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: jacco.kromkamp@nioz.nl

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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