Research
PSI Mehler reaction is the main alternative photosynthetic
electron pathway in Symbiodinium sp., symbiotic dinoflagellates
of cnidarians
Stephane Roberty1,2, Benjamin Bailleul3, Nicolas Berne3, Fabrice Franck2,4 and Pierre Cardol3,4
1
Laboratoire d’Ecologie Animale et d’Ecotoxicologie, Departement de Biologie, Ecologie et Evolution, Universite de Liege, 11 Allee du 6 Ao^
ut, B-4000 Liege, Belgium; 2Laboratoire de
Bioenergetique, Institut de Botanique, Universite de Liege, 27 Bld du Rectorat, B-4000 Liege, Belgium; 3Laboratoire de Genetique et Physiologie des Microalgues, Institut de Botanique,
Universite de Liege, 27 Bld du Rectorat, B-4000 Liege, Belgium; 4PhytoSYSTEMS, Universite de Liege, B-4000 Liege, Belgium
Summary
Authors for correspondence:
Pierre Cardol
Tel: +32 4 366 38 40
Email: Pierre.Cardol@ulg.ac.be
Fabrice Franck
Tel: +32 4 366 39 04
Email: F.Franck@ulg.ac.be
Received: 6 February 2014
Accepted: 17 May 2014
New Phytologist (2014) 204: 81–91
doi: 10.1111/nph.12903
Key words: chlororespiration, coral
bleaching, cyclic electron flow, mitochondrial
respiration, oxygen uptake, reef-building
corals, Symbiodinium, water-water cycle.
Photosynthetic organisms have developed various photoprotective mechanisms to cope
with exposure to high light intensities. In photosynthetic dinoflagellates that live in symbiosis
with cnidarians, the nature and relative amplitude of these regulatory mechanisms are a matter of debate. In our study, the amplitude of photosynthetic alternative electron flows (AEF)
to oxygen (chlororespiration, Mehler reaction), the mitochondrial respiration and the Photosystem I (PSI) cyclic electron flow were investigated in strains belonging to three clades (A1,
B1 and F1) of Symbiodinium.
Cultured Symbiodinium strains were maintained under identical environmental conditions,
and measurements of oxygen evolution, fluorescence emission and absorption changes at
specific wavelengths were used to evaluate PSI and PSII electron transfer rates (ETR).
A light- and O2-dependent ETR was observed in all strains. This electron transfer chain
involves PSII and PSI and is insensitive to inhibitors of mitochondrial activity and carbon fixation.
We demonstrate that in all strains, the Mehler reaction responsible for photoreduction of
oxygen by the PSI under high light, is the main AEF at the onset and at the steady state of
photosynthesis. This sustained photosynthetic AEF under high light intensities acts as a
photoprotective mechanism and leads to an increase of the ATP/NADPH ratio.
Introduction
Coral reefs are among the richest and the most diverse biological
ecosystems on earth. The resilience and the ecological success of
reef-building corals in tropical oligotrophic waters rely on the
symbiotic relationships between corals and photosynthetic dinoflagellates of the genus Symbiodinium (commonly referred to as
zooxanthellae). In this mutualistic relationship, the photosynthetic dinoflagellates are located in symbiosomes within the cells
of the invertebrate (Wakefield & Kempf, 2001) and translocate a
significant portion of their photosynthetic products to their host
(c. 70% in the coral Stylophora pistillata) (Tremblay et al., 2012),
thus contributing directly to its daily energetic demands (Muscatine, 1990; Tremblay et al., 2012). In return, the host supplies
the Symbiodinium cells with growth-limiting nutrients (CO2,
nitrogen and phosphate) derived from its waste metabolism (see
Yellowlees et al., 2008 for a review).
Phylogenetic studies conducted these last two decades have
revealed that the genus Symbiodinium is delineated into nine
lineages or clades (A to I) (Pochon & Gates, 2010), each comprising multiple phylotypes or species (Lajeunesse et al., 2012;
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Thornhill et al., 2014). Different Symbiodinium phylotypes can
exhibit a wide range of physiological responses to environmental variations and stress conditions (Robison & Warner, 2006;
Hennige et al., 2009, 2010; Ragni et al., 2010; McGinty et al.,
2012; Brading et al., 2013). The adaptation capacities of the
Symbiodinium strains allow their hosts to occupy a wide range
of ecological niches (Iglesias-Prieto et al., 2004; Finney et al.,
2010) and to better survive following environmental changes
(Berkelmans & van Oppen, 2006; LaJeunesse et al., 2010; Howells et al., 2012). Therefore, the host specificities along with
the external environmental conditions act as the driving forces
responsible for particular pairings between both partners (Stat
et al., 2006).
Light is likely the most important of these changing environmental factors. Although a pioneer study has provided evidence
that the coral Stylophora pistillata is able to adapt to high light
exposure or to shaded environments (Falkowski & Dubinsky,
1981), more recent studies have shown that the vertical distribution of certain coral species could be explained by their association with photosynthetic symbionts adapted to the particular
light regime. These adaptation capacities of the symbionts may
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82 Research
be associated with the lipid composition of their membranes
(Tchernov et al., 2004; Dıaz-Almeyda et al., 2011), to Photosystem II (PSII) repair mechanisms (Takahashi et al., 2009) or to
adaptation mechanisms of photosynthetic activity (Reynolds
et al., 2008; Ragni et al., 2010).
In the natural environment, the coral holobiont has to cope
with important daily variations of light intensities that sometimes
exceed the photosynthetic capacities of the Symbiodinium cells
(such as during thermal stress) (Lesser & Farrell, 2004). This
implies the existence of regulatory mechanisms when the light
absorbed is in excess of that required for CO2 assimilation. Such
mechanisms allow the dissipation of excess energy and/or to
divert excess electrons from the photosynthetic apparatus. In
addition to the linear electron flow (LEF) operating during
oxygenic photosynthesis, various types of alternative electron
flows (AEF) have been widely described in plastids of flowering
plants and microalgae. They include cyclic electron flow around
Photosystem I (PSI-CEF) as well as oxygen reduction through
various processes in the chloroplast: the Mehler reaction, chlororespiration, photorespiration and mitochondrion-dependent
re-oxidation of reducing equivalents (Asada, 1999; Badger et al.,
2000; Cardol et al., 2008, 2011).
In Symbiodinium, light-dependent O2 uptake (denoted UOL in
the following) capacity can represent up to 45% of maximum O2
evolution and is relatively insensitive to changes in CO2 concentrations (Jones et al., 1998; Leggat et al., 1999; Badger et al.,
2000). These characteristics are not consistent with a large Rubisco oxygenase activity (i.e. photorespiration) but could be
explained by the Mehler reaction. This reaction, discovered by
Mehler (1951), involves the direct reduction of O2 by PSI and
leads to the production of superoxide ion (O2•). The generated
reactive oxygen species (ROS) are rapidly converted into water
thanks to the activities of the two chloroplast-associated enzymes,
superoxide dismutase (SOD) and ascorbate peroxidase (APX)
(together grouped into the Mehler Ascorbate Peroxidase or MAP
pathway). The flow of electrons extracted from water at the PSII
level to water produced by APX, is called the water-water cycle
(WWC) (Asada, 1999).
Jones et al. (1998) proposed a model in which temperatureinduced coral bleaching begins with the impairment of the CO2
fixation mechanisms and the increase of electron flow through
the Mehler reaction in symbiotic dinoflagellates. Since then,
Tchernov et al. (2004) and, later, Suggett et al. (2008) have
reported an increase of ROS production corresponding to
light-dependent O2 consumption in different phylotypes of
Symbiodinium exposed to light and thermal stress. These results
suggest that the Mehler reaction could be the main producer of
ROS under stress conditions. However, the observed lightinduced O2 uptake could also be explained by chlororespiration
or mitochondrial respiration (Badger et al., 2000; Ulstrup et al.,
2005; Hill & Ralph, 2008; Reynolds et al., 2008). Moreover, as
among the different phylotypes investigated, Symbiodinium type
A1 possesses a higher ability to reroute electrons towards O2, it
was suggested that representatives of this clade could exhibit
enhanced capabilities for chlororespiration or PSI-CEF (Reynolds et al., 2008).
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In the current context of climate change and its impact on the
survival of symbiotic cnidarians, a better understanding of AEF
in Symbiodinium is needed. Despite the fact that the involvement
of the Mehler reaction, chlororespiration or PSI-CEF has often
been suggested in many reports to date, the nature of AEF has
never been proven in Symbiodinium species (Jones et al., 1998;
Hoegh-Guldberg & Jones, 1999; Leggat et al., 1999; Jones &
Hoegh-Guldberg, 2001; Hill & Ralph, 2008; Reynolds et al.,
2008; Suggett et al., 2008).
In this study, we have investigated in four different Symbiodinium strains the nature of photosynthetic oxygen-dependent electron transport, its light dependence and the occurrence of PSICEF during the induction phase and the steady state of
photosynthesis. This work demonstrates that PSI-CEF and chlororespiration are both inefficient in cultured Symbiodinium
strains and that the main site for light-dependent oxygen uptake
(UOL) is the Mehler reaction located at the acceptor side of
Photosystem I (PSI).
Materials and Methods
Strains and growth conditions
The strain Avir was provided by P. Furla from the University of
Nice (France). Strains FlAp1, Mf1.5b and Pd44b were obtained
from M. A. Coffroth (the BURR Culture Collection, University
at Buffalo, NY, USA). A detailed description of the Symbiodinium
strains with their host species, geographic origin and sub-clade is
given in Table 1. Cultures were grown in F/2 medium using artificial seawater (Reef Crystals Reef Salt, Instant Ocean, USA) at a
salinity of 34 g l1 under cool white fluorescent lamps (intensity
75 lmol photon m2 s1) and at a temperature of 26 0.2°C.
These experimental conditions are similar to those employed in
previous studies (Reynolds et al., 2008; Suggett et al., 2008; Hennige et al., 2009). Exponential growth was maintained by sub-culturing into fresh medium once a week. Rate of growth, cell size
and chlorophyll content are given in Table 2.
Chlorophyll concentration and cell density
Pigments were extracted from whole cells in 100% methanol in
darkness, at 4°C for 24 h. Then cellular debris was removed by
centrifugation at 10 000 g for 10 min at 4°C and chlorophyll a
and c2 concentrations were determined by spectrophotometry
according to the equations of Ritchie (2006). Cell densities were
assessed using a Beckman Coulter Counter (Brea, CA, USA). For
in vivo analyses, cell suspensions were adjusted to a chlorophyll
concentration of 15 lg ml1 in F/2 medium. Samples were supplemented with 5 mM NaHCO3 to favor the carboxylase activity
of the Rubisco and with the addition of 10% (w/v) Ficoll to prevent cell sedimentation when necessary.
Chemical treatments
Anoxia in the samples was achieved in 5 min by addition of glucose (50 mM), glucose oxidase (200 U ml1) and catalase
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Research 83
Table 1 Symbiodinium strains with host specificities, geographic origins and sub-clade memberships
Strain ID
Host
Location
Country
Ocean
cp23S typea
ITS typeb
Avir
FlAp1
Mf1.5b
Pd44b
Anemonia viridis
Aiptasia pallida
Montastrea faveolata
Porites divaricata
Villefranche-sur-Mer
Florida Keys
Florida Keys
Florida Keys
France
USA
USA
USA
Mediterranean Sea
Caribbean Sea
Caribbean Sea
Caribbean Sea
nd
B184
B184
F178
A1
B1
B1
F1
a
Based on the chloroplast 23S ribosomal DNA (rDNA) domain V (cp23S).
Based on the Internal Transcribed Spacer rDNA. Strains FlAp1 belongs to clade B1 (M. A. Coffroth, pers. comm.) although it has been described as a clade
A representative in the past (Correa et al., 2009). Strain Mf1.05b is a clade B1 representative (Granados-Cifuentes & Rodriguez-Lanetty, 2011) and Pd44b
has been described as a clade F1 (M. A. Coffroth, pers. comm.) although it has been previously described as a clade C (Granados-Cifuentes & RodriguezLanetty, 2011).
nd, not determined.
b
Table 2 Growth and cell characteristics of the Symbiodinium strains
Strain
Clade
Cell size (lm)
Growth rate (d1)
Chla (pg per cell)
Chl-c2 (pg per cell)
Chl-c2/a
Avir
FlAp1
Mf1.5b
Pd44b
A1
B1
B1
F1
9.7 1.7
10.2 1.5
9.4 0.9
9.5 1.0
0.27 0.05
0.17 0.05
0.17 0.03
0.14 0.01
1.36 0.07
0.66 0.11
0.79 0.06
0.93 0.03
0.42 0.02
0.19 0.03
0.25 0.03
0.29 0.01
0.31 0.07
0.29 0.06
0.31 0.04
0.31 0.03
Cell sizes were measured under a light microscope (BX50; Olympus, Aartselaar, Belgium) with Olympus Cell^B imaging software. Pigment contents were
measured by HPLC analysis of light adapted cells. All measurements were performed in triplicate and data are presented as means SD.
(200 U ml1) enzymes. Glycolaldehyde (GA) was prepared as
previously described (Anderson et al., 2007). Rotenone (10 mM),
antimycin A (5 mM), salicylhydroxamic acid (SHAM, 200 mM),
myxothiazol (5 mM) and oligomycin (5 mM) were prepared in
ethanol with a final concentration of ethanol that did not exceed
2% (v/v) in the cell suspension. Potassium cyanide (KCN,
200 mM) was dissolved in distilled water.
Oxygen exchange measurements
Oxygen net exchange rates (net VO2 in pmoles O2 s1 lg1
Chla) were measured using a Clark-type electrode connected to
an Oxylab oxygen electrode control unit (Hansatech Instruments,
King’s Lynn, UK). Actinic light was provided by LED light
sources peaking at 630–640 nm. Values at steady state were collected after 2 min illumination at each light intensity. By definition, net VO2 is the sum of three terms: gross oxygen evolution
by PSII (EO), oxygen uptake in the dark (UOD) and, eventually,
light-dependent oxygen uptake (UOL).
Spectrophotometry: electrochromic shift
In vivo spectroscopic measurements were performed with a JTS10 LED pump-probe spectrophotometer (Bio-logic, Claix,
France). Spectral characteristics of the electrochromic shift (ECS)
were determined as light absorption changes measured 55–85 ms
after illumination with a saturating light pulse at 640 nm in
anoxia in the presence or in the absence of a proton gradient
uncoupler (FCCP, 1.8 lM). Due to the weak contribution of cytochromes to absorption changes at 510 and 546 nm, ECS was
thereafter measured at 510–546 nm.
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Linearity of the ECS
The linearity of the ECS signal over electric field generated by
charge separations at PSI+PSII or PSI levels was determined as
described (Cardol et al., 2008). ECS changes were induced by a
series of five laser flashes fired 100 ms apart and provided by a
Nd:YAG Laser (Minilite II, Continuum), which generated one
charge separation per PS. As the dark periods between two consecutive flashes are short, the ECS decrease between two flashes is
negligible. Thus, the response of the ECS signal over the integrated electric field can be calculated (Fig. 1b and Supporting
Information Fig. S1). This response was recorded in the presence
or absence of PSII inhibitors DCMU (40 lM) and hydroxylamine
(HA, 1 mM).
PSI and PSII quantification
The ratio between active PSI and PSII centers was estimated
as described in Cardol et al. (2008). Briefly, the amplitude of
the fast phase (1 ms) of ECS was monitored upon excitation
with a laser flash. The contribution of PSII was calculated
from the decrease in the ECS amplitude after the flash upon
the addition of the PSII inhibitors DCMU (20 lM) and HA
(1 mM), whereas the contribution of PSI corresponded to the
amplitude of the ECS that was insensitive to these inhibitors
(Fig. 1b, first point of the curves). In contrast to previously
published methods (Hennige et al., 2009) our method does
not require prior knowledge of the molar extinction coefficient of P700 to estimate the ratio of PSII to PSI reaction
centres.
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ECS (∆I/I ru)
1
ECS (10–3 ∆I/I, after flash)
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(a)
0
–1
Control
+FCCP
–2
–3
437
487
537
(b)
4
3
2
no add
HA+DCMU
1
587
250
(c)
750
500
PSI
PSII
PSI+PSII
250
0
200
400
600
800
PAR (μmol photon m–2 s–1)
(d)
200
150
100
LEF+CEF (PSI+PSII)
50
0
0
2
ECS (10–3 ∆I/I, before flash)
ETR (e– s–1 PS–1)
Rate of charge separation
(s–1 PS–1)
1000
1
0
λ (nm)
CEF (PSI)
0
200
400
600
800
PAR (μmol photon m–2 s–1)
Fig. 1 Electrochromic shift in Symbiodinium (a) Spectral characteristics of the electrochromic shift (ECS) signal in Avir (A1) Symbiodinium strain in anoxia
in the presence or in the absence of a proton gradient un-coupler (FCCP, 1.8 lM). (ru, relative units). (b) ECS at 510–546 nm induced in Avir (B1) cells by a
series of five laser flashes fired 100 ms apart in the presence or absence of PSII inhibitors DCMU (40 lM) and hydroxylamine (HA, 1 mM). Gray triangle,
control (slope of linear regression = 0.97 with r2 = 1); white diamond, addition of HA (1 mM) and DCMU (40 lM) (slope of linear regression = 0.99 with
r2 = 1). (c) Rates of photon absorption (s1 PS1) in Avir (A1) cells as a function of light intensity in the presence or absence of PSII inhibitors DCMU (40
lM) and hydroxylamine (HA, 1 mM). Gray circle, PSI (slope of linear regression = 0.82 with r2 = 1); white triangle, PSII (slope of linear regression = 1.24
with r2 = 0.99); black diamond, PSI+PSII (slope of linear regression = 1.08 with r2 = 1). (d) PSI-LEF (e s1 PS1) and CEF (e s1 PSI1) in Avir (A1) cells
measured by following the relaxation of the ECS in the absence or in the presence of DCMU (20 lM), respectively. All measurements were performed in
triplicate and data are presented as means SD.
Photon absorption and photochemical rates
Photon absorption rates of PSI and PSII (s1 PS1) were measured by recording the initial rate of ECS at the onset of actinic
light, in the presence or absence of PSII inhibitors DCMU
(40 lM) and HA (1 mM) (Nagy et al., 2014). Similarly, photochemical rates (s1 PS1) were measured by following the relaxation of the ECS (Joliot & Joliot, 2002) during the first 2 ms after
switching off the actinic light. The slopes were then normalized
on ECS values corresponding to one charge separation per PS
(see earlier) (Fig. 1c).
Spectrophotometry: P700 absorption changes
P700 absorption changes were assessed with a probing light peaking at 705 nm (6 nm full width at half maximum). In order to
remove nonspecific contributions to the signal at 705 nm,
absorption changes measured at 740 nm (10 nm full width at
half maximum) were subtracted. Actinic light was provided by
LED light sources peaking at 630–640 nm. The quantum yield
of photochemical energy conversion by PSI (YI) and the quantum yield of nonphotochemical energy dissipation due to donor
side limitation (YND) or to acceptor side limitation (YNA) were
calculated as (PM0 –P)/(PMP0), (P–P0)/(PM–P0) and (PM–PM0 )/
(PM–P0), respectively (Klughammer & Schreiber, 2008). P0 is
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the absorption level when P700 are fully reduced, PM is the
absorption level when P700 are fully oxidized in presence of
20 lM DCMU upon saturating continuous illumination, P is
the absorbance level under continuous illumination and PM0 is
the maximal absorption level reached during a 200-ms saturating
light pulse (c. 3500 lmol photon m2 s1) on top of the actinic
light. The P700 concentration was estimated by using PM value
(e705 nm for P700 = 105 mM1 cm1; Witt et al., 2003).
Chlorophyll fluorescence
Chlorophyll fluorescence emission was measured using a FMS1
pulse modulated chlorophyll fluorometer (Hansatech Instruments, King’s Lynn, UK) when it was compared to oxygen evolution rates, or a JTS-10 spectrophotometer (Bio-logic) when it
was compared to PSI activity. In both experimental systems,
actinic light is provided by LED light sources peaking at 630–
640 nm. The effective photochemical quantum yield of Photosystem II (ΦPSII) and the photochemical quenching (qP) were
calculated as (FM0 –FS)/FM0 and (FM0 –FS)/(FM0 –F00 ), respectively,
where FS is the actual fluorescence level excited by actinic light,
FM0 is the maximum fluorescence emission level induced by a
150-ms superimposed pulse of saturating light (3500 lmol pho0
ton m2 s1), and F0 is the minimum fluorescence after actinic
light. It should be noted that compared to the FM value obtained
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with a series of shorter (c. 100 ls) saturating pulses (Suggett et al.,
2003; Hennige et al., 2009), FM values in low light (< 150 lmol
photon m2 s1) are slightly higher (at most 20%) in our experimental systems.
Calculating electron transport rate
The relative electron transport rates (lmol e m2 s1) through
PSII (rETRPSII) or PSI (rETRPSI) were calculated as the product
of actinic light intensity by the corresponding ΦPSII (dimensionless) or YI (dimensionless), respectively. The absolute electron
transport rates through PSII (ETRPSII) or PSI (ETRPSI) at a given
light intensity, both expressed in e s1 PS1, were obtained by
multiplying absorption rate (s1 PS1) by qP or YI, respectively.
For a direct comparison between PSI and PSII activities, ETRPSI
values were corrected by the PSI : PSII reaction center stoichiometry. Oxygen exchange rates (pmoles O2 lg1 Chla s1) were
also expressed in e s1 PSII1 according to the following calculation and assuming 4 e per O2: (pmoles O2 lg1 Chla s1) 4
(lg Chla pmoles1 P700) (PSI : PSII).
Results and Discussion
Discrepancy between net oxygen evolution and PSII
electron transport rate
Four Symbiodinium strains (Avir, Mf1.5b, Pd44b and FlAp1)
were used in this study (Table 1). They originate from the Mediterranean Sea (one strain) or from the Caribbean Sea (three
strains) and are representatives of three different sub-clades (A1,
B1, F1). Two strains have been isolated from sea anemones while
the two others originate from reef-building corals. We first conducted photosynthesis-irradiance response curves where the
apparent Photosystem II electron transfer rate (rETRPSII) was
evaluated in parallel with net O2 exchange rate (VO2) at steady
state of photosynthesis (Fig. 2a). We observed that VO2 saturated
at a lower light intensity (c. 200 lmol photon m2 s1) compared to rETRPSII (> 400 lmol photon m2 s1) (Fig. S2). The
linear relationship observed between VO2 and rETRPSII under
low light (10–50 lmol photon m2 s1) is no more valid above
100–200 lmol photon m2 s1 (Fig. 2a). This discrepancy demonstrates the existence of an alternative photosynthetic electron
flow. As the nonlinear relationship under high light was more
pronounced in A1 (Avir), this strain was mainly investigated in
the next steps of our study whereas the three other strains were
also used in key experiments.
Oxygen reduction occurs downstream of PSI in
Symbiodinium
Two mechanisms can explain why net VO2 rapidly saturated
while rETRPSII continued to increase: PSII photochemistry takes
place independently of the oxygen evolving complex activity
(PSII-cyclic electron flow involving cytb559) (Prasil et al., 1996);
or an alternative electron flow (AEF) involving O2 as a terminal
acceptor consumes part of O2 produced at the PSII level. The
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spectacularly high capacity of Symbiodinium spp. for O2 uptake
in the light (UOL) – that reaches up to c. 50% of maximum O2
evolution and is relatively insensitive to changing CO2 conditions
(Leggat et al., 1999) – has already led to the inference that these
features are not consistent with a large Rubisco oxygenase activity
(i.e. photorespiration). Rather, the plastoquinol oxidase (PTOX)
activity of chlororespiration, the activity of the Mehler reaction
associated with PSI photochemistry, and/or the mitochondrial
respiration could be involved in this process.
Because AEF to PTOX or PSII-CEF involve only PSII,
whereas other O2-dependent AEF involve both photosystems,
their occurrence will have distinct effects on ETR measured at
PSI or PSII. We thus recorded PSI and PSII photochemical
quantum yield values under steady-state photosynthesis. In
Symbiodinium, the existence of an electrochromic shift signal
(ECS) (Fig. 1a) that responds linearly to the intensity of the electric field (Fig. 1b) allowed us to quantify the ratio between active
PSI and PSII centers (Table 3), as well as the photon absorption
rates of PS (Fig. 1c). These parameters further enabled us to convert PSI and PSII yields into absolute ETR in electrons per second per PSII (e s1 PSII1; see the Materials and Methods
section). ETRPSI and ETRPSII behaved in a similar way: they
increased with increasing light intensities and saturated at c.
400 lmol photon m2 s1 (Fig. 2c,d).
Furthermore we obtained a linear relationship (r2 > 0.99, slope
c. 1) between PSI and PSII activities (Fig. 2f), which indicates
that both under low light (LEF to carbon fixation mainly) and
under high light (LEF + AEF), PSII and PSI work in series. The
observed AEF involving PSII and PSI must thus be diverted to
oxygen downstream of PSI in Symbiodinium.
The amplitude of AEF can be estimated by expressing
rETRPSII and oxygen exchange rates shown in Fig. 2(a) in electrons per second (e s1 PSII1; Fig. 2b; see the Materials and
Methods section for details). VO2 is the sum of three terms: EO
(gross oxygen evolution by PSII), UOD (dark respiration) and
UOL (light–dependent oxygen uptake). ETRPSII calculated from
PSII photochemical quantum yield is also by definition equal to
ETR derived from EO parameter. Therefore, UOL can be calculated as the difference between ETRPSII and (VO2 UOD), both
expressed in e s1 PSII1 (see the Materials and Methods section). UOL was almost null at low light intensities (< 100 lmol
photon m2 s1) but it could represent up to 50% of the total
ETRPSII at highest light intensities.
Sustained ETR under inhibition of dark respiration and
Calvin cycle activity
In order to determine whether UOL downstream of PSI is due to
mitochondrial respiration, photorespiration or PSI-Mehler activity, we measured oxygen evolution and PS activities in the presence of mitochondrial and carbon fixation inhibitors. In a
simplified view of ETR, the inhibition of AEF from PSII to O2
(i.e. UOL) will decrease ETRPSII (as well as EO proportionally)
without any change in net VO2. In deep contrast, addition of glycolaldehyde (GA, 60 mM), an inhibitor of the Calvin cycle phosphoribulokinase (Buxton et al., 2012; Bhagooli, 2013), resulted
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(a)
300
Mf1.5b
200
FlAp1
Pd44b
100
0
–40
(b)
400
AVIR
ETR (e– s–1 PSII–1)
rETRPSII (μmol e– m–2 s–1)
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–20
0
20
(net VO2 -UOD)
300
Δ (U OL)
200
100
0
60
40
ΦII-based
0
200
2
300
(c)
ETR (e– s–1 PSII–1)
ETR (e– s–1 PSII–1)
300
200
100
PSII control
PSII GA+KCN+SHAM
0
0
200
400
600
100
PSI Control
PSI GA+KCN+SHAM
0
200
GA
KCN+SHAM
ETRPSII (e– s–1 PSII–1)
rETRPSII (μmol e– m–2 s–1)
Control
–10
0
10
20
30
2
800
(f)
100
Control
GA+KCN+SHAM
0
100
200
ETRPSI (e– s–1 PSII–1)
in a reduction by c. 50% of the maximal net VO2 (Fig. 2e) while
ETRPSII was decreased by only c. 20%. Moreover, addition of the
inhibitor had almost no effect at low light intensities. This indicates that inhibition of the Calvin cycle does not prevent UOL
(i.e. ETRPSII towards O2 reduction).
We also used salicylhydroxamic acid (SHAM, 2 mM) and
potassium cyanide (KCN, 2 mM), classical inhibitors of mitochondrial alternative oxidase (AOX) and cytochrome c oxidase
(complex IV) activities, respectively. Both inhibitors act in
Symbiodinium (Oakley et al., 2014) as in other photosynthetic
organisms (Lapaille et al., 2010). In the presence of both mitochondrial inhibitors, UOD decreased by c. 80% (Fig. S3). Rotenone (mitochondrial complex I inhibitor, 1–200 lM), antimycin
A or myxothiazol (mitochondrial complex III inhibitors,
0.5–10 lM), and oligomycin (mitochondrial ATP synthase
inhibitor, 0.5–50 lM) had no effect on UOD (data not shown).
SHAM and KCN are also well-known inhibitors of photosynthesis by affecting carbon concentration mechanism and RuBiSCO
activity, respectively (Goyal & Tolbert, 1990). Moreover, KCN
is also a potent inhibitor of ascorbate peroxidase (APX) (Nakano
& Asada, 1987) and Cu/Zn superoxide dismutase (SOD) (Asada
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600
200
0
Net VO (pmol O2 s–1 μg–1 chla)
400
PAR (μmol photon m–2 s–1)
300
200
0
–20
800
200
0
800
(e)
100
600
(d)
PAR (μmol photon m–2 s–1)
300
400
PAR (μmol photon m–2 s–1)
Net VO (pmol O2 s–1 μg–1 chla)
300
Fig. 2 Electron transport rates (ETRs) at
steady-state photosynthesis of Symbiodinium
cells. (a, e). Comparison of rETRPSII (lmol e
m2 s1) and net oxygen exchange rate (net
VO2; pmol O2 s1 lg1 Chla) at 10, 25, 40,
60, 100, 200, 450 and 650 lmol photon m2
s1 (a) in four Symbiodinium strains or (e) in
Clade A1 Avir cells in the presence or absence
of glycolaldehyde (GA) (60 mM), KCN
(2 mM) and SHAM (2 mM). (b) Comparison
of ETRPSII (based on ΦPSII) and ETR based on
[net VO2 – UOD] taken from panel (a) and
expressed in e s1 PSII1 (see the Materials
and Methods section for details). ETR towards
O2 reduction (UOL) is the difference between
the two curves. (c–d) ETRPSII and ETRPSI (e
s1 PSII1) as a function of light intensity in
the presence or absence of GA (60 mM), KCN
(2 mM) and SHAM (2 mM). (f) Relationship
between ETRPSII and ETRPSI (from panel c).
Black diamonds, control (slope of linear
regression = 0.99 with r2 = 1); gray squares,
addition of GA (60 mM), KCN (2 mM) and
SHAM (2 mM) (slope of linear
regression = 1.07 with r2 = 0.99). All
measurements were performed in triplicate
and data are presented as means SD.
et al., 1974). In the presence of both SHAM and KCN, net VO2
was diminished by c. 75%, barely compensating the remaining
low UOD, while maximum ETRPSII and ETRPSI only decreased
up to 30% (Fig. 2e).
In the presence of KCN, SHAM and glycolaldehyde (GA), the
relationship between PSI and PSII activities was still linear
(Fig. 2f). This indicates that inhibition of mitochondrial respiration, Calvin cycle, and presumably SOD, APX or other possible
metabolic pathways, did not prevent ETRPSII towards O2 reduction (i.e. UOL). Altogether, these results indicate that the PSIMehler reaction is the main AEF under high light, responsible
for the light-dependent oxygen uptake (UOL) in Symbiodinium.
Limited capacity of PSI-CEF and chlororespiration in
Symbiodinium
The results mentioned above also rule out the possibility that
PSII activity under high light is independent of PSI activity
(either through PSII-PTOX or PSII-CEF activities) or vice versa
(PSI-CEF). The existence of chlororespiration has been suggested
in cultured or in symbiotic Symbiodinium. Several authors
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Table 3 Photosynthetic parameters of Symbiodinium strains
Strain
Clade
PSI/PSII
Chla/P700
PSI-CEF
(s1 PSI1)
Max NPQ
Avir
FlAp1
Mf1.05b
Pd44b
A1
B1
B1
F1
1.9 0.3
0.9 0.1
1.0 0.2
1.0 0.1
533 72
515 13
541 27
505 8
82
18 5
12 1
91
0.49 0.05
0.40 0.03
0.59 0.01
0.36 0.03
PSI/PSII ratios were estimated from PSI and PSII charge separation capacity.
Chla/P700 ratios were measured by absorption spectroscopy. PSI-CEF was
estimated by following dark re-reduction kinetics of oxidized PSI at 705–
740 nm. Nonphotochemical quenching (NPQ) was calculated as the fluorescence quenching (FM/FM’1), at steady-state photosynthesis at
650 lmol photon m2 s1. All measurements were performed in triplicate
and data are presented as means SD.
reported a diminution of the maximal photochemical quantum
yield (FV/FM) at night or during prolonged dark periods whose
amplitude varies with the O2 concentration. This led them to
postulate that this diminution was linked to a reduction of the
PQ pool in conditions where O2 was limiting for chlororespiration (Jones & Hoegh-Guldberg, 2001; Hill & Ralph, 2005,
2008; Reynolds et al., 2008). Although we identified a PTOXrelated sequence in EST database of Symbiodinium CassKB8
strain belonging to the Symbiodinium clade A (Table S1), our
current results suggest that if PTOX is expressed, its activity is
very low, like in the green alga Chlamydomonas reinhardtii where
its absence also leads to a reduction of the PQ pool in the dark
(Houille-Vernes et al., 2011).
Based on differences in chlorophyll fluorescence resulting from
a series of saturating light pulses between representatives of various clades, a previous work also suggested that among alternative
pathways, PSI-CEF could be more efficient in clade A representatives (Reynolds et al., 2008). Although we reproduced this
original observation by comparing clade A1 Avir and clade F1
Pd44b strains (Fig. S4a), PSI-CEF was stable throughout the
experiment in clade F1 Pd44b (Fig. S4b). In the same line, we
found that PSI-CEF capacity is weak, not exceeding c. 20 e s1
PSI1, in all clade representatives (including B1 representatives;
Table 3). Moreover, PSI-CEF did not exceed c. 5% of total
ETRPSI in Avir A1 strain over the whole range of light intensities
(Fig. 1d). This strongly suggests that the fluorescence signature
previously reported (Reynolds et al., 2008) is not related to the
occurrence of more efficient PSI-CEF in clade A representatives.
The values measured here for PSI-CEF in Symbiodinium strains
are in good agreement with values reported in the model green
alga C. reinhardtii under oxic conditions (Alric et al., 2010).
Finally, the presence of PGRL1, a major actor of PSI-CEF in
green plants (Tolleter et al., 2011), in Symbiodinium EST databases (Table S1) suggests that the mechanism for PSI-CEF is
conserved between these photosynthetic lineages.
anoxic conditions cannot be maintained under steady state of
photosynthesis due to oxygen evolution by PSII, especially in
high light. Therefore, we recorded ETRPSI and ETRPSII upon the
first 3 s of illumination (i.e. upon induction of photosynthesis) in
the presence or absence of oxygen.
In some microalgae, impairment of respiration following oxygen removal results in a depletion of intracellular ATP pool in
the dark, which, according to the Pasteur effect, stimulates glycolysis (with an increase of the NADPH/NADP+ ratio in the
stroma), the nonphotochemical reduction of the PQ pool and
the transfer of a pool of PSII light harvesting complexes from
PSII to PSI (state 2 transition) (Cardol et al., 2011). If this process also occurs in Symbiodinium after O2 depletion, it could
affect the electron transport rate directly by modifying the chloroplast stromal redox poise (Jones et al., 1998) or indirectly by
modifying the relationships between the electron transport rates
and the photochemical quantum yields through changes in
antenna sizes. However, state transition does not occur in
Symbiodinium (Hill et al., 2012), which was confirmed in our
experiments by the absence of changes in maximal fluorescence
of chlorophyll during the time frame (< 15 min) used for all measurements (data not shown). Similarly, no change in the redox
state of the PQ pool occurred, as judged by the constant FV/FM
value.
In oxic conditions, the saturation curves and relationship
between ETRPSI and ETRPSII (Fig. 3a–c) were similar to those
measured at steady-state photosynthesis (Fig. 2c,d,f), whereas in
anoxic conditions, ETRPSI and ETRPSII saturated at lower light
intensities (100–200 lmol photon m2 s1). The difference of
ETR between anoxic and oxic conditions, called ‘O2-dependent’
ETR, is an indirect measurement of UOL (Fig. 3a,b). ‘O2-dependent’ ETR during photosynthesis induction has the same features
as the estimated UOL at steady-state photosynthesis (Fig. 2b). Its
value is almost null under low light and represents about half the
total ETR in high light. Here, also, the relationships between
ETRPSI and ETRPSII are unchanged (Fig. 3c).
We also calculated the PSI quantum yields of nonphotochemical energy dissipation due to acceptor side limitation Y(NA) or to
donor side limitation Y(ND) (see the Materials and Methods section for details). In the presence of oxygen, Y(NA) was low (< 0.1)
and did not vary with increasing light intensities, while Y(ND)
increased with the amount of light (Fig. 3d), reflecting the limitation of ETR by cytochrome b6f (Tikkanen et al., 2012). On the
contrary, when cells were depleted in oxygen, Y(ND) remained
very low (< 0.2) across the entire range of irradiance, while Y(NA)
increased with light intensity up to 0.8 at the highest light intensities, reflecting a limitation on the acceptor side of PSI. Similar
results were obtained for the three other Symbiodinium strains
(Fig. S5, Table S2). Finally, ETRPSII and ETRPSI during photosynthesis induction in oxic conditions were also fully insensitive
to mitochondrial inhibitors (Table 4).
Oxygen acts as an efficient PSI electron acceptor upon
photosynthesis induction
Physiological roles of PSI-Mehler reaction in Symbiodinium
The light-dependent electron flow to oxygen by the PSI-Mehler
reaction must be inhibited in anoxic conditions. Obviously
The Mehler reaction leads to the formation of O2 which is rapidly detoxified thanks to the activity of superoxide dismutase
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88 Research
(a)
Control
O2-dependent
200
150
100
50
0
0
200
400
600
PAR (μmol photon
250
m–2
250
O2-dependent
-O2
100
50
200
0
400
600
800
PAR (μmol photon m–2 s–1)
1
(c)
200
(d)
0.8
150
100
Control
O2-dependent
50
0
Control
150
0
800
(b)
200
s–1)
Yield (ru)
ETRPSII (e– s–1 PSII–1)
-O2
ETRPSI (e– s–1 PSII–1)
ETRPSII (e– s–1 PSII–1)
250
0.6
Y(NA) Control
Y(ND) Control
0.4
Y(NA) -O2
Y(ND) -O2
0.2
0
0
50
100
150
200
250
ETRPSI (e– s–1 PSII–1)
0
200
400
600
800
PAR (μmol photon m–2 s–1)
Fig. 3 Photosynthetic activities of
Symbiodinium clade A1 Avir cells upon
induction of photosynthesis. (a) ETRPSII and
(b) ETRPSI (e s1 PSII1) as a function of light
intensity upon induction of photosynthesis in
presence or in the absence of O2; O2dependent ETR is the difference between ETR
upon induction in presence and in absence of
O2. (c) Relationship between ETRPSII and
ETRPSI (from panels a and b). Black diamonds,
control (slope of linear regression = 1.04 with
r2 = 0.97); grey circles, absence of O2 (slope
of linear regression = 0.91 with r2 = 0.90). (d)
PSI quantum yield of nonphotochemical
energy dissipation due to acceptor side
limitation (YNA) or to donor side limitation
(YND) in control cells and in O2-depleted cells.
(ru, relative units). All measurements were
performed in triplicate and data are presented
as means SD.
Table 4 Respiration and relative electron transport rate (rETR) of Symbiodinium strains
Strain
Clade
Respiration (UOD)
(pmol O2 s1 lg chl a1)
Avir
FlAp1
Mf1.5b
Pd44b
A1
B1
B1
F1
16.3 1.3
25.6 1.9
21.1 1.9
8.9 2.4
rETRPSIIa
rETRPSIa
Anoxia
SHAM + KCN
Anoxia
SHAM + KCN
39.4 5.5
57.2 1.4
36.5 6.2
42.7 11.6
100.7 0.5
82.9 1.1
85.6 1.7
91.6 2.3
34.8 5.7
58.1 10.1
33.8 3.0
38.2 4.9
98.6 12.1
119.3 2.8
113.3 4.2
104.0 4.4
Respiration (oxygen uptake in the dark; UOD), rETRPSII and rETRPSI upon photosynthesis induction (3 s) of four Symbiodinium strains at c. 500 lmol photon
m2 s1, in absence of oxygen (anoxia) or in presence of SHAM (1 mM) and KCN (1 mM).
a
Data are expressed as percentages of the values obtained for aerobic un-poisoned cells taken from Supporting Information Fig. S5.
(SOD), ascorbate peroxidase (APX), and monodehydroascorbate
reductase (MDHAR) (Asada, 2000), all being present in the most
recent EST libraries of Symbiodinium (Table S1). The activity of
SOD and APX in Symbiodinium have been reported since the
beginning of the nineties and an increase of their activities has
been linked to thermal and light/UV stress (Lesser et al., 1990).
According to Richier et al. (2005) at least seven different isoforms
of SOD are present in freshly isolated Symbiodinium cells of
Anemonia viridis. Interestingly, Symbiodinium strains also possess
homologous sequences for the Flv1 and Flv3 flavodiiron proteins
(FDPs) (Table S1) which in cyanobacteria both participate to
light-induced O2 uptake downstream of PSI. Flv activity, which
consists in four-electron reduction of dioxygen (Frazao et al.,
2000), does not lead to the production of reactive oxygen species
(ROS) (Allahverdiyeva et al., 2011) and thus provides protection
for PSI under fluctuating light (Allahverdiyeva et al., 2013).
Whatever the exact nature of the oxygen reduction site directly
downstream of PSI in Symbiodinium, its light dependency
(Figs 2b, 3a,b) perfectly corresponds to a photoprotective mechanism. It takes place under high light intensities when the LEF to
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CO2 fixation saturates (or is artificially inhibited), thus acting as
an efficient electron sink. Consistent with this conclusion, when
Symbiodinium are deprived of oxygen (AEF to O2 being thus prevented), ETR upon photosynthesis induction is substantially
decreased and PSI acceptor-side limitation increases (Fig. 3d).
By alleviating the excitation pressure over PSII and PSI, the
PSI-Mehler reaction must thus prevent photoinhibition and
photodamage. By acting as an efficient electron flow, it also generates an extra proton gradient across the thylakoid membranes,
without net synthesis of NADPH. It might thus promote the
synthesis of extra ATP probably required for CO2 fixation or
other cellular reactions (Asada, 1999, 2000; Eberhard et al.,
2008; Cardol et al., 2011). It is tempting to propose that the
light-dependent extra ATP synthesis supported by WWC in symbiosis compensates for the energy loss due to the export of a
substantial fraction of photosynthetic products. In addition, the
O2 consumption in chloroplasts by the Mehler reaction has to
reduce the O2 accessibility to the type II RuBisCO, thus lowering
its oxygenase activity and maximizing the carbon fixation (Quigg
et al., 2012).
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Our data moreover suggest that oxygen reduction by the
PSI-Mehler reaction operates similarly amongst A1, B1 and F1
strains. This of course does not mean that the capacity of the
MAP pathway (Mehler reaction, SOD, APX and MDHAR)
cannot vary from strain to strain. The MAP pathway possesses
only a finite capacity for protection, beyond which the rate of
ROS production will exceed the rate of ROS detoxification,
and significant damages will occur. Consequently, when
Symbiodinium cells are exposed to stress conditions (temperature, irradiance, etc.), the cellular ROS content can significantly increase (Jones et al., 1998; Tchernov et al., 2004;
Suggett et al., 2008) and cause major damage to cellular components. For instance, McGinty et al. (2012) reported that
ROS release and antioxidant activities varied significantly in
multiple Symbiodinium phylotypes in response to elevated temperatures. Among them, Symbiodinium types A1 and F2 were
the most tolerant, displaying no increase in ROS production,
and were the only types able to increase SOD activity with
temperature stress. When in symbiotic association, H2O2 produced by Symbiodinium cells can diffuse into the host tissue
(Tchernov et al., 2004; Suggett et al., 2008) and provoke damages (Lesser & Farrell, 2004). Production of H2O2 also triggers a cellular signaling cascade that will ultimately lead to
Symbiodinium cell death (Bouchard & Yamasaki, 2009)
responsible for coral bleaching (see Weis, 2008 for more
details about the cellular mechanisms leading to cnidarian
bleaching). The resulting loss of symbionts by the coral host
will have dramatic ecological consequences on the colony and
over the entire reef ecosystem (Hoegh-Guldberg, 1999). It is
noteworthy that although the Mehler reaction is largely
acknowledged as responsible for part of the initiation steps of
coral bleaching (Jones et al., 1998; Tchernov et al., 2004; Suggett et al., 2008), it was recently shown that coral bleaching
could be eventually independent of photosynthetic activity
(Tolleter et al., 2013).
Representatives of clade A are mostly present in reef colonies
inhabiting shallow waters (Toller et al., 2001), characterized by
high light conditions, and which are likely to be subject to rapid
changes in irradiance. A recent study aiming to investigate the
impact of wave lensing (transient solar irradiance of high intensity and frequency) on the photophysiology of shallow-water
coral species, suggested that corals living in this portion of the
water column should possess special abilities for mitigating
instantaneously high irradiance fluxes (Veal et al., 2010). Usually,
high light-exposed plants have more PSII units with smaller
light-harvesting units, relative to PSI (Anderson et al., 1988).
Compared to the three other strains, Symbiodinium Avir strain
(A1) has the higher PSI/PSII ratio. This observation is in good
agreement with a previous study in which A1 strains also showed
higher PSI:PSII ratios compared to F2 (but not B1) strains
(Hennige et al., 2009). It has also been previously shown that
when exposed to high light, the rates of O2 uptake are higher in
clade A1 strains than in a clade B1 strain (Suggett et al., 2008),
which is in accordance with our present results. We can thus propose that a higher PSI:PSII ratio is responsible for the higher rate
of Mehler electron transport in clade A1 strain.
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Research 89
Conclusions
In this study devoted to four Symbiodinium strains belonging
to three different sub-clades (A1, B1, F1), we present evidence
that light-dependent oxygen consumption (UOL) involves both
PSI and PSII working in series through the so-called PSI-associated Mehler reaction. Other AEF might also occur in
Symbiodinium, such as chlororespiratory PTOX activity and
PSI-cyclic electron flow, their activities are low compared to
the extent of the Mehler reaction which can account for up c.
50% of maximum photosynthetic electron transfer rate. It
should be noted that in plant leaves, the Mehler reaction does
not exceed 15–20 s1 (Joliot, 1965; Joliot & Alric, 2013)
which suggests that a specific/additional mechanism should
occur in Symbiodinium (e.g. Flavodiron proteins whose presence in Symbiodinium is reported here for the first time). In
Symbidiodium, UOL located at the acceptor side of PSI is the
main alternative electron sink at the onset and steady state of
photosynthesis. In this respect, in our opinion, the main physiological roles of the Mehler reaction in Symbiodinium are to
ensure photoprotection (decreased excitation pressure on
photosystems) and to provide additional ATP to cellular
metabolism.
Acknowledgements
We would like to thank P. Furla and M. A. Coffroth for donation of the Symbiodinium strains. We also thank P. Falkowski
and P. Joliot for helpful discussions. R. F. Matagne is acknowledged for his help in correcting the manuscript. We are also
grateful to E. Perez and D. Baurain for bioinformatic analyses.
This work was supported by grants of Fonds National de la
Recherche Scientifique (FRS-FNRS; FRFC 2.4.631.09 to F.F.
and MIS F.4520 to P.C.). S.R., P.C. and F.F. are Research Fellow, Research Associate and Senior Researcher from F.R.S.FNRS.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Photoinduced absorption changes at 510–546 nm (ECS).
Fig. S2 Light dependency of steady-state photosynthesis in four
Symbiodinium strains.
Fig. S3 Sensitivity of oxygen consumption to cyanide in the dark.
Fig. S4 Chla fluorescence and PSI-CEF estimated from kinetics of dark re-reduction of P700+.
Fig. S5 Relative ETR PSI and ETR PSII upon induction of photosynthesis in three strains of Symbiodinium.
Table S1 Components of chloroplastic AEF in Symbiodinium
Table S2 PSI quantum yield of nonphotochemical energy
dissipation
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