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Letter

Revisiting the Role of Xanthophylls in Non-Photochemical Quenching Bart van Oort, Laura Marie Roy, Pengqi Xu, Yinghong Lu, Daniel Karcher, Ralph Bock, and Roberta Croce J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b03049 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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The Journal of Physical Chemistry Letters

Revisiting the Role of Xanthophylls in Non-Photochemical Quenching

Bart van Oort1,*, Laura M. Roy1, Pengqi Xu1, Yinghong Lu2,3, Daniel Karcher2, Ralph Bock2 and Roberta Croce1

1

Biophysics of Photosynthesis, Department of Physics and Astronomy, Faculty of Sciences, and

LaserLaB Amsterdam, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands 2

Max-Planck-Institut für Molekulare Pflanzenphysiologie Wissenschaftspark Golm, Am Mühlenberg

1, 14476 Potsdam-Golm, Germany 3

present address: School of Chemical Engineering, Nanjing University of Science and Technology,

Nanjing, China *corresponding author: [email protected]

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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

Abstract Photoprotective non-photochemical quenching (NPQ) of absorbed solar energy is vital for survival of photosynthetic organisms, and NPQ modifications significantly improve plant productivity. However, the exact NPQ quenching mechanism is obscured by discrepancies between reported mechanisms, involving xanthophyll-chlorophyll (Xan-Chl) and Chl-Chl interactions. We present evidence of an experimental artefact that may explain the discrepancies: strong laser pulses lead to the formation of a novel electronic species in the major plant light-harvesting complex (LHCII). This species evolves from a high excited state of Chl a and is absent with weak laser pulses. It resembles an excitonically coupled heterodimer of Chl a and lutein (or other Xans at site L1), and acts as de-excitation channel. Laser powers, and consequently amounts of artefact, vary strongly between NPQ studies, thereby explaining contradicting spectral signatures attributed to NPQ. Our results offer pathways towards unveiling NPQ mechanisms, and highlight the necessity of careful attention to laser-induced artefacts.


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Light-harvesting is the initial step of the light-driven reactions of photosynthesis. Light-harvesting pigment-protein complexes (LHCs) absorb solar radiation, and transfer its energy to reaction centers where it induces charge separation, thereby converting it to chemical energy.1 Under illumination conditions where the rate of photon absorption exceeds the maximal rate of CO2 fixation, LHCs can act as “safety valves”, switching to a state where most absorbed energy is harmlessly dissipated as heat via non-photochemical quenching (NPQ).2 Though it was recently shown that NPQ modification can increase the productivity in tobacco by up to 15% under field conditions3, the quenching mechanism underlying NPQ remains highly debated2, thereby limiting further increases. Proposed quenching mechanisms involve either interactions between chlorophylls (Chls)4–6, energy/electron transfer between Chl and xanthophyll (Xan)7–11, or excitonic mixing of Chl and Xan12,13. This is followed by rapid relaxation to the ground state. The NPQ mechanism is extensively studied by ultrafast transient absorption and fluorescence spectroscopy, which is difficult for (near) native systems such as leaves and thylakoid membranes, due to strong scattering and many spectrally overlapping signals. Increasing excitation power can partly overcome these problems, but causes non-linear effects, such as the formation of transient quenchers and singlet-singlet (S-S) annihilation, particularly in systems of connected LHCs and photosystems (PSs).14 S-S annihilation occurs when, within the excited state lifetime, multiple photons are absorbed within a domain of connected pigments.14 Two excitons can then “meet”, forming a higher excited state on a single Chl, which rapidly relaxes to the lowest excited state, thus “annihilating” one exciton. This process distorts kinetics, and possibly also spectra, in time-resolved experiments. Though it is generally assumed that S-S annihilation does not involve intermediates living longer then hundreds of femtoseconds14, this awaits rigorous testing.



Here, we show that S-S annihilation leads to population of a short-lived (quenched) species in unquenched major plant LHC (LHCII). This species is populated via high excited states of Chls a, also when such states form by direct excitation at 360 nm, or by sequential excitation with two laser pulses (630 nm and 750 nm). Several other Xans also form this species, provided that they are at a Lut site in LHCII, most likely site L1 (nomenclature of 15). We discuss the possible implications for several NPQ studies performed under conditions with strong S-S annihilation8–11,16–21. Pump-probe (PP) data of LHCII from dark-adapted spinach leaves show main negative signals at 400-455 nm and 660-700 nm, and positive signals (excited state absorption, ESA) in between these regions (Figure 1A), typical for LHCII (see e.g. refs. 19,22,23). Early evolution-associated difference spectra (EADS) show excited Chl b, which transfers energy rapidly to Chl a. On a ps-time scale, there is relaxation to the lowest energy Chl a pigments. Xan S1 ESA (500-550 nm) appears in the PP data on a 5 ps time scale, even though Xans do not absorb at the excitation wavelength (630 nm)24. This ESA disappears in 20 ps, the same time scale at which the Chl signal decreases by 35% of Chl due to S-S annihilation (e.g. refs. 23,25,26). The Xan ESA is absent at low excitation power (grey spectrum in the inset of Figure 1A; see also the power study in Supplementary Figure 9). To separate the Xan ESA from the Chl signal loss by annihilation, we calculated the difference spectrum (∆EADSNQ) between the 20 ps and the nsEADS, each normalized to the main bleach. ∆EADSNQ shows Chl* and Xan* character (Figure 1C, blue), suggesting that it represents a novel species, that is populated during annihilation and then rapidly decays. This was tested with a simple target model (Figure 1B). In this model, energy transfer and relaxation are described by the evolution from Chl1 to Chl4, none of which show Xan ESA features. On a nanosecond timescale, 69% of Chl4 population decays to a long-lived state (Xan 4/22 ACS Paragon Plus Environment

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triplet, T). The remaining 31% describes annihilation via an intermediate species: formation of species “Q” in 16 ps (the annihilation time scale observed in ultrafast fluorescence25), and decay of Q in 10 ps. The species associated difference spectrum of Q (SADS, Figure 1C, magenta) strongly resembles ∆EADSNQ (blue), and consists of Xan ground state bleach (GSB, 450-500 nm), Xan S1 ESA (500-600 nm), Chl a GSB (around 435 nm), and a Chl bandshift feature (640-740 nm). So, contrary to common belief14, S-S annihilation in LHCII proceeds via a relatively long-lived intermediate species with Xan and Chl spectral features.

EADS [mOD]

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Chl1 Chl2 Chl3 Chl4 T 450

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Figure 1 Analysis of PP of LHCII from dark adapted spinach leaves (LHCIIsp) in β-DDM, excited at 630 nm. (A) EADS, (B) target model and resulting SADS, (C) SADS of Q from target model (magenta), ∆EADSNQ (blue), ∆EADSNQ from excitation at 360nm (black, see below), and photoproduct formed by sequential (multipulse) excitation and detected by pump-repump-probe (PRP) spectroscopy (grey, from ref27, see below). In (A) EADS1 is scaled by 0.5,and the grey spectrum in the inset is the time-gated ∆OD (multiplied by 5) in absence of annihilation (see also Supplementary Figure 9). The difference spectra in (C) are scaled to overlap with the Q SADS around 535nm. Inverse rates and relative initial populations are indicated in (A) and (B), “inf” means non-decaying on the 3ns timescale of the experiment. The fit quality is the same for both models.

0.46ps

Chl3 Chl3 6.7ps

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PP (630nm) : EADSNQ(20ps) - EADSNQ(~ns) PP (630nm) : Q SADS (10ps) PP (360nm) : EADSNQ(8.9ps) - EADSNQ(ns) PRP : photoproduct SADS (9ps)

C ∆EADSNQ , SADS [a.u.]

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Since S-S annihilation proceeds via a high excited state of Chl a, we tested whether Q also forms when such state is formed via other processes. First, we compare Q with the photoproduct formed by sequential (multipulse) excitation at 630 nm (pump) and 750 nm (repump, delayed by 100 ps to ensure full equilibration and S-S annihilation) as measured by pump-repump-probe (PRP) spectroscopy in ref27 (details in Supplementary Results 1). Indeed, the photoproduct absorption difference spectrum, and lifetime are virtually identical to those of Q (Figure 1C, grey27), indicating that they represent the same electronic species (alternative explanations for the photoproduct signal are less plausible, as discussed in Supplementary Discussion 2). Repumping at different NIR wavelengths produced the same photoproduct, with possibly higher yield at shorter wavelengths (Supplementary Results 2). A very similar species forms when high excited states of Chl a are directly populated by single-pulse excitation at 360 nm (Figure 1C black), both in absence and presence of S-S annihilation (Supplementary Results 4 and 5). In summary, the same species Q forms via several pathways that all involve a high excited state of Chl a. Next, we studied the nature of Q with PRP on several LHCII preparations (Table 1) differing in pigment composition, pH, species, oligomeric state and detergent (which affects pigment interactions 28) . Comparison of the photoproduct spectra (–DADDS1; DADDS = decay associated double difference spectra, see Experimental Methods) of all samples (Figure 2) shows that: (1) Q is formed in all samples; (2) the amount and lifetime of Q are very similar in all samples; (3) the spectrum of Q is similar in most samples.


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Figure 2 Analysis of PRP of various types of LHCII. Top Upper spectrum: SADS from simultaneous target fit of PP and PRP’27, bottom spectrum: decay-associated difference spectrum (DADS) of a caroteno-phthalocyanine dyad in toluene (“dyad10”, 670 nm excitation), attributed to an excitonically coupled state of the two moieties29. This DADS was calculated from the EADS and 25 nm blueshifted. Middle spectra: photoproduct spectra (–DADDS1) of various types of LHCII. The legend shows lifetimes (accuracy about 1-2 ps) and sample names (Table 1). Absolute amplitudes of –DADDS can be directly compared (see scaling procedure in main text). The thin black spectra are the SADS of Q from PP (Figure 1).

SADS (PRP) LHCIIspin βDDM (9ps)

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sample

description

species

name LHCIIsp

state dark adapted WT spinach

trimer

β-/α-DDM slightly higher Chl/Xan

monomer

β-DDM

slightly higher Chl/Xan

trimer

α-DDM

-

A. th

trimer

α-DDM

~50% Vio replaced by Zea and Anth

A. th

trimer

α-DDM

Neo and Vio replaced by Zea

monomer

α-DDM

lower Chl a/b, slightly higher Chl/Xan, more Vio

α-DDM

lower Chl a/b, slightly higher Chl/Xan, Vio only Xan

α-DDM

higher Chl a/b, slightly higher Chl/Xan, Asta only Xan

mLHCIIsp dark adapted WT spinach dLHCII

dark adapted WT A. th b

sLHCII

stressed WT

zLHCII

mutant npq2

Lhcb1

reconstituted c

A. th

c

A. th

Lhvb1-V LHC-asta

oligomeric detergent pigment composition difference with dLHCII

reconstituted mutant

a

N. ta

monomer d

monomer

e

Table 1. Sample description. Detailed pigment composition is in Supplementary Table 1. a Arabidopsis thaliana; b from light-stressed plants, pH 5.5; c from in vitro reconstitution; d Nicotiana tabacum; e containing a small fraction of minor LHCs



Closer inspection reveals that Q is nearly independent of: species (spinach vs. A. thaliana), detergent (α-DDM vs. β-DDM) and oligomeric state (monomeric vs. trimeric), but it depends on the Xan composition. In particular, the spectrum only changes upon substitution of the Xans at the Lut binding sites (L1 and L2 in the nomenclature of ref15): zLHCII (broadening), Lhcb1-V (blue-shift) and LHC-asta (broadening and red-shift). This suggests that, in native LHCII, Lut is the Xan contributing to Q, in agreement with the spectral resemblance of Q to Lut at site L127 (nomenclature of ref15). The assignment to L1 and not L2 is corroborated by the lack of spectral difference between monomeric and trimeric LHCII (Figure 2), whereas L2 red-shifts upon trimerization30.

The SADS of Q qualitatively resembles that of excitonically coupled chromophores14,27,29, with contributions of Chl a (GSB at 435 nm, bandshift around 680 nm) and Lut (ESA at 500-600 nm), and possibly Chl b around 650 nm. This leads to its assignment to a (Chl-Lut)* excitonically coupled state. Though Chl-Xan(S1) coupling is extremely weak in the point-dipole approximation, there are indications of such coupling in many LHCs19,23,31–37, probably enabled by conformational changes and specific local environments38.Whether the coupling is strong enough to create an excitonically mixed state, remains an open question. Theoretical prediction of the spectral shape of Q is difficult39, and we therefore rely on a physical model system: a dyad consisting of a Chl a-mimicking and a Xan-mimicking moiety, where an excitonically coupled state was reported.29 The spectrum of that state (bottom spectrum in Figure 2) strongly resembles Q, confirming the assignment. Interestingly, in the dyad, the state also shows a negative band at 650 nm suggesting that, in Q, this band may not originate from Chl b.


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In conclusion, we assign Q to an excitonically coupled state (Chl a – Lut1)*. Interestingly, also Vio and Asta can form an excitonically coupled state with Chl a, provided that they bind to the sites that usually bind Lut. This suggests that site L1 facilitates the coupling that leads to photoproduct formation, irrespective of the exact nature of the Xan. The formation of Q during S-S annihilation may have important consequences for the interpretation of experimental data. So far, annihilation is assumed to proceed without transient intermediates.14 Consequently, Q signals may have been mistakenly assigned to other species and processes. This may be particularly important when annihilation levels differ between quenched and unquenched samples, as part of the quenched minus unquenched difference signal is due to Q formation via annihilation, whereas many other PP contributions cancel out. A dramatic example is aggregationinduced quenching in isolated LHCs, where intercomplex EET dramatically increases annihilation.25 Also in intact membranes, there are indications that the functional domain size, and hence annihilation, increases upon NPQ induction40 (also different fractions of open/closed reaction centers can strongly affect annihilation). The amount of annihilation also depends on the absorption cross-section at the excitation wavelength, so absorption changes affect the amount of Q. This is particularly relevant when comparing LHCs with different pigment composition (e.g., Zea vs. Vio), and/or for small populations of quenchers (estimated for several studies in Supplementary Discussion 4). Here we take a closer look at several influential studies that used transient absorption spectroscopy to investigate the quenching mechanism in NPQ (Table 2). These studies deduce the quenching mechanism from the difference between unquenched and quenched samples, often in the presence of annihilation.



sample

fraction S-S

fraction S-T

quenching mechanism c

annihilationa annihilationb

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refere nce

thylakoid membranes +/- qE

0.6 to 1

++

ChlXan EET or (Chl-Xan)*

16


0.6 to 1

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Xan●+ (11ps)

9

CP24, CP26, CP29, reconstituted CP29

0.3

++

Xan●+ (0.5ps, 5.5ps)

11

mixed minor LHCs, reconstituted Lhcb1,2,3 0.4

++

Xan●+ (5.2ps)

10

mutants +/- Vio/Zea


1

++

Xan●+ (10ps)

10

CP24, CP26 with Vio or Zea

0.3

++

Xan●+ (5.0ps, 5.5ps)

17

LHCII aggregates

0.4 to 1

+/-

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8

LHCII aggregates vs. LHCII trimers

n.d.d

++

(Chl-Xan)*

18

LHCII aggregates

0.02 to 0.09

--

Chl-Chl charge transfer; Xan

5

not involved mixed minor LHCs with Zea as only Xan

0.4

--

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19 e


1

-

unknown and Xan●+ (10 ps)

20


0.9 to 1

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20

thylakoid membranes during qE induction

1

++

Xan●+ (15ps)

21

LHCSR1 (with Vio or Zea)

0.01

--

ChlXan EET and Xan●+

35

(30-40ps) Table 2. Estimation of annihilation in published experiments. S-S annihilation is likely to occur when more than one Chl per domain is excited per pulse. The fraction of annihilation is calculated as the Poissonian probability of absorption of >1 photons per domain divided by the ≥1 probability. Details of calculation of the numbers in this table are in Supplementary Discussion 3 and Supplementary Table 2, which includes estimated fractions of Chls a excited per pulse, estimated domain sizes, and reported laser repetition rates. a semi-quantitative assessment; b qualitative assessment; c in brackets the Xan●+ formation time; d could not be determined from the paper; e includes many more complexes

We look in more detail at the SADS of the quenching species reported in LHCII aggregates in ref8, where annihilation is treated explicitly as a multiexponential decay to the ground state. Any signal originating from Q is not included as part of the annihilation, and will therefore appear as the species assigned to the “natural” quencher in the aggregates. Indeed, the quencher’s spectrum strongly resembles Q (Figure 3, red vs. blue), suggesting that they are the same species, and may not be related to the quenching process under annihilation-free conditions. The main difference is the absence of the Chl bandshift feature, which is an inevitable consequence of the spectral zero-constraint above 620 nm in ref 8. Similar quencher spectra were obtained from aggregated vs. non-aggregated LHCII18 (Figure 3 grey) and thylakoid membranes with and without qE induction16 10/22 ACS Paragon Plus Environment

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(Figure 3 black). Interestingly, a study that carefully minimized S-S (and S-T) annihilation explicitly ruled out the role of Xans in quenching in LHCII aggregates.5 In the presence of S-S annihilation Xan features appeared (uncommented data in Supplementary Figure 3a of Müller et al.5). Figure 3 SADS of Q from PRP (light blue) and from annihilation (blue) compared with those of quenchers reported in LHCII aggregates (red, forced to zero above 620 nm, ref8; grey, ref18) and thylakoid membranes (black, ref 16). Spectra are scaled to overlap around 535 nm. Lifetimes are indicated in the legend. The two black error bars indicate standard deviations of 4/8 repeats.

Several studies report NPQ via Xan●+, mainly when using both high pulse energies (causing S-S annihilation) and high laser repetition rates9–11,16–18,20,21. We found weak indications of the formation of Xan●+ via S-S annihilation from PP with NIR probing (Supplementary Results 6). It seems plausible that additional Xan●+ may form via singlet-triplet (S-T) annihilation, which occurs when triplet states accumulate at high laser repetition rates (Table 2 and Supplementary Table 2). In solvents, the lowest energy Xan triplets act as precursors for Xan●+ 41–44. The higher energy Xan triplet states formed by S-T annihilation may be even stronger electron donors. Indeed, in LHCII aggregates strong laser pulses create long-living quenchers, probably Chl cations or other radicals, with triplet states as precursors45. Interestingly, the amount of triplet accumulation will be higher in the monomeric minor antenna complexes of PSII (1 per monomeric unit) than in trimeric LHCII (1/3 per monomeric unit, because S-T annihilation by one triplet per trimer prevents the formation of additional triplets). Consequently the amount of S-T annihilation will be higher in the minor complexes, and the therefore also the amount of Xan●+ formed via this path would be higher. This agrees with reports of Xan●+ in the minor complexes10,11,17,19, but not or far less in LHCII trimers10. 11/22 ACS Paragon Plus Environment


Interestingly, the formation time of Xan●+ is often similar to the inverse rate of annihilation within monomeric and trimeric LHCs25,31 (Table 2), in agreement with Xan●+ formation via annihilation. In larger systems at lower levels of annihilation, the population time of Q would be dominated by the natural lifetime of Q (because of inverted kinetics by slow annihilation), which is also very similar to reported formation times of Xan●+ (Table 2). We can therefore not exclude that the Xan●+ signals in refs 9–11,16,17,20,21 are (at least in part) the results of high laser intensities. The current results do not imply that Xans are never involved in Chl a quenching as there are examples in isolated pigment-protein complexes under annihilation-free conditions (Chl aXan S1 EET in Hlid36, and Chl aXan S1 and Xan●+ in LHCSR135). The transient species formed via S-S annihilation may also affect transient absorption studies other than NPQ in PSII, including Zearelated quenching in PSI46. Importantly, also time-resolved fluorescence studies are distorted in the presence of annihilation14. We therefore stress the importance of control experiments at different powers/repetition rates, and of reporting the information required to assess the extent of annihilation (and other non-linear effects) in ultrafast spectroscopy experiments. Ideally, experiments should be under annihilation-free conditions; this can greatly benefit from recent advances in scatter suppression47. If annihilation is unavoidable, it should explicitly and correctly be accounted for in data analysis and interpretation. In conclusion, in LHCII a (Chl a-Lut)* mixed state is populated via S-S annihilation and via other pathways involving high excited state of Chl a, including low power 360 nm excitation. Other Xans that bind at the Lut binding site(s) also excitonically mix with Chl a. It is not clear whether this state is physiologically relevant, but with its spectral and kinetical characterization, it will be easier to resolve it among the many overlapping signals in thylakoid membranes.


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It seems likely that the formation of (Chl a-Lut)*, and, more generally, Xan-like signals, via annihilation has affected the interpretations of previous work assigning NPQ quenching mechanisms to (Chl a-Xan)*16,18, Chl to Lut EET8,16 and quenching via Xan●+ 9–11,16–21. By explaining these contradicting results, we offer pathways towards unveiling NPQ mechanisms.

Experimental Methods Sample preparation Several types of LHCII (Table 1) were purified from fresh leaf material of Spinacia oleracea (spinach), Arabidopsis thaliana and mutant Nicotiana tabacum (tobacco) plants, or prepared by in vitro reconstitution, using published protocols.27,48–50 Pigment composition was determined as described before.24,51 Details of sample preparation and pigment analysis are in Supplementary Methods. All spectroscopic measurements were performed at room temperature in a buffer containing 50 mM Hepes (pH 7.5), 5 mM MgCl2, and 0.03% β-DDM (β-dodecyl maltoside) or 0.03% α-DDM. In LHCII from light-stressed plants (sLHCII), 10mM MES (pH 5.5) was used instead of Hepes. Ultrafast spectroscopy Multipulse transient absorption spectra (pump-repump-probe, PRP) were largely measured as before27 and described in detail in the Supplementary Methods. This gives PP, RP and PRP datasets, from interaction with, respectively, pump, repump or both pulses. For PP experiments, the repump pulse was absent. Pumping was at tpump=0 ps with 630 nm or 360 nm pulses of 80 fs duration and 500 Hz repetition rate. Repumping was at trepump=100 ps with NIR pulses (720-780 nm, 1-2 ps duration), or 675 nm (80 fs duration) and 250 Hz repetition rate. Broadband probing was at t=-50 to 3000 ps at 1 kHz repetition rate. Pump pulse energy and spot size were adjusted to get 13/22 ACS Paragon Plus Environment


∆OD≈20mOD at t=100 ps, which typically lead to about 50% S-S annihilation (≈40mOD maximal bleach, at around t=1-3 ps). For monomeric samples ∆OD at 100 ps was higher, but and annihilation lower due to the smaller domain size. The repump pulse energy and spot size were adjusted to give about 50% of de-excitation. The repump spot size was at least twice as large as the pump, which was at least twice as large as the probe. For quantitative comparison between samples, DADDS and EADDS were scaled to the amplitude of the main bleach of the ns-EADDS. This corresponds to using the amount of de-excitation as an internal standard, assuming that the oscillator strength for SE is the same in all samples. It corrects for variations in sample OD, pump/repump pulse energies and spot sizes. NIR probing transient absorption was with the same laser system and settings, but using a prism-based spectrograph with silicon CCD detector, as previously described47. The NIR spectral resolution was approximately 30-40nm, and a plateau arises above 1000 nm due to limitations of the CCD detector (Supplementary Figure 8). All experiments were in absence of oxygen27 in a shaking 1 mm quartz cuvette at room temperature, with OD of 0.4-0.5 at the maximum around 675 nm. No sample degradation was observed during the ultrafast experiments. The original data sets are presented as Supplementary Figures. Data analysis All transient absorption data were analyzed using global and target analysis, with the Glotaran and TIMP software packages.52,53 PP(λ,t) data were fitted to schemes of numerically equivalent parallel or sequential exponentially decaying components, yielding respectively decay- (DADS) or evolution-associated difference spectra (EADS)54 (λ : probe wavelength, t : probe delay). The effect of singlet-singlet (S-S) annihilation was studied by a target model in which annihilation forms a transient species (Figure 1B). In PRP data, strong coherent artefacts around trepump, were removed by subtracting RP, yielding PRP’≡PRP-RP.27 PRP data were analyzed by parallel exponential fitting of ∆∆OD≡PP-PRP’, yielding decay-associated double difference spectra (DADDS). This approach 14/22 ACS Paragon Plus Environment

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extracts the effect of the repump pulse, because ∆∆OD is nonzero only when there is a repump pulse induced effect. –DADDS1 represents the photoproduct spectrum and its lifetime, as described in detail in Supplementary Methods and ref 27 (see also red and blue spectra in Figure 2). The variation of the lifetime of –DADDS1 between technical and biological replicates was 1-2 ps.

Supporting information Electronic Supporting information is available as a pdf file, providing: •

Methods: details on sample preparation, ultrafast spectroscopy methods and data analysis

•

Results: PRP of LHCII from dark adapted spinach leaves, PRP with repumping 675 nm and at different NIR wavelengths, PP at 360 nm, PP at 630 nm with NIR probing

•

Discussions: alternative explanations of the photoproduct signal, details of the estimation of the amounts of annihilation in previous studies (Table 2), details of the estimation of the amounts of Xan●+ in previous studies

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Original PP and PRP datasets

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Acknowledgements B.v.O and P.X were supported by the Netherlands Organization of Scientific Research (NWO) Earth and Life Sciences (ALW), through a Vici grant to R.C., L.M.R. by a FOM program to R.C. Additionally, B.v.O. was supported by NWO through a VENI grant to B.v.O. We thank Henny van Roon for preparation of LHCII trimers and monomers from spinach. We thank Dr. John Kennis of the Department of Physics and Astronomy (Vrije Universiteit Amsterdam) for use of the transient absorption setup.


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