Light-Harvesting Strategy during CO2-Dependent Photosynthesis in

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Light-Harvesting Strategy During CO-Dependent Photosynthesis in the Green Alga Chlamydomonas Reinhardtii Yoshifumi Ueno, Ginga Shimakawa, Chikahiro Miyake, and Seiji Akimoto J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b03404 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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The Journal of Physical Chemistry Letters

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Light-Harvesting Strategy during CO2-dependent Photosynthesis in the Green Alga

2

Chlamydomonas reinhardtii

3 4

Yoshifumi Ueno1, Ginga Shimakawa2, Chikahiro Miyake2, and Seiji Akimoto1,*

5 6

1

Graduate School of Science, Kobe University, Kobe 657-8501, Japan

7

2

Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan

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*

Corresponding author:

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Dr. Seiji Akimoto

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Graduate School of Science, Kobe University, Kobe 657-8501, Japan

12

Fax: +81-78-803-5705

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E-mail: [email protected]

14 15 16

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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To maximize the efficiency of photosynthesis, photosynthetic organisms must properly

3

balance their light-harvesting ability and CO2 utilization. However, the molecular

4

mechanisms of light harvesting under various CO2 conditions remain unclear. To reveal

5

these mechanisms, we performed a new analysis on cells of the green alga Chlamydomonas

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reinhardtii under different CO2 conditions. The analysis combines three kinds of

7

fluorometries: pulse-amplitude modulated fluorescence, steady-state fluorescence with

8

absolute intensity, and time-resolved fluorescence. Under low CO2 conditions, the main

9

regulatory mechanism was migration of light-harvesting chlorophyll-protein complex

10

(LHC) II from photosystem (PS) II to PSI. However, under CO2-deficient conditions with

11

carbon supplementation, some of the LHCII separated from the PSI and aggregated with

12

quenching. These different light-harvesting abilities of LHCII may play an important role in

13

the regulation of light harvesting in C. reinhardtii under various CO2 conditions.

14

15 16

Table of Contents Graphic

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1

Oxygenic photosynthetic organisms capture light energy by their light-harvesting antennas,

2

and transfer it to the reaction center (RC) of their photosystems (PSs), in which

3

photochemical reactions occur. The photochemical reaction in the RC induces a

4

photosynthetic electron flow and a proton gradient across the thylakoid membrane,

5

generating NADPH and ATP for CO2 assimilation in the Calvin–Benson cycle.1–3 To ensure

6

efficient oxygenic photosynthesis, the energy transfer from the light-harvesting antennas

7

must be balanced between two PSs (PSI and PSII). In nature, the constantly changing light

8

and CO2 conditions unbalance the excitation between the PSs, and reactive oxygen species

9

accumulate.4 To adapt to changing environments, photosynthetic organisms have developed

10

a variety of photoprotective mechanisms.5,6

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Energy-dependent quenching (qE) is a type of photoprotective mechanism that

12

depends on the ∆pH across the thylakoid membrane7, and results in the heat dissipation of

13

the excess energy that is not required for photosynthesis.5,6 In the green alga

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Chlamydomonas reinhardtii, a major qE effector consists of two light-harvesting complex

15

stress-related (LHCSR) proteins (LHCSR3 and LHCSR1).8 Both proteins connect to

16

light-harvesting chlorophyll-protein complex (LHC) II and function as a quencher.9–11 State

17

transition is another type of photoprotective mechanism, as first proposed in red and green

18

algae in 1969.12,13 In green algae, state-transition quenching (qT) occurs when LHCII

19

isolates from PSII, and the detached LHCII migrates to maintain the excitation balance

20

between both PSs.14 State transition is controlled by the redox state of the plastoquinone

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pool and is related to the phosphorylation of LHCII proteins.15

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Light-harvesting ability is closely related to CO2 concentration.16,17 Under

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CO2-deficient conditions, the PSII antenna size is reduced and the PSI/PSII fluorescence

24

intensity increases.18,19 Addition of a carbon source under low-CO2 conditions immediately

25

restores the photosynthetic activity and relaxes the non-photochemical quenching (NPQ).20



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As deficient CO2 slows the reoxidation of NADPH and leads to over-reduction of the

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plastoquinone pool, it is thought to induce a state transition. However, the molecular

3

mechanisms of light harvesting under various CO2 conditions remain unclear. In the present

4

study, we examined how the green alga C. reinhardtii regulates its light-harvesting ability

5

in

6

pulse-amplitude modulated (PAM) fluorometry, we measured the absolute intensities of all

7

fluorescence bands in the steady-state fluorescence spectra, and acquired the time-resolved

8

fluorescence (TRF) spectra.

response

to changing CO2 concentration.

Besides the

conventionally used

9

Figure 1 exhibits the time courses of the relative chlorophyll (Chl) fluorescence

10

and [O2] in HEPES–KOH (50 mM; pH 7.5) containing C. reinhardtii cells (30 µg Chl

11

mL−1) without NaHCO3 addition. The minimal fluorescence (Fo) was determined by

12

illumination with a measuring light (ML). The dark respiration rates and maximal Chl

13

fluorescence (Fm) of the cells were measured at time point A (dark condition). The actinic

14

light (AL) was then turned on to monitor the induction phase of photosynthesis. During this

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phase, the [O2] increased in the medium and the relative Chl fluorescence increased from its

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minimum (Fo) to steady-state (Fs). After determining the O2 evolution rates and the

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maximum variable fluorescence (Fm′) at B (the CO2-saturated condition), we opened the top

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of the reaction chamber, allowing the medium to equilibrate with air. Thereafter, the relative

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Chl fluorescences and O2 concentrations were monitored at regular intervals of saturated

20

pulse illumination (10 min; Figure 1). After reaching steady-state photosynthesis, the [O2]

21

decreased continuously and eventually stabilized at C (the CO2-limited condition). After

22

CO2 addition (10 mM NaHCO3), the [O2] was restored at D (the CO2-recovery condition).

23

When obtaining the O2 evolution rate at C and D, the top of the reaction chamber was

24

temporarily closed. The Fm′ (Fm), O2 evolution rates and relative quantum yields of PSII

25

(Y(II)) under the different conditions are summarized in Table 1. The O2 evolution rate and


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Y(II) decreased from B to C and increased at D, indicating that CO2 limitation suppressed

2

the photosynthesis but activity was recovered by adding CO2. The maximum fluorescence

3

did not obviously differ between A and B. The Fm′ decreased at C but recovered at D,

4

reaching approximately the same level as A and B. These results confirm that NPQ is

5

induced under low CO2 conditions and is relaxed by adding a carbon source, as previously

6

reported.16,19,20

7

To examine the energy distribution of the two PSs, we measured the low

8

temperature steady-state fluorescence spectra of cells at time points A, B, C and D in Figure

9

1. Figure 2 shows the steady-state fluorescence spectra of C. reinhardtii cells under

10

different conditions and 480-nm excitation. The spectra are normalized to the intensity of

11

the excitation light absorbed by the sample. The fluorescence peak at 688 nm originates

12

from PSII, and the peak at 714 nm derives from PSI. The PSI/PSII fluorescence intensity

13

ratios and absolute fluorescence intensities of PSII and PSI are summarized in Table 2. The

14

changes in the PSI/PSII ratios under CO2-limited and CO2-recovery conditions are

15

consistent with previous studies.17–20 The PSII and PSI intensities were almost identical at

16

time points A and B. At C, the PSII and PSI intensities decreased and increased,

17

respectively. The PSI intensity decreased at D, reaching the intensity of A and B, while the

18

PSII intensity increased above that of A and B. These results indicate that extra energy is

19

transferred to PSI under CO2-limited conditions and that the responses relax under

20

CO2-recovery conditions.

21

To better understand the origins of the absolute changes of the steady-state

22

fluorescence spectra under different conditions, we measured the TRF spectra at the four

23

time points and globally analyzed them to obtain the fluorescence decay-associated (FDA)

24

spectra. Figure 3 shows the FDA spectra of the C. reinhardtii cells under different

25

conditions. To fit the fluorescence decay and rise curves, we required six time constants:
5 ns. We first discuss the origin

2

of each FDA spectrum. The first (< 10 ps) and second (40–60 ps) FDA spectra,

3

corresponding to fast and slow energy transfer respectively, exhibit both positive and

4

negative amplitudes. In the first spectrum, the pairs of positive and negative amplitudes

5

below and above 690 nm indicate the energy transfer within the PSII–LHCII and PSI–LHCI

6

complexes, respectively. The positive amplitude below 680 nm is much larger than the

7

negative amplitude, suggesting that the light-harvesting antennas are rapidly quenched. In

8

the second FDA spectrum, the positive peak at 676 nm and negative peaks at 688 and 710

9

nm correspond to energy transfer from LHC to PSII or PSI, respectively.21 The third FDA

10

spectra (130−140 ps) display two positive peaks. The first peak (at 686 nm) originates from

11

energy trapping on PSII,22 and the second (at 704 nm) originates from PSI red-Chl. At D,

12

the third spectrum exhibits a negative amplitude around 725 nm. Therefore, over this time

13

constant, the energy transfer from LHC to red-Chl occurs.23 The subsequent FDA spectra

14

exhibit only positive amplitudes, indicating fluorescence decay. The peak wavelengths in

15

the PSII and PSI fluorescence are red-shifted from the fourth (440–480 ps) to the sixth (> 5

16

ns) spectra, suggesting the presence of red-Chls with different energy levels within PSII and

17

PSI.

18

Next, we discuss the changing energy transfer under different CO2 conditions.

19

When transiting to CO2-saturated conditions (A→B), the negative amplitude in the PSII

20

fluorescence band decreases and increases in the first and second FDA spectra, respectively,

21

whereas that in the PSI fluorescence band increases and decreases. These results suggests

22

that the total amount of energy transferred to the PSs is almost identical at A and B (Tables

23

1 and 2 and Figure 2), although the contributions of the fast and slow energy transfers from

24

LHC to PSs are changed by the light irradiation. The transition to CO2-limited conditions

25

(B→C) enhances the negative amplitude in the PSI fluorescence band (c.f. second FDA


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1

spectra of B and C). Coupled with this behavior, the positive amplitude in the PSI

2

fluorescence band becomes more pronounced in the fourth to sixth FDA spectra.

3

Conversely, the positive amplitude in the PSII fluorescence band reduces in the third and

4

fourth FDA spectra. The intensities of PSII and PSI decreased and increased, respectively,

5

during the transition (Table 2). Previous studies examined only the changes in relative

6

fluorescence intensities from the PSs.17–20 Whereas these studies confirmed that the

7

PSII/PSI ratio decreases under low CO2 conditions, the present study identified the

8

responses of the individual PSII and PSI fluorescences, and confirmed the migration of

9

LHCII from PSII to PSI, under such conditions. This was achieved by combining the

10

absolute fluorescence intensity measurements with TRF measurements. LHCSR proteins

11

are the main qE component in C. reinhardtii,8 but their effect under CO2-limited conditions

12

is probably restricted. This has been evidenced by decreased time constants and changes in

13

the relative amplitudes, which suggest qE quenching by the LHCSR proteins.9–11 However,

14

neither of these changes were observed in the present study (Figure 3). Our measurements

15

were performed before the LHCSR3 proteins could appreciably accumulate;24 moreover,

16

LHCSR proteins do not accumulate under AL and other red light sources (620 < λ < 695

17

nm).25,26 When the photosynthetic activity is reduced, the reduction of PSII fluorescence

18

might arise through photoinhibition of PSII. However, the photosynthetic activity is rapidly

19

relaxed by adding NaHCO3 (Table 1). If the reduction of PSII fluorescence were related to

20

PSII photoinhibition, the relaxation would take many minutes to several hours.27 Moreover,

21

there is no large difference in light intensity between AL (220 µmol photons m−2 s−1) and

22

growth light (150 µmol photons m−2 s−1). Therefore, we infer that LHCII migration is the

23

main regulatory mechanism of light harvesting under CO2-limited conditions. When

24

recovering the CO2 conditions by adding NaHCO3 (C→D), the negative amplitude

25

decreases in the PSI fluorescence band of the second FDA spectra, and the positive



1

amplitude decreases in the third to fifth FDA spectra. Collectively, these changes reduce the

2

PSI fluorescence in the steady-state fluorescence spectra (Table 2). At this time, LHCII

3

detaches from PSI. Meanwhile, the PSII fluorescence amplitude reduces in the second and

4

third FDA spectra, indicating that less energy is transferred from LHCII to PSII during CO2

5

recovery than CO2 depletion. These results suggest that when LHCII separates from PSI, it

6

does not always connect to PSII. During the C→D transition, the amplitudes of the PSII

7

fluorescence bands increase in the fourth to sixth FDA spectra. Such enhancement of the

8

long-lived fluorescence components in the PSII fluorescence band increases the Fm′ and

9

PSII intensities in the steady-state fluorescence spectra (Tables 1 and 2). In the closed state

10

of PSII RC, the contribution of the long-lived fluorescence components will increase.28

11

However, the oxygen evolution rate and Y(II) are larger than under the CO2-saturated

12

condition (Table 1), which excludes a closed state of PSII RC. The LHCII monomer

13

exhibits a fluorescence band around 680 nm when detached, but a red-shifted fluorescence

14

peak when aggregated.21,29,30 Therefore, by combining PAM fluorometry, absolute

15

fluorescence intensity measurements, and TRF measurements, we can deduce that the

16

long-lived fluorescence components under the CO2-recovery condition originate from

17

LHCII aggregations. Consistent with this conclusion, recent studies on C. reinhardtii 21,31,32

18

have reported higher quenching ability in LHCII aggregates than in monomeric and trimeric

19

LHCII.29,30 Moreover, the quenching ability of LHCII aggregates dynamically responds to

20

light.33,34 By executing two kinds of light-harvesting ability in its LHCII, the green alga C.

21

reinhardtii might adapt its light harvesting to various CO2 conditions. The combination of

22

fluorometries used in this study will be a useful analysis to reveal light-harvesting abilities

23

in the photosynthetic organisms.

24 25

EXPERIMENTAL METHODS


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Cells of the green alga C. reinhardtii were maintained under a fluorescence lamp (25 °C, 50

2

µmol photons m−2 s−1) on tris-acetate-phosphate agar plates. Prior to measurement, the cells

3

were inoculated into a high-salt liquid medium,35 and photoautotrophically grown with

4

agitation at 100 rpm under high CO2 (2%) in light/dark conditions (25 °C, 16 h light at 150

5

µmol photons m−2 s−1 from fluorescence lamp; 23 °C, 8 h dark).

6

The oxygen evolution and Chl fluorescence were measured simultaneously in a

7

reaction mixture (2 mL) containing C. reinhardtii cells (30 µg Chl mL−1) in 50 mM

8

HEPES–KOH (pH 7.5). The total Chl was determined by extraction with 100% methanol.36

9

During the measurements, the reaction mixture was continuously stirred with a magnetic

10

microstirrer. The cells were illuminated with AL (red light, 620 < λ < 695 nm: 220 µmol

11

photons m−2 s−1) at 25 °C. The [O2] was continuously monitored by an oxygen electrode

12

(Hansatech, King’s Lynn, UK). The cuvette was maintained open to allow O2 and CO2

13

diffusion between the medium and atmosphere,20,37 with temporary closures (1–3 min) for

14

measuring the O2 evolution rates. The relative Chl fluorescence was measured by a

15

PAM-Chl fluorometer (PAM-101, Walz, Effeltrich, Germany).20,38 Pulse-modulated

16

excitation was sourced from a LED lamp with peak emission at 650 nm. The modulated

17

fluorescence was measured at λ > 710 nm (Schott RG9 long-pass filter). The Fm was

18

determined by subjecting dark-acclimated cells to 1000-ms pulses of saturated light (10,000

19

µmol photons m−2 s−1). The Fs was monitored under AL, with saturating light applied at

20

arbitrary intervals to determine the Fm′. Y(II) was defined as (Fm′ − Fs) / Fm′. The

21

fluorescence terminology in this study follows that of Reference 39.

22

The steady-state fluorescence spectra at 77 K were measured by a

23

spectrofluorometer equipped with an integrating sphere (JASCO FP-6600/ILFC-543L). The

24

excitation wavelength was set to that of LHC (480 nm). The TRF spectra were measured by

25

a time-correlated single-photon counting system, also operated at 77 K40 at an excitation



1

wavelength of 408 nm. The repetition rate of the pulse train was 5 MHz. The time interval

2

for data acquisition was set to 2.44 ps/channel (total time window = 10 ns). The

3

fluorescence kinetics were measured at 2-nm intervals (660–750 nm). The measured

4

fluorescence rise and decay curves were fitted globally by sums of exponentials with

5

common time constants as follows:

6

6  t  F(t, λ ) = ∑ An (λ ) exp  −   τn  n=1

(1)

7

Here, An(λ) is the amplitude (as a function of detection wavelength) and τn is the time

8

constant. By plotting the amplitudes of these exponential components versus the emission

9

wavelength, we obtained the FDA spectra.

10


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1

ACKNOWLEDGEMENTS

2

This work was supported by JSPS KAKENHI (Grant No. 21570041 to C.M. and G.S., and

3

No. 16H06553 to S.A.), and by JST CREST (Grant No. AL65D21010 to C.M.). We thank

4

Leonie Pipe, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this

5

manuscript.

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Quenching in Aggregates of the LHCII Chlorophyll-Protein Complex: a Time-Resolved

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Fluorescence Study. Biochim. Biophys. Acta, Bioenerg. 1993, 1141, 23–28.

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Quenching of Chlorophyll a Fluorescence in the Aggregates of LHCII: Steady State

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Petroutsos, D.; Garab, G.; Finazzi, G.; et al. Chloroplast Remodeling During State


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Transitions in Chlamydomonas reinhardtii as Revealed by Noninvasive Techniques in vivo.

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1

Table 1. Photosynthetic parameters of C. reinhardtii cells under dark (A),

2

CO2-saturated (B), CO2-limited (C), and CO2-recovery (D) conditions

3

O2 exchange rate

Sample

Fm′ (Fm)

A

10 ± 1

−10 ± 2

0

B

10 ± 1

27 ± 3

0.42 ± 0.03

C

8.3 ± 0.5

3.1 ± 1.4

0.33 ± 0.04

D

9.9 ± 0.4

42 ± 5

0.48 ± 0.03

(µmol O2 [mg Chl]−1 h−1)

Y(II)

4 5

The O2 uptake rates under dark conditions (time point A in Figure 1) were determined

6

before ML illumination. The O2 evolution rates and Chl fluorescences were simultaneously

7

measured at time points B, C, and D in Figure 1. Y(II) was calculated for the corresponding

8

measurements of Fm′ as (Fm′ − Fs) / Fm′. All values are the means ± SDs of six independent

9

measurements.

10



Page 18 of 22

1

Table 2. PSI/PSII fluorescence intensity ratios and fluorescence intensities of PSII and

2

PSI in C. reinhardtii cells under dark (A), CO2-saturated (B), CO2-limited (C), and

3

CO2-recovery (D) conditions

4

Sample

PSI/PSII

A

Intensity PSII

PSI

1.08 ± 0.36

11.8 ± 2.5

14.4 ± 0.8

B

1.07 ± 0.26

11.6 ± 1.8

14.4 ± 1.3

C

1.27 ± 0.21

10.4 ± 1.0

15.1 ± 1.1

D

0.93 ± 0.16

13.1 ± 1.6

14.6 ± 0.9

5 6

The PSI fluorescence intensity was determined by subtracting 0.2 × PSII fluorescence

7

intensity from the observed fluorescence intensity at the PSI peak, as described

8

previously.41 The PSI/PSII fluorescence intensity ratios and fluorescence intensities of PSII

9

and PSI are the averages of six biological replicates. Individual values are based on Figure

10

2.

11


Page 19 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

FIGURE CAPTIONS

2 3

Figure 1. Time courses of [O2] (red) and relative Chl fluorescence (black) in C. reinhardtii

4

cells. The media containing the cells (30 µg Chl mL−1) was initially maintained in the dark,

5

then successively illuminated with measuring light (ML) and red actinic light (AL; 220

6

µmol photons m−2 s−1) as indicated by the arrows. In the blue shaded regions, the oxygen

7

electrode chamber was closed and the measurement time scale was reduced to 1/10.

8

NaHCO3 (10 mM) was added as indicated. Shown are representative data of six

9

experiments.

10 11

Figure 2. Steady-state fluorescence spectra of C. reinhardtii cells under different conditions

12

at 77 K. The excitation wavelength was 480 nm. The spectra are normalized to the intensity

13

of the excitation light absorbed by the sample. The cells were sampled under dark (black),

14

CO2-saturated (red), CO2-limited (green), and CO2-recovery (blue) conditions. The spectra

15

are the averages of six biological replicates.

16 17

Figure 3. Fluorescence decay-associated spectra of C. reinhardtii cells under different

18

conditions at 77 K. The excitation wavelength was 408 nm. The amplitudes are normalized

19

to the integrated intensity of the steady-state fluorescence spectrum reconstructed from the

20

fluorescence decay-associated spectrum. The cells were sampled under dark (black),

21

CO2-saturated (red), CO2-limited (green), and CO2-recovery (blue) conditions. The spectra

22

are the averages of three biological replicates.



Fig. 1 202x124mm (72 x 72 DPI)

ACS Paragon Plus Environment

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Fig. 2 194x150mm (288 x 288 DPI)



Fig. 3 173x156mm (288 x 288 DPI)


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Light-Harvesting Strategy during CO2-Dependent Photosynthesis in

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