Defining the Far-Red Limit of Photosystem II in Spinach C W

Anders Thapper, Fikret Mamedov, Fredrik Mokvist, Leif Hammarstrom, and Stenbjorn Styring1

Department of Photochemistry and Molecular Science, Angstrom Laboratory, Uppsala University, SE-751 20 Uppsala, Sweden

The far-red limit of photosystem II (PSII) photochemistry was studied in PSII-enriched membranes and PSII core

preparations from spinach (Spinacia oleracea) after application of laser flashes between 730 and 820 nm. Light up to 800

nm was found to drive PSII activity in both acceptor side reduction and oxidation of the water-oxidizing CaMn4 cluster. Far-

red illumination induced enhancement of, and slowed down decay kinetics of, variable fluorescence. Both effects reflect

reduction of the acceptor side of PSII. The effects on the donor side of PSII were monitored using electron paramagnetic

resonance spectroscopy. Signals from the S2-, S3-, and S0-states could be detected after one, two, and three far-red

flashes, respectively, indicating that PSII underwent conventional S-state transitions. Full PSII turnover was demonstrated

by far-red flash-induced oxygen release, with oxygen appearing on the third flash. In addition, both the pheophytin anion

and the Tyr Z radical were formed by far-red flashes. The efficiency of this far-red photochemistry in PSII decreases with

increasing wavelength. The upper limit for detectable photochemistry in PSII on a single flash was determined to be 780 nm.

In photoaccumulation experiments, photochemistry was detectable up to 800 nm. Implications for the energetics and

energy levels of the charge separated states in PSII are discussed in light of the presented results.

INTRODUCTION

The action spectrum of oxygenic photosynthesis has been

extensively studied, and the overall quantum yield sharply de-

creases at wavelengths above 680 nm (Emerson and Lewis,

1943; Emerson et al., 1957; Blankenship, 2002). This red drop is

considered to be due to an unbalance of excitation between

photosystem I (PSI) and photosystem II (PSII). PSII is less

efficient in using long wavelength light than is PSI. Therefore,

there is a limited supply of electrons available for PSI above 680

nm, decreasing the photosynthetic yield in the far-red region.

The antenna system of PSI contains a small amount (3 to 10%)

of so-called red or low energy chlorophylls. These have their

absorption maxima in the red region (the Qy band) shifted to

beyond 700 nm (Gobets and van Grondelle, 2001; Karapetyan

et al., 2006). Apart from capturing far-red photons, they have

been ascribed a role in photoprotection (Gobets and van

Grondelle, 2001). The red chlorophylls originate from intermo-

lecular interactions between chlorophyll a molecules forming

strongly excitonically coupled dimers (or larger aggregates). This

intermolecular interaction does not require that the chlorophylls

are in contact with each other, only that they are electronically

interacting. The localization of the red chlorophylls in PSI is not

unambiguously determined (Jordan et al., 2001; Karapetyan

et al., 2006). There is no known corresponding system with such

far-red absorbing chlorophylls in PSII, and the available x-ray

crystal structures have not revealed any strongly coupled chlo-

rophylls in the PSII antenna (Ferreira et al., 2004; Loll et al., 2005).

Instead, the common view is that the primary donor chlorophylls,

referred to as P680, define the red-edge limit of excitation and,

therefore, the practical energy limitation for driving water oxida-

tion and plastoquinone reduction.

Some organisms can use light of longer wavelengths for

photosynthesis. For example, >95% of the antenna pigments

of PSII in the cyanobacterium Acaryochloris marina are chloro-

phyll d molecules that absorb light further to the red than

chlorophyll a (Miyashita et al., 1996, 1997, 2003). The nature of

the chlorophylls in the PSII reaction center of A. marina is still

debated. This also holds for the absorption associated with the

primary donor that has been placed at 713 nm (Tomo et al., 2007)

or at 725 nm (Itoh et al., 2007).

Surprisingly, oxygen evolution from PSII has also been re-

ported above 700 nm for green algae (GreenbaumandMauzerall,

1991) and higher plants (Pettai et al., 2005a, 2005b), both of

which have the more common chlorophyll a/b containing an-

tenna. For the green algae Chlorella vulgaris, 723-nm light

promoted the same maximum oxygen evolution (within 10%)

as shorter wavelengths (Greenbaum and Mauzerall, 1991). Oxy-

gen evolution at 723 nm followed the same light saturation

behavior as at shorter wavelengths, but a much higher photon

flux was needed to achieve maximal activity, indicating that the

effective optical cross section for the antenna wasmuch smaller.

The physiological significance is also clear, and oxygen evolution

and variable chlorophyll a fluorescence have been observed in

sunflower (Helianthus annuus) and bean (Phaseolus vulgaris) leaves

using wavelengths up to 780 nm (Pettai et al., 2005a, 2005b).

Charge separation can also be induced in PSII core complexes at

cryogenic temperatures up to 730 nm (Hughes et al., 2006a).

Here, we used spinach (Spinacia oleracea) PSII preparations,

with different degrees of purification, to focus on far-red light–

induced PSII activity in vitro. Well-defined laser flashes between

1Address correspondence to stenbjorn.styring@fotomol.uu.se.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Stenbjorn Styring(stenbjorn.styring@fotomol.uu.se).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.108.064154

The Plant Cell, Vol. 21: 2391–2401, August 2009, www.plantcell.org ã 2009 American Society of Plant Biologists

730 and 820 nm were used to study PSII photochemistry and

electron transfer. Variable chlorophyll a fluorescence and elec-

tron paramagnetic resonance (EPR) spectroscopy were used to

follow reactions on both the acceptor and donor side of PSII. We

also detected a flash-dependent oxygen evolution pattern using

far-red laser flashes. Implications for the energetics in PSII to

accommodate the presented results are discussed.

RESULTS

Reduction of the Acceptor Side: Effect of Far-Red

Preillumination on Variable Fluorescence

Appearance of variable fluorescence (FV) is a consequence of the

charge separation in PSII that results in reduction of QA- (Renger

et al., 1995). Analysis of the flash-induced FV decay kinetics

provides information about the activity of the PSII acceptor side

(Joliot et al., 1971; Crofts and Wraight, 1983; Robinson and

Crofts, 1983; Renger et al., 1995). In our samples, the decay

kinetics could be resolved in three phases (Figure 1A; t1 = 1.4 ms

[38%], t2 = 26 ms [38%], and t3 = 4.8 s [24%], with relative

amplitudes within brackets). These phases are normally as-

signed to electron transfer from QA- to QB (QB

-), electron transfer

fromQA- to QB in a PSII center whereQB has to bind to theQB site

prior to electron transfer, and recombination of QA- with the

donor side, respectively. The two fast phases, t1 and t2, involved

;75% of the FV.

The application of a series of visible light flashes to PSII

membranes builds up an increased FV, compared with the effect

of only one flash (Mamedov et al., 2000; Pospısil and Dau, 2000).

This flash-dependent enhancement of FV is not observed in

intact thylakoidmembranes and has been attributed to reduction

of the limited plastoquinone (PQ) pool in the PSII membranes

leading to inefficient forward electron transfer from QA-.

We used far-red monochromatic laser flashes (730 to 820 nm)

to determine the upper limit of this flash-dependent build-up of

FV. In the experiment, preillumination with laser flashes of varying

wavelengths was used to induce the effect. This was thereafter

observed as FV induction and decay in a standard fluorescence

setup (see Methods).

Figure 1A shows the FV decay kinetics recorded in a control

(dark adapted) sample and a sample preilluminated with 100

laser flashes at 750 nm. The FV obtained after the preillumination

was about 2 times higher than that of the control sample andwas

much closer to the maximal FV level that can be obtained by

reduction with 10mMdithionite. The initial fluorescence level (F0)

also increased by about 2 times after the preillumination with 100

flashes at 750 nm.

The wavelength dependence of this preillumination-induced

enhancement of FV and F0 was studied in the 650- to 820-nm

region (Figure 1B). In samples preilluminated with 10 flashes

(25 mJ) between 650 and 740 nm, the FV was enhanced to near

the maximum level, while the effect decreased between 740 and

810 nm. Importantly, the enhanced FV was observed above

760 nm if the number of preillumination flashes was increased

(Figure 1B). Above 810 nm, no FV enhancement could be

observed. The same wavelength dependence also holds for

the increase of F0 by the preillumination flashes (Figure 1B). As

will be shown below, far-red flashes of 25 mJ (the maximum

power available with our laser for the whole wavelength interval)

are saturating for wavelengths up to 750 nm, but not for the

wavelengths above.

Preillumination with the far-red laser flashes also changed the

decay kinetics of FV (Figure 1A) substantially. When FV was

induced to its maximal level, three decay phases could still be

resolved (t1 = 1.0 ms [27%], t2 = 200 ms [17%], and t3 = 2.5 s

[56%]). The relative amplitude of the first two phases had

decreased from ;75% in the dark control to <50% of FV in the

preilluminated sample. In addition, t2 had increased by almost

one order of magnitude. The slow phase, which reflects recom-

bination between QA- and the water oxidizing complex (WOC),

was dominating the fluorescence decay (56%).

If the sample was incubated in the dark for 20min at 208C after

the preillumination, the effect of the far-red flashes completely

disappeared, and FV, F0, and Fv decay kinetics returned to

control values.

Although these complex effects on FV and F0 induced by far-

red preillumination of PSII have not been described earlier, they

have direct analogies to the effects seen by preillumination with

visible light on similar PSII preparations (Mamedov et al., 2000;

Pospısil and Dau, 2000). This allows assignment of the effects,

and it is highly likely that both the considerable increase of the FVand F0 and the slowed decay kinetics of QA

- reflect that the

limited PQ pool has been reduced by far-red laser flashes. Ten

laser flashes atwavelengths up to 740 nmwere enough to reduce

the PQ pool. The PQ pool was fully reduced by 900 flashes at

wavelengths up to 775 nm and partly reduced with 900 flashes

up to 800 nm. Thus, we conclude that the application of far-red

laser flashes up to 800 nm induces sufficient electron transport to

reduce both the acceptor side of PSII and the PQ pool.

This conclusion was further verified by the addition of ferricy-

anide prior to the far-red laser flashing. This completely abolished

Figure 1. Changes in FV after Preillumination of PSII Membranes with

Laser Flashes.

(A) Flash-induced fluorescence decay traces before (closed circles,

dark) and after (open circles, 750 nm) the application of 100 flashes at

750 nm. The arrow indicates the time of the actinic flash.

(B) F0 (closed squares) and FV (open squares) levels in samples that were

exposed to 10 (left) or 900 (right) laser flashes at the indicated wave-

lengths. The F0 (dotted line) and FV (broken line) levels before the

application of flashes are indicated.

2392 The Plant Cell

the enhancement of FV and the slowing of the FV decay kinetics

(see Supplemental Figure 1 online). We interpret this to reflect

ferricyanide functioning as an effective oxidant of QA-, thereby

preventing reduction of the acceptor side and the PQ pool. This

maintains FV on a low level and leaves the QA- reoxidation kinetics

unaffected.

Direct Detection of Donor Side Reactions: Oxidation of the

CaMn4 Cluster

The observation of far-red-induced photochemistry on the ac-

ceptor side of PSII prompted us to look for electron transfer at

the donor side as well. Many intermediates in the water oxida-

tion S-cycle of the CaMn4 cluster can be trapped by freezing,

followed by EPR at cryogenic temperatures. The well-studied

multiline EPR signal from the S2-state is detected at liquid helium

temperatures (Dismukes and Siderer, 1981; Miller and Brudvig,

1991). This also holds for the so-called split EPR signals origi-

nating from the S1-, S3-, and S0-states, which are inducible by

visible or IR (S3 only) illumination at 5 to 10K of PSII samples

poised in the respective S-state (Nugent et al., 2002; Zhang

and Styring, 2003; Zhang et al., 2004; Petrouleas et al., 2005;

Havelius et al., 2006; Su et al., 2007). We studied the S-cycle

advancement in samples set in the S1-state and then exposed to

0 (dark), 1, 2, or 3 flashes at 750 nm at 208C. The different

S-states were then recognized by the observation of their re-

spective EPR signals. We also exposed a sample to 10 flashes at

750 nm to scramble (if possible) the S-states.

Figure 2A shows the S2-state multiline EPR signal in samples

exposed to 1, 2, 3, or 10 flashes. One flash at 750 nm resulted in a

significant multiline EPR signal (Figure 2A, 1 fl) corresponding to

45% 6 5% of the maximally inducible signal (using one saturat-

ing 800-mJ laser flash at 532 nm), indicating that the flash at 750

nm induced the S2-state in;45%6 5% of the PSII centers. The

rest of the centers remained in theS1-state, whichwas confirmed

by the observation of a large split EPR signal from the S1-state

(Figure 2B, spectrum 1). The split S1 EPR signal corresponded

well to the 55% 6 5% of centers that had remained in the

S1-state when compared with the split S1 EPR signal from a

sample poised in the S1-state (Figure 2B, 0 fl). No sign of the split

EPR signal from the S3-state was observed in this sample (Figure

2C, 1 fl). In a sample exposed to two flashes, the Split S1 (Figure

2B, 2 fl), the S2-state multiline (Figure 2A, 2 fl), and the split S3

EPR signals (Figure 2C, 2 fl) all were observed. This reflects

turnover all the way to the S3-state.

In addition to the split EPR signal from the S3-state, there are a

few other EPR signals connected to the S2- and S3-states of the

CaMn4 cluster that are reported to be induced by excitation or

spin transitions by far-red or IR wavelengths. However, these

effects were reported only at cryogenic temperatures (<150K) at

conditions in which no charge separation can occur (Boussac

et al., 2005). Our observed effect does not involve spin transitions

in the CaMn4 cluster at low temperatures. This is further sub-

stantiated bymeasurements of far-red-induced electron transfer

in PSII preparations lacking the CaMn4 cluster (see below).

In the sample exposed to three flashes, split EPR signals from

all of the S1-, S3-, and S0-states states (Figures 2B and 2C, 3 fl)

were observed together with the multiline EPR signal from the

S2-state (Figure 2A, 3 fl), clearly indicating that the full S-cycle

could be completed by only a few flashes at 750 nm. In the

sample that was given 10 flashes at 750 nm, all of the EPR signals

had considerable amplitudes (Figure 2, 10 fl spectra). Clearly, the

10 flashes at 750 nm led to almost complete scrambling of the

S-states in the WOC. The miss factor on these far-red flashes is

Figure 2. EPR Signals from PSII Membranes after Far-Red Turnover Flashes, Representing Intermediates in the S-State Cycle in the WOC.

(A) Light minus dark difference spectra of the multiline EPR signal from the S2-state in samples exposed to 1, 2, 3, or 10 flashes (fl) at 750 nm. The

asterisks indicate the peaks used for signal quantification.

(B) EPR spectra from the S1- (Y) and the S0-states (*). The samples were exposed to 0, 1, 2, 3, or 10 flashes at 750 nm. Contributions from the QA--Fe2+

signal are indicated with bars.

(C) The split EPR signal from the S3-state (the asterisk indicates the trough of the signal) in samples exposed to 0, 1, 2, 3, or 10 flashes at 750 nm.

The EPR spectra in (B) and (C) were obtained as described in Methods. EPR conditions were as follows: temperature, 7K (A) and 5K ([B] and [C]);

microwave power, 10 mW (A) and 25 mW ([B] and [C]); modulation amplitude, 20 G (A) and 10 G ([B] and [C]); microwave frequency, 9.28 GHz.

The Far-Red Limit of Photosystem II 2393

high. This reflects the fact that the PSII sample absorbs ineffi-

ciently at 750 nm and that our laser power is too low to allow light

saturation in the concentrated EPR samples. Previously, we have

reported our initial observations of WOC turnover using exten-

sive 750-nm laser flashing. In samples exposed to >300 laser

flashes, we detected centers in the S1-, S2-, and S0-states by

EPR spectroscopy (Thapper et al., 2008). Thus, the early results

involved studies of only scrambled S-states, not allowing quan-

titative analysis of single turnover photochemistry.

Single flashes between 730 and 790 nm were applied to PSII,

and the multiline EPR signal from the S2-state was measured to

investigate further the far-red edge of the action spectrum of PSII

(Figure 3A). The S1 to S2 turnover in a single flash was high at

730 nm (85% 6 5% compared with the multiline signal induced

by a saturating flash at 532 nm) but decreased as the wavelength

was increased. At 790 nm, the amount of themultiline EPR signal

from the S2-state was hardly detectable after one flash (see

Supplemental Figure 2 online). In a separate sample given 100

flashes at 790 nm, both the S2-state multiline (Figure 3A, 790 nm

100 fl) and the split S3 EPR signals (Figure 3A, inset) were induced

to significant extents. By contrast, in a sample given 100 flashes

at 810 nm, only the split EPR signal from the S1-state, and no

S2-state multiline EPR signal (Figure 3A, 810 nm 100 fl), could be

detected, indicating that the CaMn4 cluster did not turn over at

this wavelength. This puts the far-red limit for photochemical

oxidation of the CaMn4 cluster using multiple flashes at around

800 nm. This is very similar to the limit for the photochemical

reduction of the acceptor side of PSII obtained from the fluores-

cence experiments (Figure 1B).

Minimal PSII core preparations from plants with fully active

oxygen evolution contain the D1/D2 heterodimer, the chlorophyll

binding proteins CP43 and CP47, cyt b559, the psbO protein, and

a set of small subunits (Smith et al., 2002). The PSII core

preparation does not contain LHCII or the minor antenna com-

ponents CP24, CP26, or CP29. To elucidate further the location

of the far-red absorbing species in PSII, we exposed a PSII core

sample to 1 and 100 flashes at 750 nm. In both cases, we

observed the induction of the multiline EPR signal from the

S2-state (Figure 3B), which demonstrated that far-red photo-

chemistry occurs also in PSII core preparations. The multiline

EPR signal from the S2-state after one flash corresponded to 10

to 15% of the maximum inducible signal (Figure 3B, top spec-

trum). One hundred flashes gave ;30% of the multiline EPR

signal from the S2-state (Figure 3B, bottom spectrum). Seem-

ingly, the turnover capability is considerably lower (no acceptor

was used in this experiment) than the almost 50% turnover that

was observed after a single 750-nm flash in the PSII membranes.

However, in the context of this study, the observation of any

S2-state multiline signal at all is important because it shows that

light at 750 nmcan lead to photochemical oxidation of theCaMn4cluster in a PSII core preparation lacking a large part of the

chlorophyll antenna.

Direct Detection of Donor Side Reactions: Tyr Z Oxidation

PSII contains two redox active Tyr residues, YZ and YD. The light-

induced formation and subsequent decay of the radical from YZ,

YZd

, can be studied by EPR spectroscopy in PSII membranes in

Figure 3. The Multiline EPR Signal from the S2-State after Application of

Far-Red Flashes of Different Wavelengths.

(A) The multiline EPR signal from the S2-state in PSII membrane samples

exposed to one flash (25 mJ) at 730, 750, and 770 nm and in samples

exposed to 100 flashes at 790 and 810 nm. The vertical lines indicate the

position of five peaks in the multiline EPR signal, and the asterisks

indicate the three peaks used for signal quantification. The amounts of

PSII centers in the S2-state after a single flash at 730, 750, or 770 nm

were 87% 6 5%, 45% 6 5%, and 14% 6 10%, respectively (the S2-

state multiline EPR signal induced by a saturating flash at 532 nm is taken

as 100%). The inset shows the split EPR signal from the S3-state

(obtained as in Figure 2C) from the sample exposed to 100 flashes at

790 nm.

(B) The multiline EPR signal from the S2-state in PSII core samples

exposed to one flash (25 mJ) or 100 flashes at 750 nm. EPR conditions

were as follows: temperature, 7K, (inset 5K); microwave power, 10 mW

(inset 25 mW); modulation amplitude, 20 G, (inset 10 G); microwave

frequency, 9.28 GHz.

2394 The Plant Cell

which theCaMn4 cluster has been removed by Tris washing. This

treatment significantly (100-fold) slows down YZd

reduction and

allows its detection by EPR spectroscopy at room temperature

(Babcock et al., 1976, 1989; Roffey et al., 1994). We used far-red

laser flashes to induce YZ oxidation in Tris-washed samples

between 730 and 790 nm (Figure 4A). A 730-nm flash induced a

YZd

radical EPR signal corresponding to 55% 6 3% of the YZd

EPR signal induced by a 532-nm flash (70 mJ). The amount of

radical induced decreased with increasing wavelength and,

at 780 nm, the signal was at the detection limit, even after

averaging 250 traces (Figure 4A, inset, cyan trace). Both the

shape of the YZd

signal and the decay kinetics of the signal in the

dark were identical to a YZd

signal induced by a 532-nm laser

flash (Figure 4A).

Thus, the application of far-red light resulted in YZd

radical

induction in Tris-washed PSII membranes in which the CaMn4cluster was destroyed and removed. This rules out the possibility

of Mn excitation and Mn-driven reactions as an explanation for

the far-red photoactivity.

Direct Detection of Acceptor Side Reactions: FV Induced by

Single Far-Red Laser Flashes

It is also possible to induce directly QA reduction using far-red

laser flashes and measure the resulting FV. The power depen-

dence of FV induction by a single flash, in the far-red region

(Figure 4B) and at 532 nm (see Supplemental Figure 3 online),

was measured using a double modulation fluorometer. The laser

flash was used as the actinic flash to induce PSII photochem-

istry, and the fluorometer’s internal LEDs were used as measur-

ing flashes. The measurements were done in the presence of

DCMU. For all wavelengths up to 760 nm, the power depen-

dence is linear at the lower laser powers and saturates at the

higher powers; ;20 times stronger flashes must be used to

reach saturation at 730 nm compared with 532 nm. Above 760

nm, the level of FV induction is very low even when the strongest

available laser flash is used and saturation cannot be reached

with our laser system (Figure 4B, 770 nm, orange, and 780 nm,

cyan). The limit at which the effect of a single flash could be

observed was determined to be 780 nm. Importantly, the FV level

at saturation decreases strongly with increasing wavelength (see

Discussion).

Reduction of the Acceptor Side: Photoaccumulation of the

Pheophytin Anion

Addition of dithionite in millimolar concentrations to PSII mem-

branes efficiently reducesQA. This treatmentmakes it possible to

accumulate a pheophytin anion (Phe-) under intense illumination

at room temperature because forward electron transfer to the

Figure 4. Characterization of Far-Red-Induced Redox Activity in PSII.

(A) Kinetic EPRmeasurements of the YZ

d

radical induced by far-red flashes in Tris-washed PSII membranes. The traces were induced by laser flashes at

532 nm (black), 730 nm (red), 740 nm (green), 750 nm (blue), 760 nm (pink), 770 nm (orange), 780 nm (cyan), and 790 nm (grey). The inset shows an

expanded view of YZ

d

induction at higher wavelengths (760 to 790 nm). The traces are normalized for the laser flash power and number of

accumulations. Details of the experiment are given in Methods. EPR conditions were as follows: temperature, 293K; microwave power, 20 mW;

modulation amplitude, 10 G; time constant, 2.56 ms; microwave frequency, 9.76 GHz.

(B) The fraction of FV induced after a single flash in the presence of DCMU as a function of the laser flash power at 730 nm (red), 740 nm (green), 750 nm

(blue), 760 nm (pink), 770 nm (orange), or 780 nm (cyan). The maximum FV (100%) was obtained by a 639-nm flash (LEDs, 50 ms). The full lines are a fit of

the expression FV = FV(sat) (1� e-N) to the experimental data. FV(sat) is the saturating FV signal, andN = fabs nhy/nPSII, where fabs is the fraction of photons

absorbed, nhy is the number of photons in a flash, and nPSII is the number of PSII reaction centers (seeMethods). The dashed lines are straight lines given

as an estimation of the FV induction at 770 and 780 nm.

(C) Photoaccumulation of Phe- radical in dithionite-reduced PSII membrane samples detected with EPR after 650 flashes at 532 nm (black), 4000

flashes at 730 nm (red), 4000 flashes at 750 nm (blue), and 10000 flashes at 770 nm (orange). The inset shows the fraction of the induced Phe- radical (as

a percentage of PSII centers) at 532 nm (black circles), 730 nm (red triangles), 750 nm (blue squares), and 770 nm (orange diamond). EPR conditions

were as follows: temperature, 15K; microwave power, 1.3 mW; modulation amplitude, 3.5 G; microwave frequency, 9.28 GHz.

The Far-Red Limit of Photosystem II 2395

now doubly reduced QA is not possible (Klimov et al., 1980;

Rutherford and Zimmermann, 1984; Styring et al., 1990). We

investigated the formation of Phe- at room temperature using far-

red flashes. After application of laser flashes, the samples were

frozen rapidly inN2(l), andEPR spectra from thePhe- radical were

recorded. As shown in Figure 4C, we were able to accumulate

Phe- under these conditions. It should be noted that the exten-

sive flashing resulted in an initial increase and subsequent

decrease of the amount of Phe- radical (Figure 4C, inset). This

decrease of Phe- is due to photoinhibition of PSII, which takes

place rapidly at reduced conditions at room temperature. The

amount of the Phe- that could be photoaccumulated dropped

with increasing wavelength, and at 780 nm no Phe- could be

observed.

The three measurements of electron transfer components,

reflecting stable charge separation, themultiline EPR signal from

the S2-state (Figure 5, black), the YZd

radical (Figure 5, red), and

the FV induction (Figure 5, green), all gave comparable wave-

length dependence curves even though the measurement

method and the state of the samples varied greatly. Thus, the

final state after light absorption and primary charge separation is

the same, independent of the wavelength. These wavelengths

dependences are compared with the P680* fluorescence exci-

tation spectrum at 684 nm after excitation between 725 and

780 nm (Figure 5, full line). This fluorescence intensity decreases

more quickly with increasing excitation wavelength than do all of

the other measurements. This difference is interesting and sug-

gests that excitation of P680 is not sufficient to explain the

wavelength dependence of the far-red photochemistry.

Flash-Induced Oxygen Evolution

Given that we observe photochemical electron transfer at both

the donor and the acceptor sides of PSII with far-red light, would

it also be possible to observe flash dependent oxygen evolution?

The bottom traces in Figure 6 show the results in thylakoid

membranes. Green flashes (532 nm; Figure 6B, bottom) induced

the known period four oscillation pattern with the oxygen

appearing on the third flash. Far-red flashes at 750 nm (Figure

6A, bottom) also yielded the first oxygen release on the third

flash. However, the period four oscillation cannot be observed,

and the oxygen yield instead reaches a steady state level with

approximately the same amount of oxygen being formed at each

flash. This lack of oscillation could be simulated by assuming a

miss factor of ;50%, similar to what we observed for the

formation of themultiline EPR signal from the S2-state and the FVinduction.

Similar results were obtained for the PSII membranes (Figure

6, top traces). No oxygen is formed on the first two flashes, and

oxygen formation occurs on the third flash, indicating that the

S-state chemistry works as normal. At 750 nm (Figure 6A, top),

the oscillation is less pronounced than at 532 nm (Figure 6B, top)

and is dampened faster due to low light absorption and to the

limited PQ pool in the PSII membrane preparations. The impor-

tant fact, however, is that in both PSII membranes and thylakoid

membranes, we observe the first oxygen formation at the third

flash when light at 750 nm is used.

DISCUSSION

This study has shown that far-red light up to 800 nm is able to

drive PSII electron transfer. We observed (1) formation of Phe-

and QA-, and multiple electron transfer to PQ on the acceptor

side; (2) formation of the YZd

radical and complete S-state

turnover in the CaMn4 cluster on the donor side that lead to (3)

a flash-induced oxygen evolution pattern. Our study has thereby

confirmed an earlier prediction (Trissl, 2006a) that far-red pho-

tosynthesis and photochemistry should be possible to observe in

isolated chloroplasts or thylakoid membranes. We were able to

prove this experimentally and have extended the observations to

PSII-enrichedmembranes and purified core preparations of PSII.

Thus, except for the low yield that is mainly due to the low light

absorption, PSII functions at wavelengths as high as 800 nm. The

OD in this region is low (OD ;0.01 at 730 nm at the concentra-

tions used for our fluorescence measurements) when measured

in an integrating sphere and decreases with increasing wave-

length (see Supplemental Figure 4 online).

The origin of the earlier observed far-red photosynthesis in vivo

has been discussed in terms of three different mechanisms. The

first proposal, that far-red forms of chlorophylls could also exist

in the PSII antenna, was discussed in the original publication

(Pettai et al., 2005a). A second mechanism, in which far-red light

Figure 5. Correlation between Wavelength Dependence of Different

Far-Red-Induced Signals in PSII.

Wavelength dependence of the amplitude of the multiline EPR signal

from the S2-state (black circles) from Figure 3A, the amplitude of the EPR

signal from YZd

(red triangles) from Figure 4A, and the fraction of light

absorbed (fabs) in the FV induction experiment (green squares) from

Figure 4B. The values at 770 and 780 nm are estimations using the fabsvalue at 760 nm and the difference in FV induction for these three

wavelengths. The solid line shows the excitation spectrum of PSII

membranes at 684 nm after excitation at far-red wavelengths. All data

are normalized to the value at 730 nm.

2396 The Plant Cell

directly excites vibrational sublevels of PSII chlorophyll a mole-

cules, was debated in a discussion of that article (Pettai et al.,

2006; Trissl, 2006a, 2006b). The third proposed mechanism

involves reversed (uphill) spillover from the far-red chlorophyll

excitations in the PSI antenna to the PSII antenna (Pettai et al.,

2005a).

There are no reported far-red chlorophylls in the PSII antenna.

The long wavelength components from chlorophyll a species

that have been observed in the PSII absorption spectrum have

absorption bands just above 680 nm. Studies of the absorption

spectrumof LHCII isolated fromplants at room temperature have

identified low energy bands with peaks at 684 and 693 nm

(Zucchelli et al., 1990). Other studies performed at low temper-

ature, however, have not identified any significant absorption

bands higher than 677 to 678 nm in LHCII (Brown and Schoch,

1981; van Dorssen et al., 1987).

Spectral hole burning measurements at low temperatures on

spinach PSII core complexes have revealed that QA- formation

can be observed up to 730 nm (Hughes et al., 2006a, 2006b).

Two different photochemical events leading to QA reduction

could be distinguished. An absorption band, with a maximum at

689 nm,was linked to the lowest energy chlorophyll apigments in

CP47, and stretches out to 704 nm at 1.7K. For a second

absorption with a maximum at 705 nm, 80 and 30% of QA could

still be reduced using 708- and 728-nm light, respectively

(Hughes et al., 2006a, 2006b). This absorption was proposed

to be linked directly to a charge separating state of the special

pair chlorophylls (Hughes et al., 2006a, 2006b). No QA- formation

could be observed with illumination at 750 nm at 1.7K (Hughes

et al., 2006a).

When the three earlier suggestions for the origin of the far-red

absorbing species are reviewed in the light of this work, the

proposed uphill reversed spillover from PSI can immediately be

ruled out. The amount of PSI in PSII-enriched membranes is

insignificant and cannot account for the 45% S1 to S2 turnover

observed with a single laser flash at 750 nm. The fact that we see

an appreciable turnover on a single flash at 750 nm also in the

PSII core particles, which does not contain PSI, strengthens this

conclusion.

For the proposal of far-red chlorophylls in the PSII antenna, it is

interesting to study the PSII core particles that completely lack

the outer antenna components. Since far-red photosynthesis

was observed also in PSII cores, it is not (only) dependent on the

presence of these antenna components. If far-red chlorophylls

are present in PSII and act as the absorbing species, at least one

of these special chlorophylls (or chlorophyll dimers) has to be

located in the PSII core complex. Furthermore, the S1 to S2

turnover following a single flash in the PSII core preparation was

less efficient than in the PSII membranes. This could, in turn,

indicate that the outer antenna complex does contribute to the

absorption of the far-red light. However, estimations by Pettai

et al. (2005a) suggested on average only one far-red chlorophyll

per PSII. Another way of explaining the observed photochemistry

would be that a two-photon process is involved at longer

wavelengths to produce an excited state of sufficient energy to

initiate charge separation. In that case, we would expect the PSII

activity, as measured for example by the induction of FV, to rise

with the square of the applied laser power. However, the ob-

served power dependence does not give any support for this

explanation (see Supplemental Figure 5 online).

In the following, we discuss the consequences our data and

these considerations have for our understanding of the energet-

ics in PSII. Formation of P680*, which is the excited state of the

primary electron donor in PSII, from the excited antenna normally

initiates the primary charge separation to form P680+ and a Phe-,

which subsequently lead onwards to S-cycle advancement and

quinone reduction. However, it does not seem that this pathway

can explain our data in the far-red region. The photochemical

action spectra in Figure 5 are markedly red shifted also com-

pared with the 705-nm absorption band of Hughes et al. (2006a),

more shifted than can be explained by the higher temperature in

our measurements. For example, at 750 nm, we obtain almost

50% yield of the S2-state in a single flash (Figure 5, black). The

thermal energy (kBT) corresponds to only 10 nm at these wave-

lengths. Thus, only vibrational sublevels of P680 at several times

kBT of thermal energywill be possible to excite with 750-nm light.

Therefore, the fractional population of these sublevels will be

very small (<<1%) and would give only insignificant yields of, for

example, the S2-state compared with what we observe.

Direct excitation of vibrational sublevels in a single chlorophyll

a molecule in the antenna would be equally improbable, but the

fact that PSII membranes contain ;200 chlorophylls per PSII

center could give a combined contribution to the far-red absorp-

tion. However, when exciting in the far-red region, the steady

state fluorescence measured at 684 nm from PSII membranes

does not have the same excitation wavelength dependence as

our EPR and FV induction experiments (Figure 5). The FV induc-

tion indicates that efficient electron transfer to QA can be driven

at longer wavelengths than can induce fluorescence at 684 nm,

suggesting that different processes are involved in the two

phenomena. In addition, the maximal inducible FV is lower at

the far-red wavelengths. That stronger flashes must be used to

reach saturation at longer wavelengths is explained by the lower

absorbance. However, the saturation value at 532 and 730 nm is

95% 6 5% of the value obtained with a red (639 nm, 50 ms)

saturating actinic flash, while the saturation value at 760 nm is

only around 30%. The fact that the yield is lower at higher far-red

Figure 6. Demonstration of Oxygen Evolution Induced by Far-Red Light.

Flash-induced oxygen evolution patterns using 750-nm (A) and 532-nm

(B) laser flashes on PSII membranes (top) or thylakoid membranes

(bottom). The arrows indicate the first five flashes.

[See online article for color version of this figure.]

The Far-Red Limit of Photosystem II 2397

wavelengths, although saturating excitation is obtained, points

to a different intrinsic yield of charge separation in PSII for the far-

red light.

Therefore, we propose a model in which an excited state, X*,

different from P680*, can be generated by far-red light (Figure

7A). The different intrinsic yields of charge separation suggested

by the FV saturation data supports an alternative charge sepa-

ration pathway for far-red excitation. It is important to point out

that we are not suggesting that X is an unknown chromophore in

PSII, but rather that X* is an excited state from a known chro-

mophore that has not been previously observed and linked to

PSII activity. Direct measurement of the absorption spectrum of

PSII at wavelengths above 700 nm and at room temperature to

identify X is difficult due to the strong scattering by PSII mem-

branes (Hughes et al., 2006a). We have used an integrating

sphere to reduce scattering effects in the measurements of the

absorption spectrum of PSII membranes. In the region of interest

(730 to 800 nm), we have a substantial absorption (OD 0.005 to

0.01 in a 10-mg chlorophyll/mL sample) that could contain a

chromophore responsible for the far-red photoactivity of PSII

membranes (see Supplemental Figure 4 online). To account for

the photochemical yield obtained still at 780 nm, we estimate

that the energy of the first excited state of X* should not be higher

than ;1.65 eV (photon energy at 750 nm) above the ground

state. The much lower energy than P680* suggests that X* may

not be efficient in populating P680* by uphill energy transfer

(Figure 7A). This is supported by the fact that the red flank of the

684-nm fluorescence excitation spectrum (Figure 5, full line)

decreases more steeply with wavelength than the variable fluo-

rescence, the YZd

signal, and the multiline signal from the

S2-state, showing that population of P680* from X* cannot be

quantitative. As the OD above 700 nm was moderate and

identical in the measurement of the fluorescence excitation

spectrum and the variable fluorescence measurement, this dif-

ference cannot be attributed to optical saturation effects. It

should be noted that the multiline EPR signal from the S2-state

and the Phe- signal were induced at high sample concentration,

the YZd

signal at high concentration in a flat cell with short path

length, and the variable fluorescence in a sample with low

concentration, all of whichmakes direct quantitative comparison

difficult. If X* is not able to populate P680* directly, we suggest

that X* might undergo charge separation directly to form

P680+Phe- or an alternative primary charge separated state

that we denote X+Phe- (Figure 7A). Irrespective of which is the

primary charge separated state, our results indicate that it leads

to the same final YZd

QA- state because we observe efficient QA

reduction, YZd

formation (in Tris-washed samples), and S-state

turnover (in intact samples). Importantly, our data and the

scheme we propose put further constraints on the upper energy

limit of the primary charge separated state, be it P680+Phe- or

X+Phe-.

In Figure 7B, we compare estimated energies of different

states in PSII with the energy of photons at a few wavelengths.

Generally, P680* is assumed to be 1.83 V above the level of P680.

The energies of photons at 750 nm (1.65 eV), where we have

observed oxygen evolution, and at 790 nm (1.53 eV), close to our

outer edge for detected photochemistry, are also shown. Figure

7B also shows the proposed free energy of two charge separated

states in PSII. Earlier estimations of the free energy for the

primary charge separated state, P680+Phe-, that were based on

direct titration of the Phe/Phe- couple and equilibrium constants

between redox centers within PSII, were placed around 1.77 eV

(Klimov et al., 1978, 1979; Rutherford et al., 1981; Holzwarth,

1986) (Figure 7B, P680+Phe- i)), very close to the value assumed

for P680*. This has been questioned in two more recent mea-

surements of the charge recombination of the S2QA- state

(Rappaport et al., 2002) and the free energy difference between

an excited antenna state and the YZd

QA- pair (Grabolle and Dau,

2005). Both studies place the P680+Phe- pair around 1.65 V

(Figure 7B, P680+Phe- ii)). The latter assignment (P680+Phe-

;1.65 eV) is better in line with our results that far-red photons

can drive efficient charge separation. However, the observation

of light-driven electron transfer as high as 790 nm (photon energy

1.53 eV) might indicate that further adjustments of the primary

charge pair level could become necessary.

It is interesting to discuss which redox component(s) in PSII

in that case would be most likely to be adjusted. The YZd

QA- pair

has a considerably lower free energy (Rappaport et al., 2002)

(Figure 7B, ;1.29 V) than the P680+Phe- pair, mainly reflecting

the large free energy drop in the electron transfer fromPhe- toQA.

Consequently, there might exist some thermodynamic freedom

in the placement of the Phe/Phe- couple. By contrast, YZd

and the

higher S-states are all placed around +0.9 to 1.1 V to allow water

Figure 7. Proposal of an Alternative Charge Separation Pathway in PSI

Using the Energy of Far-Red Photons.

(A) Energy level scheme and reactions for the states involved in the

primary charge separation of PSII. We propose the existence of a state X*

that is directly generated by far-red light and that lies substantially lower

in energy than P680*. We further suggest that X* undergoes primary

charge separation to form either P680+Phe- or the new state X+Phe- and

ultimately leads to the same YZd

QA- state as with P680 excitation.

(B) Energies of photons of different wavelengths in comparison with

proposed free energies of charge separated states in PSII. From top to

bottom: energies of photons of at 680, 750, and 790 nm. Proposed free

energy for the P680+Phe- state; P680+Phe- i) based on Klimov et al. (1978,

1979), Rutherford et al. (1981), and Holzwarth (1986) and P680+Phe- ii)

based on Rappaport et al. (2002) and Grabolle and Dau (2005). Proposed

free energy for the YZ

d

QA- state (Rappaport et al., 2002).

2398 The Plant Cell

oxidation (;0.8 V). These values are critical for function, and

there are no large drops in free energy on the donor side of PSII.

This probably indicates that our results will lead to few readjust-

ments of the oxidation potentials among these donor-side com-

ponents.

In conclusion, we have observed photosynthetic activity in

PSII, in vitro, using long wavelength light up to 790 nm. The

efficiency of the far-red photons to induce full charge separation

and electron transfer leading to water oxidation leads us to

tentatively introduce a low energy excited state X* in PSII that can

be induced by far-red light. This excited state nevertheless leads

to the same charge separated YZd

QA- state as obtained when

exciting P680 with light below 700 nm. This suggests that the

action spectrum of PSII is wider than has been previously

thought and that PSII operates at wavelengths beyond those

that have been considered to be able to only drive PSI reactions.

The photosynthetic reactions in PSII in the far red occur at all

levels, from very purified PSII core samples to intact leaves

(Pettai et al., 2005a, 2005b). There is no reason that it would not

occur under natural conditions. Although the absorption is low,

an absorbed photon at, for example, 750 nm will drive PSII

electron transfer rather efficiently. It would thus be a valuable

addition for an organism living under conditions in which most of

the photosynthetically important radiation is lacking, for exam-

ple, in the shade of a dark forest. Of course, if such an organism is

exposed to normal sunlight, it will use this instead. Furtherwork is

needed to address the physiological consequences of this new

finding. It is also necessary to assess how the two photosystems

cooperate at these longer wavelengths.

METHODS

Growth Conditions

Spinach (Spinacia oleracea) was grown hydroponically at 208Cunder cool

white fluorescent light with light–dark periods of 12 h and with the light

intensity of 300 mE m22 s21. As a light source, dysprosium lamps of the

type Osram Power star HQI-E 400W/DV, relatively close to daylight, were

used.

PSII Membrane Preparation

PSII (BBY)membraneswere prepared fromspinach as described (Berthold

et al., 1981; Volker et al., 1985) and resuspended in 25 mM MES-NaOH,

pH 6.1, 400 mM sucrose, 15 mM NaCl, and 3 mM MgCl2 for storage at

2808C. For the EPR measurements, the salt composition was changed

to 10 mM NaCl, 10 mM MgCl2, and 5 mM CaCl2. Thylakoid membranes

were prepared as described (Mamedov et al., 2000) and used in a 10 mM

phosphate buffer, pH 7.4, with 100 mM sucrose and 5 mMNaCl. PSII core

particles were prepared as described (Smith et al., 2002).

Tris washing to remove the WOC was performed by suspending the

PSII membranes in 1.0 M Tris, pH 9.1, and incubating in light on ice for

30 min. After centrifugation, the pellets were washed twice in the

measuring buffer before storage at 2808C. This procedure removed

>90% of the bound Mn and the three extrinsic proteins from PSII

(Gadjieva et al., 1999).

Fluorescence Excitation Spectra

Steady-state fluorescence excitation spectra were obtained with a SPEX

Fluorolog 3 at 208C on PSII membrane samples (10 mg chlorophyll/mL).

Excitations were made between 725 and 780 nm, and the emission was

measured at 684 nm. The data were collected in 1-nm steps with an

integration time of 5 s at each point, and 30 scans were averaged. The

excitation and emission monochromator slits were 3 and 2 nm, respec-

tively.

Laser Light Source

A Spectra Physics Quanta Ray MOPO 730 optical parametric oscillator

pumped by a Spectra Physics PRO-290 Q-switched Nd:YAG laser (6-ns

flashes, 5 Hz) was used as the laser source. The bandwidth of the MOPO

730was60.1 nm.Control experimentsweremade to assure the quality of

the laser light as described in the Supplemental Methods online.

Flash-Induced Oxygen Evolution

The oxygen evolution pattern was measured on a Joliot-type bare

platinum electrode with 50 mM KCl. The samples (750 mg chlorophyll/

mL) were dark adapted on the electrode for 3 min prior to the measure-

ments. Laser flashes from theMOPO730 triggered at 1Hzwere usedwith

a power of ;4 mJ for the 532-nm flashes and ;2.5 mJ for the 750-nm

flashes.

Application of Laser Flashes for Fluorescence Measurements

A thermostated (208C) and stirred quartz cell was filled with 3.5 mL of PSII

membranes at 25 mg chlorophyll/mL and exposed to 1 to 900 flashes of

;25 mJ at indicated wavelengths using the MOPO 730.

Variable Fluorescence Decay Kinetics

First, the PSII sample was exposed to the far-red laser flashes as

described above. One minute after the last far-red flash, the induction

and subsequent decay of the FV wasmeasured. Themeasurements were

done in a double modulation fluorometer (PSI Photon Instruments). After

the actinic flash of the fluorometer (LEDs at 639 nm, 50 ms), eight

measuring light pulses (2.0ms) were applied per decade in the range from

150ms to 180 s. Fittings of a triple exponential function to the decay traces

were made as described (Mamedov et al., 2000).

Power andWavelength Dependence of FV Induced by Single

Far-Red Flashes

With the same laser systemas above, the far-red flashwas used to induce

directly FV in a cuvette with 1.5 mL of PSII membranes at 10 mg

chlorophyll/mL inhibited by 10 mM DCMU. The level of fluorescence

was recorded with a measuring light pulse every 10 ms. The normalized

FV (nFV) was corrected using FV(corrected) = nFV/(1 2 0.5(1 2 nFV)) to

account for the nonlinear relationship between FV and [QA-] (Joliot and

Joliot, 1964). The corrected fluorescence data can thereby be used as a

measure of the level of reaction centers in which QA is reduced. Assuming

independent photon absorption events by the reaction center pigments,

the fraction of reaction centers that are not excited by a single flash will be

given by a Poisson distribution: P(0) = e-N, whereN is the average number

of photons absorbed per reaction center. Then, the power dependence

becomes: FV = FV(sat) · (1-e-N), where FV(sat) is the saturating FV signal. N

can also be expressed as fabs/nPSII · nhy, where fabs is the fraction of light

absorbed, nPSII is the number of PSII reaction centers in the sample (;0.1

nmol), and nhy is the number of photons in a flash. From a plot of FV as a

function of nhy, it is then possible to determine FV(sat) and fabs.

Application of Laser Flashes for EPR Spectroscopy of the S-States

Synchronized S1-state samples (1.8 mg chlorophyll/mL) were prepared

as described (Zhang and Styring, 2003). Phenyl-p-benzoquinone (50 mM

in DMSO, final concentration 1 mM) was used as electron acceptor.

The Far-Red Limit of Photosystem II 2399

Thereafter, laser flashes from the MOPO 730 (25 mJ) of the desired

wavelength were applied to the EPR tubes at 208C. The samples were

frozen within 1 to 2 s in a dry-ice ethanol bath and transferred to N2(l). In

experiments with the PSII core preparation, the dark-adapted EPR

sample (1.0 mg chlorophyll/mL) was used in the absence of phenyl-

p-benzoquinone. The sample was given one and 100 flashes at 750 nm at

208C and was then directly frozen.

For the Phe- measurement, PSII-enriched membranes (2.0 mg chloro-

phyll/mL) were fully reduced using sodium dithionite (Na2S2O4, final

concentration 50 mM). Laser flashes from the MOPO 730 of desired

wavelength were applied to the EPR tubes at 208C. The samples were

frozen within 1 s in a dry-ice ethanol bath and transferred to N2(l) before

the measurements.

YZd

measurements at room temperature were performed in Tris-

washed PSII membranes (2.0 mg chlorophyll/mL) in the presence of the

ferricyanide (K3[Fe(CN)6])/ferrocyanide (K4[Fe(CN)6]) couple (10 mM).

EPR Spectroscopy

EPR spectra were recordedwith an ELEXSYSE500 spectrometer (Bruker

Biospin) equipped with a SuperX bridge and a SHQE4122 cavity. For the

low temperaturemeasurements, anOxford InstrumentCryostat and ITC-4

temperature controller were used. The split EPR signal from the S3-state

was induced by IR illumination (830 nm) for 30 min at 5K as described (Su

et al., 2007). This was followed by visible illumination for 10 min at 5K to

induce the split EPR signals from the S0- and S1-states (Su et al., 2007).

EPR spectra were analyzed with the Bruker Xepr 2.1 software. The split

EPR signal from the S3-state was obtained as the IR light minus dark

difference spectra. The split EPR signals from the S1- and S0-states were

obtained as the visible light illuminated minus IR light illuminated differ-

ence spectra.

For the kinetic measurements of YZ

d

at room temperature, the sample

was placed in an EPR flat cell (250 mL). The laser flashes were directed

directly into the EPR cavity and were synchronized with EPR acquisition

using a home-built triggering system. The data were obtained at the low

field peak of the YZ

d

radical EPR spectrum (3345 G). Fifty or 250

accumulations were used at 532 or 730 to 790 nm, respectively.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Effect of Ferricyanide on FV in a Far-Red-

Treated PSII Membrane Sample.

Supplemental Figure 2. The Multiline EPR Signal from the S2-State

Induced by 730- and 790-nm Light.

Supplemental Figure 3. The Fraction of FV Induced after a Single

Flash at 532 nm.

Supplemental Figure 4. The Absorption Spectrum of PSII Mem-

branes at 208C Measured Using an Integrating Sphere.

Supplemental Figure 5. Power Dependence of FV Induction at 750

nm Fit with Functions Representing One- or Two-Photon Processes.

Supplemental Methods. Control Experiments with Optical Filters.

ACKNOWLEDGMENTS

This article is dedicated to Achim Trebst (Ruhr Universitat Bochum,

Germany) on the occasion of his 80th birthday. Paul Smith, Ron Pace,

and Elmars Krausz (The Australian National University, Canberra,

Australia) are acknowledged for providing the PSII core preparation.

This work was supported by the Swedish Research Council, the Knut

and Alice Wallenberg Foundation, and the Swedish Energy Agency.

Received October 31, 2008; revised July 8, 2009; accepted August 4,

2009; published August 21, 2009.

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The Far-Red Limit of Photosystem II 2401

DOI 10.1105/tpc.108.064154; originally published online August 21, 2009; 2009;21;2391-2401Plant Cell

Anders Thapper, Fikret Mamedov, Fredrik Mokvist, Leif Hammarström and Stenbjörn StyringDefining the Far-Red Limit of Photosystem II in Spinach

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