diatoms.
ANDREW E. ALLEN
Ecole Normale Supérieure, Organismes
Photosynthétiques et Environnement,
46 Rue D’Ulm, 75320 Paris, France,
e-mail [email protected].
Abbreviations: CCM, carbon concentrating mechanism; GDCT, glycine decarboxylase; GSII, glutamine synthetase II; NPQ, non-photosynthetic
quenching; NR, nitrate reductase; PGP, phosphoglycolate phosphatase; SBP, sedoheptulose 1,7bisphosphatas
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