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REGULAR PAPER
State-transitions facilitate robust quantum yields and causean over-estimation of electron transport in Dunaliella tertiolectacells held at the CO2 compensation point and re-supplied with DIC
Sven Ihnken • Jacco C. Kromkamp •
John Beardall • Greg M. Silsbe
Received: 6 April 2013 / Accepted: 1 October 2013 / Published online: 18 October 2013
� Springer Science+Business Media Dordrecht 2013
Abstract Photosynthetic energy consumption and non-
photosynthetic energy quenching processes are inherently
linked. Both processes must be controlled by the cell to
allow cell maintenance and growth, but also to avoid
photodamage. We used the chlorophyte algae Dunaliella
tertiolecta to investigate how the interactive regulation of
photosynthetic and non-photosynthetic pathways varies
along dissolved inorganic carbon (DIC) and photon flux
gradients. Specifically, cells were transferred to DIC-
deplete media to reach a CO2 compensation before being
re-supplied with DIC at various concentrations and dif-
ferent photon flux levels. Throughout these experiments we
monitored and characterized the photophysiological
responses using pulse amplitude modulated fluorescence,
oxygen evolution, 77 K fluorescence emission spectra, and
fast-repetition rate fluorometry. O2 uptake was not signif-
icantly stimulated at DIC depletion, which suggests that O2
production rates correspond to assimilatory photosynthesis.
Fluorescence-based measures of relative electron transport
rates (rETRs) over-estimated oxygen-based photosynthetic
measures due to a strong state-transitional response that
facilitated high effective quantum yields. Adoption of an
alternative fluorescence-based rETR calculation that
accounts for state-transitions resulted in improved linear
oxygen versus rETR correlation. This study shows the
extraordinary capacity of D. tertiolecta to maintain stable
effective quantum yields by flexible regulation of state-
transitions. Uncertainties about the control mechanisms of
state-transitions are presented.
Keywords Dunaliella tertiolecta � DIC depletion �Photoacclimation � Non-photochemical quenching �State-transitions � Photoprotection
Introduction
Knowledge of the carbon acquisition affinities of photo-
trophic primary producers is essential to understand how
continual ocean acidification may alter the ecology and
physiology of marine phytoplankton (Raven et al. 2011).
The affinity for dissolved inorganic carbon (DIC) can be
determined in photosynthesis versus DIC response curves
where cells are suspended in DIC-free medium while
exposed to saturating light, and consecutive additions of
increasing DIC concentrations are applied (e.g., Amoroso
et al. 1998). When cells are at CO2 compensation, photo-
synthetic activity of the Calvin–Benson–Bassham cycle is
limited to photorespiration and/or re-fixation of CO2 that is
released from mitochondrial respiration. The exceptionally
low photochemical activity at CO2 compensation results in
a large excess of absorbed quanta; this energy has to be
dissipated through non-photochemical pathways to avoid
damaging photosynthetic reaction centers. Thus, examining
phytoplankton physiologically at CO2 compensation is
interesting because photoprotective mechanisms are chal-
lenged and alternative energy quenching mechanisms are
paramount. Theoretically, autotrophic cells can acclimate
to CO2 compensation by employing and regulating a
variety of mechanisms and physiological pathways. Even
in the absence of DIC photosystem II (PSII) can exhibit
linear electron transport through the regulation of Mehler
S. Ihnken � J. C. Kromkamp (&) � G. M. Silsbe
Netherlands Institute for Sea Research, NIOZ, Postbus 140,
4400 AC Yerseke, The Netherlands
e-mail: [email protected]
J. Beardall
School of Biological Science, Monash University, Clayton,
VIC 3800, Australia
123
Photosynth Res (2014) 119:257–272
DOI 10.1007/s11120-013-9937-8
reaction and consecutive water–water cycle (Asada 2000;
Heber 2002), electron donation to O2 via plastid terminal
oxidases (PTOXs; Cournac et al. 2002; Joet et al. 2002;
Rochaix 2011), photorespiratory activity (Peterhansel and
Maurino 2011), or elevated mitochondrial activity. The
latter would supply CO2 to RuBisCO and allow a respira-
tion—photosynthesis short circuit loop. Cyclic electron
flow around PSII also can avoid damage of PSII (Miyake
and Okamura 2003; Prasil et al. 1996).
When cells are exposed to DIC-limiting conditions the
common non-photosynthetic energy quenching mecha-
nisms (NPQ) are challenged and the photoprotective
potential might be exploited. NPQ is commonly described
as a sum parameter for photoinhibition (qI), state-transi-
tions (qT) (absent in diatoms), and thermal energy
quenching (qE; Lavaud 2007; Muller et al. 2001). In higher
plants, diatoms and chlorophyta, qE is considered the most
effective photoprotective component of NPQ (Lavaud
2007; Muller et al. 2001). qE allows conversion and dis-
sipation of the quantum’s energy in the form of heat. Full
qE activation requires a suitable trans-thylakoid-pH-gra-
dient (DpH gradient), which is linked to H? translocation
by PSII and/or PSI activity, its sensing by the Psbs protein
(Li et al. 2004, 2002) (but see Johnson and Ruban (2011)
and violaxanthin de-depoxidation (Demming-Adams 1990;
Nilkens et al. 2010). When H? and zeaxanthin bind to PSII,
the light-harvesting complexes shift from an energy-
transfer to an energy-dissipating state due to a change in
their conformation (Perez-Bueno et al. 2008; Ruban et al.
2007). Zeaxanthin de-epoxidation requires some minutes
(Nilkens et al. 2010; Niyogi 1999; Niyogi et al. 1997),
while a fast component of qE, which is not initiated by
xanthophyll cycle activation (e.g., Moya et al. 2001), can
quench energy seconds after light exposure (Ihnken et al.
2011; Li et al. 2009). qT can photoprotect PSII, especially
in cyanobacteria (Campbell et al. 1998), but also in green
algae (Finazzi and Forti 2004). State-transitions are con-
trolled by a signal cascade, which involve binding of
plastoquinol at the Qo site of the cytochrome b6f complex
and activation of the Stt7/STN7 kinase, which phospory-
lates light-harvesting-complex-proteins (LHCPII) at PSII
(Lemeille and Rochaix 2010). The absorption cross-section
of both PS is therefore plastic and can react in timescales of
minutes. PSI can extinguish excess energy by cyclic elec-
tron transport (Bukhov and Carpentier 2004) even when
linear electron transport is limited as under low DIC con-
ditions. When cells are at the CO2 compensation point one
would expect a high-PSI and low-PSII absorption cross-
section because PSII is more susceptible to photodamage.
Subsaturating DIC conditions elevated the PSI cross-sec-
tion in Chlamydomonas (Iwai et al. 2007; Palmqvist et al.
1990), presumably to provide ATP for CO2 concentrating
mechanism (CCM) operation (Giordano et al. 2005), which
acquire CO2 and HCO3- actively (Amoroso et al. 1998,
1996). With increasing DIC concentrations PSII can
quench increasing amounts of energy through linear elec-
tron transport and its cross-section will increase to balance
the ATP/NADPH ratio production in the photosynthetic
unit (Campbell et al. 1998; Finazzi and Forti 2004; Niyogi
et al. 2001). CCMs can quench excess ATP due to a CO2
efflux-re-acquisition loop (Giordano et al. 2005; Sukenik
et al. 1997). This mechanism might contribute to dissipate
energy provided by high cyclic electron transport around
PSI and entailed high ATP production. CO2 will easily leak
out of the cell and can be re-acquired under energy con-
sumption (Sukenik et al. 1997). There is no evidence for an
ATP-consuming CCM in Dunaliella tertiolecta, which
heavily relies on external carbonic anhydrase (Amoroso
et al. 1996; Beardall and Giordano 2009) and might induce
an anion transport CCM (Young et al. 2001). Alternatively,
this species might quench excess ATP by elevated flagella
movement when DIC limitation prohibits photochemical
quenching and gain therefore an advantage to alternative
energy quenching at CO2 compensation. The CCM acqui-
sition-efflux-acquisition cannot operate effectively at CO2
compensation, which might enhance energy dissipation
needs for species that rely on non-photosynthetic energy
quenching by this system.
The study cells at CO2 compensation allow insight into
the cell’s capacity to photoacclimate and photoprotect.
Activation of alternative pathways, such as Mehler reaction
or PTOX activity, can potentially lead to erroneous DIC
affinity measures during photosynthesis versus DIC curve
measurements. In addition, slow photoacclimative and
photoprotective response kinetics might falsify results if
acclimation times are not explicitly considered. Moreover,
excessive photon flux (PF) during P versus DIC assays can
affect measurements and lead to a misinterpretation of the
measurements due to confusion of DIC affinity and pho-
toinhibition/repair activities. Physiological studies using
DIC deprivation and a single DIC addition as in the present
study are mostly restricted to cyanobacteria or higher
plants (e.g., Badger and Schreiber 1993; Miller et al. 1996;
Sivak and Walker 1983) and Chlamydomonas reinhardii
(Sultemeyer et al. 1989). Chlamydomonas shows fluores-
cence responses to DIC addition, which are similar to
higher plants (marginal increase in F, strong elevation of
Fm
0) (Sultemeyer et al. 1989) although it has a high capacity
for state-transitions (Delosme et al. 1996; Finazzi et al.
2002; Palmqvist et al. 1990). Higher plants show less
flexible qT (Minagawa 2011).
In the present study, we used a large set of methods
(simultaneous pulse amplitude modulated [PAM] and O2
evolution measurements, FRRf, 18O2 uptake by MIMS,
77 K fluorescence emission spectra) to characterize the
photophysiological response of the chlorophyte flagellate
258 Photosynth Res (2014) 119:257–272
123
D. tertiolecta at the CO2 compensation point and to repe-
ated DIC additions of various concentrations. The data
show that flexible photoacclimation is mainly achieved by
qT, which facilitated stable effective quantum yields, and a
balanced QA- and PQ pool reduction state. Stable quantum
yields in parallel with state-transitions served to decouple
relative electron transport rates (rETRs) from net oxygen
evolution rates. However, multiplication of rETR with
minimal fluorescence F0, a proxy of the PSII cross-section
which is sensitive to state-transitions (Oxborough et al.
2012), yielded in congruent oxygen and fluorescence-based
photosynthetic rates. Across all experiments, photoinhibi-
tion occurred only to a small degree.
Materials and methods
Culture conditions
The chlorophyte D. tertiolecta (CSIRO strain CS-175) was
grown in 500 mL conical glass flasks with a 200 mL head-
space under constant irradiance (incident 100 lmol pho-
tons m-2 s-1, Cool White light, Silvania fluorescent tubes),
constant temperature (18 �C), and aeration. Cells were kept
in their exponential growth phase (l * 1) by means of
daily dilutions with F/2 medium (pH 8.0). The pH was
allowed to rise to maximal pH 9.0 and cell densities were
kept below 1 9 106 cells mL-1.
Before measurements, cells were washed by gentle
centrifugation and re-suspended in DIC-depleted F/2
medium. To drive out DIC from the F/2 media, it was
acidified to approximately pH 4.0, vigorously bubbled with
N2 gas for a minimum of 30 min and the pH adjusted to pH
8.2 with freshly made NaOH. DIC-depleted medium was
kept in a sealed glass container until usage on the same
day. Media were pH buffered with 20 mM 4-(2-hydroxy-
ethyl) piperazine-1-ethanesulfonic acid (HEPES) or
20 mM tricine (both Sigma-Aldrich, USA) in case of
combined PAM and oxygen measurements. Lower cell
densities for the FRRf and MIMS measurements precluded
the need for buffers.
Oxygen evolution and PAM fluorescence
Washed and concentrated cell solutions (1–1.5 9 107
cells mL-1) were either directly exposed to experimental
conditions or kept air tight under low photon flux (PF)
(*50 lmol photons m-2 s-1, i.e., 50 % of the growth PF
conditions for a maximum of 2.5 h) until the measurements
were started. PAM chlorophyll fluorescence measurements
and oxygen evolution measurements were carried out
simultaneously in the same sample. A 4 mL Oxygraph
Perspex cylinder served as a measurement chamber and O2
concentrations were measured using a Clark-type electrode
(Oxygraph, Hansatech, U.K.). The optical fiber of a Div-
ing-PAM (Walz GmbH, Germany) was attached to the side
of the chamber, and a slide projector halogen light source
provided 270 lmol photons m-2 s-1 (*29 light satura-
tion parameter Ek), while the temperature was kept con-
stant at 18 �C. The PF was carefully chosen to avoid
photoinhibition under deplete DIC conditions, where pho-
tosynthetic energy quenching is minimized to CO2 com-
pensation. Cells were exposed to light until the CO2
compensation point was detected by the absence of O2
evolution after approximately 10–30 min.
Oxygen production was calculated as the slope over 30 s
measurement intervals and normalized to the chlorophyll a
(chla) concentration of the sample. Samples were bubbled
with N2 gas when O2 concentrations approached approxi-
mately 450 lM, which was only necessary in the repeated,
1,300 lM, DIC addition. Chlorophyll concentrations were
calculated after (Jeffrey and Humphrey 1975) overnight
extraction in 90 % acetone at 4 �C. To compare fluores-
cence-based measures of photosynthetic electron transport
and oxygen production we calculated rETR by multipli-
cation of the effective quantum yield (DF/Fm
0) with the
applied photon flux (270 lmol photons m-2 s-1).
Fluorescence emission spectra at 77 K
State-transitions were interpreted as changes of the fluo-
rescence yield at F685 and F715 for PSII light-harvesting
antennae, and PSI, respectively, using a spectrofluorometer
(Hitachi F7500, Japan; excitation 440 nm (slit width
10 nm), emission slit width 2.5 nm) at 77 K. We used
higher cell concentrations (1 9 108), a larger measurement
chamber (8 mL), and a higher PF (660 lmol photons
m-2 s-1). Samples were taken by pipetting 300 lL into
Pasteur pipettes that have been sealed at the bottom, and
plunging these into liquid nitrogen, where samples were
stored in darkness until measurement. Sample handling
times were B3 s and 3–5 spectra were averaged into a
single value. These spectra were baseline corrected in
OPUS (Bruker Optic GmbH, Germany), de-convoluted
(PeakFit 4.12, SeaSolve Software Inc.), and peaks forced
thru F685(PSII reaction core), F695, F702 (unknown, see
Ihnken et al. (2011)), F715, and F730 nm (vibration). For
more details and an example for low temperature fluores-
cence spectra refer to Ihnken et al. (2011).
Culture conditions and FRRF and MIMS measurements
Steady state grown cells of D. tertiolecta cells subjected to
fast-repetition rate fluorometry (FRRF—FastTracka-I, Chel-
sea Technology Group Ltd, UK) or membrane inlet mass
spectrometer (MIMS, Balzers Omnistar) were grown in
Photosynth Res (2014) 119:257–272 259
123
1.6-L flat-faced glass vessels (*5 cm light path), under
constant aeration and irradiance (100 lmol photons
m-2 s-1, 400 W Philips high pressure HPIT E40 lamp) at
18 �C. Cells were kept in a steady state by means of con-
tinuous dilution (flow rate 64 mL h-1, giving a dilution rate
of *0.95 day-1) with fresh F/2-enriched seawater medium
(pH 8.2) at a cell density of 7.6 ± 1 9 105 cells mL-1 and a
pH of 8.7 ± 0.2 inside the culture vessel. For additional
details of FRRf measurements refer to Ihnken et al. (2011).
Quantum efficiencies for photochemistry (UPSII), thermal
energy dissipation (UNPQ), and fluorescence should (Uf,d)
equal one when values are summed up. We have calculated
the quantum efficiencies of the different quenching param-
eters from FRRf using the approach of Hendrickson et al.
(2004) as explained in Ihnken et al. (2011, results represented
in Fig. 9).
MIMS measurements
MIMS measurements yield the total O2-uptake in the
light (400 lmol photons m-2 s-1) after enrichment with
18O2-gas. Oxygen concentrations were normalized to
Argon to improve the signal-to-noise ratio (Kana et al.
1994). Photosynthetic rates were calculated by linear
regression of the change in oxygen concentrations with
time using 2-min intervals as detailed by Peltier et al.
(1985) and Claquin et al. (2004).
Results
Fluorescence responses to DIC additions
Figure 1 documents fluorescence kinetic measurements in
DIC-depleted media subjected to a range of DIC injections.
Across all DIC concentrations, F0 and Fm
0(the latter visible
as the maximum fluorescence caused by the saturating
pulses—‘‘spikes’’) quickly increased when supplied with
DIC. The 20, 40, and 160 lM additions showed similar
fluorescence response kinetics. After a rapid F0 rise, fluo-
rescence signals peaked after approximately 2–3 min,
dependent on the concentration of DIC added with highest
peaks in the strongest addition. Fluorescence then
decreased until a new CO2 compensation point was
reached. When high DIC concentrations were added
(1,300 lM—*� of DIC replete conditions) a markedly
different fluorescence response was observed. Although
values also rose quickly, maximal signals were recorded
after an additional 12 min in the fluorescence measures
compared to the lower DIC additions.
200
300
400
500
600
F' [
rela
tive]
20 µM 40 µM
0 10 20 30 40 50
200
300
400
500
600
time [min]
F' [
rela
tive]
160 µM
0 10 20 30 40 50
time [min]
1300 µM
Fig. 1 DIC concentration dependent fluorescence recoded with a
PAM fluorometer. Cell suspensions were at CO2 compensation, i.e.,
the media depleted of DIC so that the photosynthetic carbon fixation
activity is limited to re-fixation of CO2. DIC was added by injection
of NaHCO3- solution (arrows) to a final concentration as shown in
the graphs. DIC injections were repeated on the same sample (not
shown in case of 1,300 lM). Vertical ‘‘spikes’’ show maximal
fluorescence (Fm
0) during a saturating light pulse. The photon flux was
270 lmol photons m-2 s-1, almost triple of the growth PF and
sufficiently high to saturate photosynthesis under DIC replete
conditions. DF/Fm
0were approximately 0.1–0.15 when cells were at
CO2 compensation. Chlorophyll a concentrations were approximately
12 mg L-1 (1.2 9 107 cells mL-1). Data show a representative
sample from n C 3
10 5 0 5 10 15 20
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
time [min]
F/F
m'
0.0
0.5
1.0
1.5
2.0
2.5
NP
Q
NPQyield
Fig. 2 Effective quantum yields (DF/Fm
0) and NPQ ((Fm-Fm
0)/Fm
0) at
CO2 compensation, during a 160 lM DIC addition (t = 0), and a
following dark phase. High Fv/Fm after 10 min recovery in darkness
show the absence or low degree of photoinhibition caused by
experimental treatment. Data show mean ± SD (n = 3). Cell densi-
ties were approximately 1.2 9 107 cells mL-1. Cells were transferred
into darkness as indicated by the arrow
260 Photosynth Res (2014) 119:257–272
123
Figure 2 shows fluorescence responses to a single
160 lM DIC addition and recovery after 10 min in the
dark. Effective quantum efficiencies increased rapidly,
while NPQ was lowered by DIC addition. DF/Fm
0values
were stable, but decreased slightly after approximately
5 min. NPQ increased already after 2.5 min, values were
less stable than quantum yields. Incubation in darkness for
10 min relaxed NPQ, while Fv/Fm increased to high values,
which suggests a low degree of photoinhibition. That
photodamage was not a predominant fluorescence quencher
can be seen by high Fv/Fm recovery rates after the different
treatments (Table 1).
Oxygen evolution and electron transport measurements
Figure 3 shows parallel net oxygen evolution rates and
relative electron transport rates (rETR = DF/Fm
09 PF)
measured by a Clark-type oxygen electrode and PAM
fluorescence in the same chamber during 40, 160, and
1,300 lM DIC additions (A, C, and E, respectively). Net
oxygen evolution quickly reached maximal values in the 40
and 160 lM additions, but a slower response was noticed
in the 1,300 lM injection. Relative ETR show similar
trends as oxygen evolution, however, values deviated from
a linear relationship after maximal photosynthetic rates
were reached. A good linear rETR versus O2 correlation
was found at the highest DIC concentration (1,300 lM)
(Fig. 3 e, f). Correlation coefficients for linear model fits
for net oxygen evolution and rETR range between very
good values of 0.96 and weaker correlations and values of
0.50 (Table 2). In general, rETR coincided with net oxygen
production to a degree, but deviation from linearity was
clearly visible. The correlation fits were improved when
rETR values were multiplied by F0 measured directly
before the saturation pulse was applied. F0 values are
subjected to changes in NPQ, but also to changes in the
absorption cross-section and the concentration of light-
harvesting pigments associated with PSII and the reduction
state of QA (Oxborough et al. 2012) and it was demon-
strated in this paper that F0 is proportional to the concen-
tration of reaction center II ([RCII]) over the functional
cross-section of PSII (rPSII). Absolute electron transport
rates can be estimated from fluorescence measures as F0
theoretically and empirically covaries with the concentra-
tion of light-harvesting pigments associated with PSII
(Oxborough et al. 2012) and its multiplication with rETR
provides an improved proxy for true ETR. For example,
rETR 9 F0 correlated by 0.90 ± 0.01 with net O2 evolu-
tion, an improvement by 0.4 units compared to rETR
versus net O2 correlation at a 160 lM DIC pulse (Table 2,
Fig. 3c, d). Indeed, the correlation between net oxygen
evolution and fluorescence measurements increased in all
cases when rETR was multiplied by F0 (Table 2). Oxygen
production might represent photosynthetic processes
incorrectly if elevated O2 consuming processes mask pro-
duction rates. We therefore employed MIMS measure-
ments to test if oxygen uptake rates were responsible for
weak rETR versus net O2 evolution correlations.
Membrane inlet mass spectrometry
Net oxygen evolution measurements confirmed data
achieved by the Clark-type oxygen measurement. Before
the addition of DIC, respiration and oxygen evolution were
similar, corroborating the fact that the cells were at the DIC
compensation point where the rate of C-fixation is deter-
mined by the rate of respiration. Net oxygen production
increased 323 ± 83 % after the first DIC addition and
151 ± 55 % after the 2nd DIC addition (Fig. 4a). O2
uptake rates estimated from the uptake of 18O2 decreased
by 0.0076 ± 0.0070 mg O2/mg chla/min after DIC addi-
tion. Hence, the oxygen uptake in the light decreased by
only 14 ± 12 %. The decrease in oxygen uptake after the
2nd DIC addition was even smaller (7 %) and a one-way
ANOVA analysis on the data just before and after the DIC
addition showed that these decreases in oxygen uptake
were not significant (p = 0.17 and 0.06 respectively). O2
uptake under replete DIC conditions was similar to samples
kept under DIC-deplete conditions (not shown).
State-transitions
As mentioned above, we observed changes in NPQ. To see
if state-transitions were involved as a possible driver of
these changes we measured 77 K fluorescence spectra and
investigated the F685/F715 fluorescence ratios. In DIC-
depleted samples, a high fraction of the light-harvesting
complexes were associated with PSI as indicated by the
low F685/F715 ratios (Fig. 5a). When 160 lM DIC was
Table 1 Fv/Fm measured 10 min after treatment measured by PAM
(row 1–5) and FRRf (row 6, 7)
Photon flux
(lmol m-2 s-1)
DIC addition (lM) Fv/Fm n
270 40 0.53 ± 0.086 5
270 160 0.63 ± 0.042 3
270 1,300 0.64 ± 0.103 4
70 160 0.74 ± 0.035 2
1,550 160/1,300 0.53 ± 0.006 4
440 160 0.51 ± 0.004 3
440 Replete 0.55 ± 0.003 3
FRRf causes a PSII single turnover and results in naturally lower
values compared to multiple turnover measurements using PAM. Fv/
Fm in replete conditions were measured 10 min after the light phase
of similar duration has ended
Photosynth Res (2014) 119:257–272 261
123
added the F685/F715 ratio increased, which is indicative of a
state II–state I transition (LHCP funnel increasing amounts
of energy toward PSII). This response was rapid with an
observable effect after 30 s that continued until 90 s after
the DIC addition. However, the 28 % increase in the
F685/F715 ratio during the 90 s after the DIC addition was
not significant (One-Way Repeated Measures ANOVA,
p = 0.275). Data in Fig. 5b show the amplitude of DIC
concentration induced state-transitions. F685/F715 ratios
were lowest in the dark (1.7 ± 0.53), slightly higher at
CO2 compensation (2.2 ± 0.03), while more LHCP funnel
energy to PSII in the presence of deplete DIC (4.2 ± 0.06).
0.0
0.5
1.0
1.5
5000
1000
015
000
2000
0
rela
tive
ET
R *
F'
1020
3040
50
rela
tive
ET
R
0 5 10 15
0.0
0.5
1.0
1.5
time [min]
010
000
2000
030
000
4000
0
rela
tive
ET
R *
F'
020
4060
8010
012
014
0
rela
tive
ET
R
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
5000
1000
015
000
2000
025
000
rela
tive
ET
R *
F'
1020
3040
5060
70
rela
tive
ET
R
O2rETRrETR * F'
0 50 100 150
0.0
0.5
1.0
1.5
relative ETR
0 1 2 3 4relative ETR * F'/10000
O2 vs rETRO2 vs rETR * F'/10000
A
C
D
E
B
F
0 50 100 150
0.0
0.5
1.0
1.5
relative ETR
0 1 2 3 4relative ETR * F'/10000
0 50 100 150
0.0
0.5
1.0
1.5
relative ETR
0 1 2 3 4relative ETR * F'/10000
40 µM1601300
O2
evol
utio
n [n
mol
O2/
µg c
hla
/min
]ne
tO
2 ev
olut
ion
[nm
ol O
2/µg
chl
a/m
in]
net
O2
evol
utio
n [ n
mol
O2/
µg c
hla
/min
]ne
tgr
oss
O2
evol
utio
n [n
mol
O2/
µg c
hla
/min
]ne
tgr
oss
O2
evol
utio
n [n
mol
O2/
µg c
hla
/min
]ne
tO2
evol
utio
n [n
mol
O2/
µg c
hla
/min
]ne
t
Fig. 3 Fluorescence and net
oxygen evolution measured
simultaneously at CO2
compensation and a single DIC
addition at time = 0. DIC
additions were 40, 160, and
1,300 lM in a, c, e,
respectively, for net O2
evolution (squares), relative
electron transport (rETR)
(effective quantum yield
multiplied by photon flux—
triangles), and rETR multiplied
by F0 (minimal fluorescence in
light—circles). b, d, f show
scatter plots of net O2 evolution
versus rETR (squares) and net
O2 evolution versus rETR 9 F0
(circles) for 40, 160, and
1,300 lM, respectively. For
graphical clarity, rETR 9 F0
values were divided by 10,000.
Lines represent linear
correlation for net O2 evolution
versus rETR (dashed line) and
net O2 evolution versus
rETR 9 F0/10,000 (solid line).
Please note different y-axis
scales in a, c, e. Inset figure in e
shows net O2 evolution from a,
c, e on the same scale for
convenient comparison. Plots a,
c, and e show mean ± SD
(n C 2). For correlation
parameters of net O2 evolution
versus fluorescence
measurements refer to Table 2
Table 2 Correlation coefficient (r2) of linear fits from net oxygen
evolution and fluorescence parameters rETR (effective quantum yield
multiplied by photon flux) or rETR 9 F0 as shown in Figs. 3, 8
Photon flux (lmol
photons m-2 s-1)
DIC
addition
(lM)
O2 vs. Retr r2 O2 vs.
rETR 9 F0
r2
n
270 40 0.84 ± 0.044 0.93 ± 0.008 3
270 160 0.50 ± 0.108 0.90 ± 0.091 3
270 1,300 0.96 ± 0.019 0.97 ± 0.000 2
70 160 0.50 ± 0.108 0.71 ± 0.002 2
1,550 160 0.02 ± 0.020 0.10 ± 0.073 3
262 Photosynth Res (2014) 119:257–272
123
This value represents almost the upper end of the state-
transition, i.e., highest possible PSII functional cross-sec-
tion by qT as shown by far-red light exposure for 15 min
(F685/F715 = 4.4 ± 0.32). Cells that were treated with
DCMU maintained high ratios (F685/F715 = 4.6 ± 0.1).
Cells that were treated with DCMU in the dark and then
transferred to the light in the absence of DIC elevated the
F685/F715 ratio slightly (?1.14 units) (Fig. 5c), though this
change was not significant (p = 0.063, dark F685/F715 =
1.51 ± 0.51 and 2.65 ± 0.58). Nevertheless, it is note-
worthy that cells locked LHCP in state II in the absence of
electron transport in PSII and a DCMU-induced oxidized
PQ pool. However, the DIC addition induced state II–state
I transition increased the absorption cross-section of PSII
(rPSII0) as measured using single turnover fluorescence
measures (Fig. 6d).
FRRf measurements in single 160 lM DIC addition
Figure 6 shows fluorescence responses to DIC addition
using FRRf. NPQ, rETR, F0, and Fm
0were similar to PAM
measurements where a lower PF flux was used (170 lmol
photons m-2 s-1 less—Fig. 1, 3). Notice that when NPQ
starts to increase again *8 min after the DIC addition, that
this does not change rETR, but that the this change in NPQ
is reflected in our proxy of absolute ETR (rETR 9 F0),again demonstrating that this gives a better representation
of the rate of oxygen evolution than rETR. Due to lower
cell densities used in FRRf measurements DF/Fm
0remained
stable after the DIC injection while other parameters
acclimated. DIC addition increased QA- re-oxidation
kinetics, which shows that QA and/or the PQ pool is
reduced when cells are at CO2 compensation. QA- re-oxi-
dation (sPSII) remained constant after a brief acclimation
phase when DIC was introduced. The functional absorption
cross-section rPSII’ increased to highest values in the light
1 min after DIC addition. After the DIC addition Fm
0values
were as high as Fm. F0 and Fm
0correlated well in all
experimental phases (Table 4). Initial slopes during
160 lM DIC injection were similar to experiments where
PAM was used (0.78 and 0.77 for FRRf and PAM
respectively—Table 3). The same was found for correla-
tion coefficients at CO2 compensation. When cells were
transferred to the dark, F0 and Fm correlated with an initial
slope of 0.44 (±0.01).
Connectivity parameter p values were high at CO2
compensation (0.57 ± 0.01) and decreased upon DIC
addition (0.25 ± 0.02) suggesting a high degree of ener-
getically connected PSII in the absence of DIC and a
separation due to DIC addition. Connectivity was inversely
connected to rPSII’. Energy partitioning analysis showed
an inverse correlation between UNPQ and photosynthesis
(DF/Fm
0). Surprisingly, however, was the strong response of
‘‘constitutive’’ energy quenching Uf,D. Values mirrored
regulated UNPQ and increased upon DIC addition.
Effect of light intensity on DIC additions
When the light intensity was lowered to 70 lmol photons
m-2 s-1, DIC addition caused a rapid F0 and Fm
0rise to
quasi maximal values within approximately 2 min (Fig. 7),
which coincides with measurements made at higher PF.
Thereafter signals were either stable for approximately
15 min or oscillated slightly, which lead to a deviation
from F and Fm
0linearity.
When cells were exposed to high PF (1,550 lmol pho-
tons m-2 s-1) F and Fm
0responded in a linear fashion to
DIC additions (Fig. 6c; Table 3). However, the variable
fluorescence was low, saturation pulses could only mar-
ginally increase the fluorescence signal (Fig. 7). DF/Fm
0val-
ues were low before the DIC addition (0.012 ± 0.006),
during the first (160 lM) DIC addition (0.024 ± 0.011) and
the consecutive 1,300 lM addition (0.053 ± 0.014). The
DIC additions caused similar fluorescence oscillations as in
lower PF (Fig. 7, 1), however, an overall decrease in the
fluorescence signal was clearly visible. After the light was
switched off, a fluorescence oscillation was visible that is
mainly caused by a state-transition (Casper-Lindley and
Bjorkman 1996; Ihnken et al. 2011). Low Fm values 10 min
after cells been transferred to the dark were possibly caused
by xanthophyll cycle induced qE, which does not relax in the
first minutes of darkness in this species (Casper-Lindley and
Bjorkman 1998) and LHCP are possibly in state II. Fv/Fm
Ane
t 16O
2 p
rodu
ctio
n
0.00
0.02
0.04
0.06
0.08
0.10
0.12
B
time [min]
-10 0 10 20 30 40 50 60
18O
2up
take
-0.07
-0.06
-0.05
-0.04
-0.03
Fig. 4 Net 16O2 evolution (a) and 18O2 uptake measured by
membrane inlet mass spectroscopy (b) under DIC-deplete conditions
and after addition of 160 lM DIC at t = 0 and 28 min. Plotted is the
mean and standard deviations of three replicates. To compensate for
small offset changes (different initial values) we shifted the 2nd and
3rd replicate to the initial value of the first replicate. By this way, the
pattern and the standard deviations are not influenced by these offset
changes. Notice the difference in the Y-axis scales between a and b
Photosynth Res (2014) 119:257–272 263
123
was 0.53 ± 0.06 (n = 3) after the experimental treatment,
which shows that despite the very high PF the cells have
experienced, they did not suffer from photoinhibition. This is
corroborated by oxygen evolution measurements, which
coincide with measurements performed at lower, but still
saturating PF (Fig. 3).
Figure 8A shows net O2 evolution and rETR 9 F0 in
high and low PF treatments. In low PF conditions values
coincide initially, but deviated 5 min after DIC additions.
rETR 9 F0 correlated better than rETR with net O2 evo-
lution (r2 = 0.71 ± 0.00, and r2 = 0.50 ± 0.11 for O2 vs.
rETR 9 F0 and O2 vs. rETR, respectively), but weaker
compared to other treatments.
Both proxies of photosynthesis, fluorescence, and oxy-
gen measurements correlated poorly at high PF
(1,550 lmol photons m-2 s-1) during a 160 lM DIC
addition (Fig. 8) due to noisy fluorescence signals.
F0 over Fm
0correlation
Figure 9 and Tables 3, 4 show the strong linear correlation
between F0 and Fm
0during a DIC addition. A correlation to
this degree was unexpected. F0 values are affected by NPQ
and the QA oxidation state which is mainly a function of
photosynthetic electron transport processes, the absorption
cross-section, and the PF. During Fm
0measurements, QA is
0 30 60 90
time after 160 µM DIC addition [s]
F68
5/ F
715
emis
sion
01
23
45A B
20 5 20time of treatment [min]
F68
5/ F
715
emis
sion
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
conditions:
DIC, +DCMU
C
DIC
15
min
+D
IC 6
0
DIC
1.3
mM
DIC
10
+D
IC 1
5
+D
IC&
DC
MU
5
+D
IC&
FR
15
F68
5/ F
715
emis
sion
0
1
2
3
4
5
Fig. 5 Ratios of fluorescence emission spectra measured at low
temperature (77 K). Data show ratios from LHCP-II
(F685 nm = PSII) and PSI (F715 nm) during DIC depletion and at
30, 60, and 90 s after DIC addition (a, p = 0.045 for t = 0 vs.
t = 90 s). b PSII/PSI fluorescence emission ratio for various treat-
ments measured in a separate experiment. Samples have been dark
acclimated (filled bars) or taken in the light (open bars) after times
denotes as number in X-axis (in minutes). ?DIC: replete DIC
(*2.2 mM), FR far-red light, DCMU 3,4 dichlorophenyl-1,1-dimeth-
ylurea. (c): PSII PSI ratio from cells treated with DCMU and kept
under DIC-deplete conditions in the dark (filled bar) and light (open
bars). Data in (c) are statistically not quite significantly different
(p = 0.063, t test); note different y-axis scale compared to a and
b. Photon flux was 660 lmol photons m-2 s-1. This higher light
intensity was chosen to account for much higher cell densities needed
for fluorescence emission detection. Data show mean (n C 3) ± SD
264 Photosynth Res (2014) 119:257–272
123
fully reduced, and mainly non-photosynthetic fluorescence
quenching mechanisms are responsible for depressing
values below Fm. A strong correlation between both values
indicates that the photosynthetic energy quenching mech-
anisms or rather processes that affect the QA reduction state
in actinic light, are subservient for fluorescence values
measured in a DIC addition response. It appears that pro-
cesses that have an effect on both F and Fm
0are responsible
for changes in variable fluorescence measurements. State-
transitions could be such processes.
Additions of 40 and 160 lM DIC resulted in different
initial slopes of F0 over Fm
0for the duration of the DIC
addition (0.62 ± 0.023 and 0.77 ± 0.017 for 40 and
160 lM DIC, respectively) and a very high correlation
coefficient (0.99 in both cases). A consecutive DIC addi-
tion of the same concentration resulted in slightly lower
initial slopes, the correlation, however, was similarly
strong (Fig. 9b; Table 3). Deviation from linearity was
found at high DIC additions (1,300 lM) and under low
light (70 lmol photons m-2 s-1). In the former case, the
Fig. 6 FRR fluorescence parameters during CO2 compensation, a
single 160 lM DIC addition and darkness. Minimal and maximal
fluorescence during the flashlet sequence fluorescence induction
(F0 and Fm
0, respectively) and QA
- re-oxidation parameter (sPSII)
measured every 13 s (a). b rETR and non-photochemical quenching
(NPQ) during the light phase. In addition, rETR from replete DIC
measurements is shown (triangle). Energy partitioning of absorbed
quanta UNPQ: regulatory NPQ; Uf,D: constitutive energy quenching;
Yield: DF/Fm
0and Fv/Fm during the light- and dark-phase, respec-
tively; sum: values of all three parameters added (c). Effective (light),
maximal (dark) absorption PSII cross-section (rPSII) as well as p, a
parameter indicative of flexible energy sharing over energetically
connected PSII centers (d). A representative measurement was chosen
from 3 independent experiments (a, c, d) or means presented ±SD
(n = 3; b). Actinic photon flux was 440 lmol photons m-2 s-1 and
the light was switched off 12 min after the DIC addition (downward
facing arrow). Note the different time axis in panel (b). Lower cell
densities were used in FRRf, compared to MIMS, or combined PAM
and oxygen measurements. This affects the DIC consumption during
photosynthesis, leading to a lower decline over time with lower cell
densities
Photosynth Res (2014) 119:257–272 265
123
primary phase after DIC addition was characterized by
comparably stronger increase in Fm
0than in F0 for
approximately 9 min and therefore a lower initial slope of
F0 over Fm
0(left hand side triangles in Fig. 9a, 1 1,300 lM
approximately [15 \25 min). Samples exposed to LL
conditions showed a reduced F0 increase when DIC was
added compared to other PF conditions. A deviation from
F0 to Fm
0linearity was shown in one replicate. Here F0
correlated inversely with Fm
0during the first *5 min after
DIC addition. F0 values decreased in this phase, while a
soft increase in Fm
0was noticed.
Repetition of DIC addition
Figure 10 describes the photosynthetic response to two
consecutive DIC additions. Oxygen evolution showed a
similar response, although data presented were variable. A
repeat study consolidated data presented (not shown,
n = 3). Oxygen uptake rates were constant during the
second DIC addition (Fig. 4). rETR was higher in the
second addition compared to the first one due to higher
effective quantum yields. Differences between first and
second addition were less pronounced in rETR 9 F0.
Discussion
Non-photosynthetic response to DIC additions
Higher plants, held at CO2 compensation and re-supplied
with CO2, show a decrease in fluorescence due to inter-
active regulation of photosynthetic and thermal energy
quenching (qE) (Dietz et al. 1985; Sivak and Walker 1985).
In higher plants, state-transitions (qT) are not a predomi-
nant NPQ mechanism, but rather employed to regulate ratio
of photosynthetically generated ATP and NADPH (Dietzel
et al. 2008; Finazzi and Forti 2004; Lemeille and Rochaix
2010). Conversely in cyanobacteria qT is considered to be a
primary photoprotective mechanism (Campbell et al. 1998;
Campbell and Oquist 1996). When the cyanobacterium
Synchococcus was re-supplied with DIC at CO2 compen-
sation, fluorescence decreased as fluorescence did in higher
plants (Miller et al. 1991). Species that can carry out qT can
be expected to decrease their PSII absorption cross-section
under DIC limitation to lower the risk of photodamage in
PSII due to the low photosynthetic energy quenching. If
this the case, re-supply of DIC to cells that are held at CO2
compensation should induce a state II–state I transition,
thereby increasing the absorption cross-section at PSII to
fuel increasing energy demands of the Calvin–Benson–
Bassham cycle.
Interestingly, DIC addition did not induce state-transi-
tions in cyanobacteria, and cells were already in state I (the
phycobilisomes funnel energy to PSII) in the absence of
DIC (Miller et al. 1996). On a regulatory level this appears
Table 3 linear correlation of F0 versus Fm
0during CO2 compensation and a repeated DIC addition as shown in Fig. 9
Photon flux
(lmol m-2 s-1)
DIC addition
(lM)
First DIC addition Second DIC addition
F0 vs. Fm0
initial slope
F0 vs. Fm0
correlation
coefficient r2
F0 vs. Fm0
initial slope
F0 vs. Fm0
correlation
coefficient r2
270 40 0.62 ± 0.023 0.99 ± 0.023 0.57 ± 0.022 0.97 ± 0.014
270 160 0.77 ± 0.017 0.99 ± 0.002 0.73 ± 0.025 0.10 ± 0.000
270 1,300 0.25 ± 0.023 0.83 ± 0.040 0.39 ± 0.007 0.33 ± 0.062
70 160 0.37 ± 0.017 0.95 ± 0.042 n.t. n.t
1,550 160/1,300 1.01 ± 0.010 1.00 ± 0.001 1.02 ± 0.027 0.99 ± 0.003
Only a single DIC addition was performed under low light (70 lmol photons m-2 s-1)
n.t. not tested
0 10 20 30 40 50
100
300
500
time [min]
F' [
rela
tive]
70 µE
0 10 20 30 40 50
time [min]
1550 µE
Fig. 7 Fluorescence in DIC and photon flux dependent measure-
ments. Cell suspensions were at CO2 compensation, when DIC was
added at approximately 8 min (open arrow heads, final 160 lM). DIC
was injected once in the low light treatment (70 lE = 70 lmol
photons m-2 s-1). In the high light treatment (1,550 lE) first160 lM
were added, a higher DIC concentration was chosen for the second
DIC addition (1,300 lM DIC, double arrow heads). The light was
switched off as indicated by filled arrows and the saturation pulse
train was interrupted from this point on. A saturation pulse 10 min
after the light was switched off shows maximal fluorescence in the
high light treatment (Fv/Fm = 0.53 ± 0.06). Chla concentrations
were approximately 12 mg L-1 (1.2 9 107 cells mL-1). Data show a
representative sample from n C 2
266 Photosynth Res (2014) 119:257–272
123
reasonable as electron transport via the PQ pool is very
low. An oxidized PQ pool would induce a connection of
phycobilisomes toward PSII. Cyanobacteria would risk a
high degree of photodamage if photoprotection would
merely be accomplished by qT and the phycobilisomes rest
in state I in the absence of DIC. However, photoprotection
in cyanobacteria can also be facilitated by energy quencher
in the phycobilisome antennae related to the orange
carotenoid protein (Bailey and Grossman 2008) which is
present in most phycobilisome containing cyanobacteria
(Kirilovsky and Kerfeld 2012), which can explain a low
degree of photodamage even though the absorption cross-
section of PSII is large when cells are at the CO2 com-
pensation point. However, despite the fact that Synecho-
coccus PC7942 did not show state-transitions after DIC
addition when in DIC-deplete conditions (Miller et al.
1996), it also does not contain an OCP ortholog (Kirilovsky
and Kerfeld 2012), suggesting that the OCP mechanism is
apparently not the only mechanism cyanobacteria can
invoke to protect itself against excess irradiance.
Increasing fluorescence signals upon DIC addition in the
present study show a different photoresponsive strategy in
D. tertiolecta than in higher plants and cyanobacteria, but
is in agreement to measurements using Chlamydomonas
reinhardtii (Iwai et al. 2007; Miller et al. 1996; Sultemeyer
et al. 1989). DIC deprivation in the light shifted LHCP
toward PSI to avoid excess photon absorption in PSII and
high risk of photodamage. PSI can quench photon’s energy
effectively by cyclic electron transport and is less prone to
photodamage. Re-supply of DIC clearly shifted LHCP to
PSII, which can increase linear electron transport and water
splitting activity. State-transition measurements by 77 K
fluorescence emission spectra show this clearly and func-
tional absorption cross-section changes measured by FRRf
confirm this finding.
However, qT was not the mere form of NPQ. That
energy-dependent quenching also contributed to NPQ can
be seen by the rapid increase in Fm, seconds after cells
were transferred to the dark. This rapid increase can be
explained by relaxation of DpH gradient dependent thermal
energy quenching qE. This fast component of qE appears to
be very efficient in D. tertiolecta, where the xanthophyll
cycle component of qE is less pronounced and slower
compared to other species (Casper-Lindley and Bjorkman
1998). Poisoning cells with dithiothreitol (DTT), which
prohibits the activation of the xanthophyll cycle, did not
0 20 40 60 80 100
0.0
0.5
1.0
1.5
relative ETR
0.0 0.5 1.0 1.5 2.0
relative ETR * F'/10000
10 15 20 25 30 35 40
0.0
0.2
0.4
0.6
0.8
relative ETR
0.0 0.5 1.0 1.5
relative ETR * F'/10000
O2 vs rETRO2 vs rETR * F'/10000
A
C
D
B
0 5 10 15
0.0
0.5
1.0
1.5
time [min]
5000
1000
015
000
rela
tive
ET
R *
F'
1020
3040
5060
70
rela
tive
ET
R
0 10 20 300.
00.
20.
40.
60.
8time [min]
2000
4000
6000
8000
1000
0
rela
tive
ET
R *
F'
510
1520
2530
35
rela
tive
ET
R
O2
rETRrETR * F'
O2
evol
utio
n [n
mol
O2
/µg
Chl
a/m
in]
net
O2 e
volu
tion
[nm
ol O
2/µ g
chl
a/m
in]
net
O2
evol
utio
n [n
mol
O2/
µg c
hla
/min
]ne
t
O2
evol
utio
n [n
mol
O2/
µg c
hla
/min
]ne
t
Fig. 8 Fluorescence and net
oxygen evolution measured
simultaneously at CO2
compensation and a 160 lM
single DIC addition at time = 0.
Photon fluxes were 70 lmol
photons m-2 s-1 (a, low light)
and 1,550 lmol photons
m-2 s-1 (c, high light),
respectively for net oxygen
production (squares), relative
electron transport (rETR), and
rETR multiplied by F0. b,
d show scatter plots of net O2
evolution versus rETR (squares)
and net O2 evolution versus
rETR 9 F0 divided by 10,000
(circles) for low light and high
light treatments, respectively.
Lines represent linear
correlation for net O2 evolution
versus rETR (dashed line) and
net O2 evolution versus
rETR 9 F0/10,000 (solid line).
Plots a and c show mean ± SD
(n C 2). For correlation
parameters of net O2 evolution
versus fluorescence
measurements refer to Table 2
Photosynth Res (2014) 119:257–272 267
123
cause differentiable fluorescence responses in the present
study (not shown) and is in agreement with results by
Casper-Lindley and Bjorkman (1998). This suggests that
xanthophyll cycle induced qE was probably marginal if
present at all in the present study, with the exception of the
high light experiments. In high PF exposure F0 decreased
continuously with the exception for a short perturbation
during DIC injections. This fluorescence decrease is pre-
sumably due to xanthophyll cycle dependent qE component
as qT is not possible (LHCPII in stateII at t = 0) and qI
was not substantial. Xanthophyll cycle activation can
potentially be facilitated by further acidification of the
thylakoid lumen (Kramer et al. 1999) compared to the state
reached when cells are at CO2 compensation. It is possible
that lumen acidification is accelerated by combined linear-
and cyclic-electron transport in high PF, especially after a
1,300 lM DIC addition, where elevated linear electron
transport would promote an increased DpH gradient.
At medium PF, however, absorption changes at 535 nm
were visible, which confirms the contribution of a qE to
NPQ in the present study (not shown) (Heber 1969; Ilioaia
et al. 2011; Ruban et al. 1993). Compared to other algae D.
tertiolecta has unusual high lutein concentrations and
might exhibit plant-like qE despite the fact that xanthophyll
cycle activation responds unusually (Casper-Lindley and
Bjorkman 1998). Lutein is a carotenoid found in PSII
antennae and is involved in DpH gradient induced NPQ by
causing conformational changes within PSII which is
comparable to the effect of zeaxanthin (Johnson et al. 2009,
2011).
Nevertheless, DIC re-supply to D. tertiolecta cells at
CO2 compensation triggered a substantial state-transition in
the present study. Only small changes in the effective
quantum yields were visible when DIC was supplied
(Fig. 6), leading to the assumption that qT and not
150
200
250
300
350
400
450
F' [
rela
tive]
square = 40 µMround = 160 µM triangle = 1300 µM
200 300 400 500 600
Fm' [relative]
A
C
B
200 300 400 500 600
150
200
250
300
350
400
450
Fm' [relative]
F' [
rela
tive]
round = 1550 µEsquare = 70 µE
Fig. 9 Minimal fluorescence in
the light-acclimated state (F0)versus maximal fluorescence
during a saturation pulse (Fm
0) at
CO2 compensation and DIC
addition as shown in Figs. 1, 7.
a circles 160 lm, squares
40 lM and 1,300 lM
(triangles) during the first DIC
addition or a second DIC
addition (b). Photon flux were
270 lmol photons m-2 s-1 a, b,
or 70 lmol photons m-2 s-1
(squares—c) and 1,500 lmol
photons m-2 s-1 (circles—c).
DIC additions were 160 lM in
(c). Different fill of symbols
show individual measurements.
For correlation coefficients refer
to Table 3
Table 4 Initial slope and linear correlation coefficient for linear fits
from F0 versus Fm
0and F0 versus Fm measured before and after a
160 lM DIC addition in the light (440 lmol photons m-2 s-1) and
subsequent darkness using FRRf as shown in Fig. 6
Treatment F0 vs. Fm0 initial slope F0 vs. Fm
0 correlation
coefficient r2
Deplete 0.67 ± 0.110 0.88 ± 0.135
160 lM DIC 0.78 ± 0.026 0.95 ± 0.026
Dark 0.44 ± 0.012 0.98 ± 0.011
Data show mean ± SD (n = 3)
268 Photosynth Res (2014) 119:257–272
123
regulative mechanisms within PSII was the main regula-
tory system. Stable QA- re-oxidation kinetics (sPSII) and
strong linear correlations between F0 and Fm
0within each
phase of the experimental treatments corroborate this. By
maintaining a low PSII absorption cross-section in the
dark, D. tertiolecta cells minimize potential photodamage
when suddenly exposed to light. State-transitions are con-
trolled by a signal cascade, which involve binding of PQH2
to Qo located at the lumen side of cytochrome b6f complex
and Stt7/STN7 kinase facilitated phosphorylation of
LHCPII (Lemeille and Rochaix 2010). In addition, PSII
core proteins are suggested to be phosphorylated by Stl1/
STN8 (Minagawa 2011; Rochaix et al. 2012). However,
phosphorylation is controlled by the reduction state of the
PQ pool (Dietzel et al. 2008; Wollman 2001) or more
specifically the binding of plastoquinol at the Qo site
(Lemeille and Rochaix 2010). D. tertiolecta cells showed a
high association of LHCP to PSI in the dark where the PQ
pool should theoretically be oxidized due to the absence of
photosynthetic electron transport. A reduced PQ pool in
darkness can be maintained by chlororespiratory activity
(Peltier and Cournac 2002). When cells were transferred to
the light, a fraction of the LHCP shifted toward PSII. This
was not a result of photosynthetic electron transport. Cells
that were treated with DCMU and were DIC was depleted
also shifted a fraction of the LHCP from PSI toward PSII
(Fig. 5c). Under these conditions electron transport via the
PQ pool from PSII should be prohibited, yet, a small state-
transition can be seen, which shows that state-transitions
were regulated by means other than PSII-mediated electron
transport. In contrast to D. tertiolecta, cyanobacteria are in
stateI when held at CO2 compensation (Miller et al. 1996).
It is not obvious what keeps the PQ pool reduced when D.
tertiolecta cells are at CO2 compensation. Low electron
transport rates in PSII, a high cross-section at PSI, and
therefore high electron drain in the electron carrier of the
photosynthetic unit, theoretically suggest an oxidized and
not a reduced PQ pool. Theoretically, cells could have
elevated mitochondrial respiration to re-supply CO2 to the
Calvin–Benson–Bassham cycle and allow photosynthetic
energy quenching. Oxygen uptake measurements, however,
did not support the theory of elevated respiratory activity to
provide substrate for carbon fixation.
Photosynthetic response to DIC additions
Effective quantum yields and rETR were rapidly up-regu-
lated when cells were supplied with DIC. Values remained
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
gros
s O
2 ev
olut
ion
[nm
ol O
2/µg
chl
a/m
in]
gros
s O
2 ev
olut
ion
[nm
ol O
2/µg
chl
a/m
in]
A
C
B
0 5 10 15
5000
1000
015
000
2000
0
time [min]
rela
tive
ET
R *
F'
2030
4050
60
rela
tive
ET
R
0 5 10 15
time [min]
open: first DIC additionclosed: second DIC addition
Fig. 10 Net O2 evolution (a),
rETR (b), and rETR 9 F0
(c) for the first 160 lM DIC
addition (open symbols) and a
consecutive 160 lM DIC
addition (closed symbols). DIC
was added at t = 0. Data show
mean ± SD (n C 2)
Photosynth Res (2014) 119:257–272 269
123
surprisingly high when oxygen evolution decreased some
minutes after DIC has been added. That oxygen evolution
was representative for photosynthesis can be seen by only
slightly increased, or even, O2 uptake under DIC depletion
and DIC additions. Oxygen uptake by Mehler reaction,
increased photorespiratory activity, or electron donation
and water formation at the PTOX are possible. However,
these processes are only employed to a small degree by
algae when exposed to limiting DIC concentrations
(Franklin and Badger 2001; Hanson et al. 2003; Kaplan and
Berry 1981; Sultemeyer et al. 1987, 1989) compared to
higher plants and cyanobacteria (Dietz et al. 1985; Miller
et al. 1996; Peterhansel and Maurino 2011; Sivak and
Walker 1983). In the present study, O2 evolution rates were
representative for assimilatory photosynthesis as shown by
the low degree of 18O2 uptake dependency on the DIC
concentration. Deviation from linearity between O2 and
rETR in relation to the DIC concentration was shown
before, but could not be resolved (Carr and Bjork 2003). It
is possible that cyclic electron transport in PSII facilitates
higher electron transport and a deviation from linearity
with oxygen evolution.
Following the principle of Oxborough et al. (2012) we
multiplied rETR with F0, which resulted in a very good fits
between this parameter and O2 evolution rates. We prefer
this estimator of absolute ETR above the use of the rPSII as
this approach can be used by any fluorometer measuring
variable fluorescence. Usage of F0 as a proxy for the PSII
light-harvesting cross-section resulted in a strong linear
correlations of F0 9 rETR with net O2 production. This
further suggests that state-transitions, and not cyclic elec-
tron transport in PSII, were the predominant photoregula-
tory mechanism. The improvable rETR versus O2
evolution correlation also shows that the mere usage of
effective quantum yields for rETR or true ETR (under
consideration of absorption factors), bears the potential of
inaccurate estimation of photosynthesis in species that
carry out significant state-transitions. ETR is frequently
presented under assumption of a fixed PS stoichiometry,
which can lead to erroneous interpretation of electron
transport rates in PSII. While the usage of rETR 9 F0 as a
proxy for photosynthesis resulted in an improved fit with
net O2 evolution, we note that the correlations coefficients
between the parameters are PF dependent, but more
accurate compared to rETR in all cases.
The present study shows a strong plasticity in distribu-
tion of harvested light energy toward PSI and PSII with
significant effects on variable fluorescence. State-transi-
tions facilitated stable effective quantum yields in PSII by
regulating its absorption cross-section rapidly and effec-
tively under extreme conditions of DIC depletion and DIC
re-supply. Compared to higher plants, D. tertiolecta
appears to retreat to state-transitions as a major NPQ
mechanism. Cyanobacteria, where state-transitions are a
major regulatory mechanism, keep a large PSII absorption
cross-section in the absence of DIC, while D. tertiolecta
uses qT to lower the light-harvesting area of PSII. The
reason for the different responses to DIC deprivation is
likely to be related to PQ pool reducing and oxidizing
processes. Here, the oxidation state of the PQ pool appears
to be affected by non-photosynthetic electron flow pro-
cesses in the species used in the present study.
The data presented show that electron transport rates and
energy partitioning parameters can be erroneous if species
exhibit a strong state-transitional regulation. Under con-
sideration of the absorption cross-section, which was esti-
mated by the fluorometer at room temperature, electron
transport correlated strongly with net oxygen evolution.
This shows that variable fluorescence measurements can be
used to measure photosynthesis even in the presence of
considerable state-transitions. The substantial capacity of
D. tertiolecta to cope with high and variable PF can be
explained by its plastic and rapid capacity to regulate the
absorption cross-section of PSII by state-transitions.
Acknowledgments SI was funded by Monash Graduate Scholarship
and Monash International Postgraduate Research Scholarship.
Experiments at JB’s laboratory were funded by the Australian
Research Council. Kevin Oxborough made constructive and helpful
comments to the data presented, which is very much appreciated.
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