BBA - Bioenergetics 1861 (2020) 148206

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BBA - Bioenergetics
journal homepage: www.elsevier.com/locate/bbabio

Harvesting far-red light: Functional integration of chlorophyll f into T

Photosystem I complexes of Synechococcus sp. PCC 7002
Martijn Trosa, Luca Bersaninia, Gaozhong Shenb, Ming-Yang Hob,c, Ivo H.M. van Stokkuma,
Donald A. Bryantb,d, Roberta Crocea,
⁎

a
Department of Physics and Astronomy and LaserLaB, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, the Netherlands
b
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
c
Department of Life Science, National Taiwan University, Taipei 10617, Taiwan
d
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA

ARTICLE INFO ABSTRACT

Keywords: The heterologous expression of the far-red absorbing chlorophyll (Chl) f in organisms that do not synthesize this
Photosynthesis pigment has been suggested as a viable solution to expand the solar spectrum that drives oxygenic photo-
Light harvesting synthesis. In this study, we investigate the functional binding of Chl f to the Photosystem I (PSI) of the cya-
Pigments nobacterium Synechococcus 7002, which has been engineered to express the Chl f synthase gene. By optimizing
Time-resolved fluorescence
growth light conditions, one-to-four Chl f pigments were found in the complexes. By using a range of spectro-
Excitation energy transfer
scopic techniques, isolated PSI trimeric complexes were investigated to determine how the insertion of Chl f
affects excitation energy transfer and trapping efficiency. The results show that the Chls f are functionally
connected to the reaction center of the PSI complex and their presence does not change the overall pigment
organization of the complex. Chl f substitutes Chl a (but not the Chl a red forms) while maintaining efficient
energy transfer within the PSI complex. At the same time, the introduction of Chl f extends the photo-
synthetically active radiation of the new hybrid PSI complexes up to 750 nm, which is advantageous in far-red
light enriched environments. These conclusions provide insights to engineer the photosynthetic machinery of
crops to include Chl f and therefore increase the light-harvesting capability of photosynthesis.

1. Introduction minimal energy required to support photochemical oxidation of water

[9,13–15]. However, it was found that the cyanobacterium Acaryo-
Photosystem I (PSI) is a light-driven plastocyanin/cytochrome c6: chloris extends this limit by using Chl d [16], a pigment absorbing at
ferredoxin/flavodoxin oxidoreductase composed of eleven or twelve 30 nm longer wavelengths than Chl a [17,18]. More recently, Chl f, the
polypeptides [1,2]. The PSI core complex binds 95 chlorophyll (Chl) a longest-wavelength chlorophyll known (max Qy = 706 nm in organic
molecules, 22 β-carotenes, 2 phylloquinones (or menaquinone-4), and solvents) [19,20], and the enzyme responsible for its synthesis from Chl
three [4Fee4S] clusters [3–7]. The Chls are exclusively Chl a in algal, a or Chlide a (Chl f synthase [21]) were discovered. Chl f is produced by
plant and most cyanobacterial PSI core complexes [5,8]; 89 Chl a mo- some terrestrial cyanobacteria when they grow in environments en-
lecules form the core antenna, while the additional six participate in the riched in far-red light (FRL) (λ = 700–800 nm [22]) such as soils,
electron transport chain. PSI is remarkably efficient in converting light rocks, caves, microbial mats, and under the shade of plants. These or-
energy into stored chemical potential energy, with an overall quantum ganisms are able to perform Far-Red Light Photoacclimation (FaRLiP)
efficiency close to 100% [9–12]. PSI possesses photochemically active [22–24], which involves the synthesis of Chl d and f, in addition to the
pigments absorbing at 700 nm. This wavelength has for a long time common Chl a, and substitution of pigment-binding subunits with
represented the “red limit” of oxygenic photosynthesis, meaning the products of gene variants from a 21-gene cluster [22,24]. These

Abbreviations: CS, charge separation; Chl, chlorophyll; CD, Circular Dichroism; DAS, decay-associated spectra; EAS, evolution associated spectra; EET, excited
energy transfer; ETC, electron transport chain; FaRLiP, far-red light photoacclimation; FRL, far-red light; FWHM, full-width at half max; GL, green light; HPLC, high
performance liquid chromatography; IRF, instrument response function; LL, low-intensity white light; PAR, photosynthetically active radiation; PSI, Photosystem I;
PSII, Photosystem II; RC, reaction center; RT, room temperature; SAS, species associated spectra; WT, wild type; WL, cool-white fluorescent light
⁎
Corresponding author.
E-mail address: [email protected] (R. Croce).

https://doi.org/10.1016/j.bbabio.2020.148206
Received 22 January 2020; Received in revised form 1 April 2020; Accepted 14 April 2020
Available online 17 April 2020
0005-2728/ © 2020 Elsevier B.V. All rights reserved.
M. Tros, et al. BBA - Bioenergetics 1861 (2020) 148206

combined changes lead to extensive remodeling of the photosynthetic Pigments were extracted from purified PSI complexes and analyzed by
apparatus and allow FaRLiP organisms to perform oxygenic photo- procedures described in Shen et al. [35]. The HPLC data were processed
synthesis using FRL. In fact, PSI and PSII gain absorbance up to 800 nm using Agilent ChemStation software (revision B.02.01-SR1 6100 series).
due to the presence of Chls d and f associated with paralogous PSI and The number of Chl f molecules per PSI complex was calculated from the
PSII subunits [7,23,25]. Recent studies have shown that Chl f is asso- % composition of Chl a and f in the complexes as determined by re-
ciated with both FRL-PSI (8%) and FRL-PSII (11.4–12.7%), while Chl d versed-phase HPLC analysis and by assuming that a PSI monomer
is exclusively associated with FRL-PSII (2.9–4%) [25,26]. contains 95 total Chl molecules [7,40].
The discovery of Chl d and Chl f in cyanobacteria [16,19,27,28],
and in particular of a Chl f synthase enzyme [21], has opened the way 2.4. Absorption, Circular Dichroism and steady state fluorescence
to the production of these pigments in algae or higher plants, with the measurements
potential to enhance photosynthetic efficiency and crop productivity. It
was estimated that by expressing Chl f, the photosynthetically active Room temperature (RT) absorption spectra of isolated PSI com-
radiation (PAR) could be expanded into the FRL region [12,21], in- plexes were recorded with a Cary 4000 spectrophotometer (Agilent
creasing the number of available photons by 19% when extended to Technologies, Santa Clara, CA United States). The Circular Dichroism
750 nm [29,30]. The suggested approach, following a smart canopy (CD) spectra were measured using a Chirascan CD Spectrophotometer
concept [31], could increase the light-harvesting capacity in the lower (Applied Photophysics, Leatherhead, United Kingdom) with samples at
leaves of the canopy or at the bottom of a pond/bioreactor, that mainly OD 0.5 at the maximum of the Qy absorption band. The buffer condi-
receive light enriched in far-red wavelengths [29,31–33]. The expan- tions of the samples were kept the same as the stock solutions of the
sion of the photosynthetically active radiation could also provide an purified PSI complexes, i.e. in 50 mM MES buffer (pH 6.5) containing
advantage in non-saturating light conditions. Recent evidence demon- 10 mM CaCl2, 10 mM MgCl2, 0.05% (w/v) β-DM and 5% (w/v) gly-
strated that the efficiency of photosynthetic energy storage in a cya- cerol. The fluorescence emission spectra were recorded at 77 K and RT
nobacterium producing red-shifted Chls is comparable or higher than using a Fluorolog 3.22 spectrofluorimeter (Jobin Yvon-Spex,
that in Chl a-containing species [34]. However, whether photosynthesis Longjumeau, France). The excitation wavelength was 500 nm and
would be efficient in genetically engineered organisms expressing Chl d emission was detected in the 600–850 nm range. Excitation and emis-
or f, in combination with their WT photosynthetic proteins is still an sion bandwidths were set to 3 nm. All fluorescence spectra were mea-
open question. Recently, the Chl f synthase was successfully expressed sured at OD 0.05 at the maximum of the Qy absorption band. For 77 K
in the non-FaRLiP cyanobacterium Synechococcus sp. PCC 7002 (here- measurements a liquid nitrogen cooled device was used (Nitrogen Cold
after Synechococcus 7002) [21,35]. Depending on the growth light finger).
conditions, the PSI of strains expressing the chlF gene accommodate a
different number of Chl f pigments depending upon strain background 2.5. Time-resolved fluorescence measurement
and growth conditions [35]. In this work, by means of time-resolved
fluorescence spectroscopy, we quantitatively investigated how the in- Time-resolved fluorescence decays of isolated PSI complexes were
serted Chls f are functionally connected to the bulk Chl within PSI and recorded with a Hamamatsu C5680 synchroscan streak camera, com-
how their presence influences the quantum efficiency of PSI. bined with a Chromex 250IS spectrograph. A grating of 50 grooves per
mm and blazed wavelength of 600 nm was used. The central wave-
2. Materials and methods length was set at 720 nm, giving a detection range from 590 nm to
860 nm and a time range from 0 to 350 ps (TR2 temporal response of
2.1. Strains and growth conditions 5–6 ps) was chosen. The 400 nm excitation light was vertically polar-
ized, the spot size diameter was typically ~100 μm, and the laser re-
Synechococcus sp. PCC 7002 (hereafter Synechococcus 7002) wild petition rate was 250 kHz [41]. The laser power was set to 200 μW,
type (WT) and the engineered strain WT::ChlF with heterologous ex- after a careful power-dependent study confirmed the absence of anni-
pression of the chlF gene of Fischerella thermalis PCC 7521 (hereafter hilation in these conditions (see power-dependent studies in Supple-
Ft7521 [35]) were grown in liquid A+ medium under standard condi- mental Fig. S2). Approximately 500 μL sample with optical density of
tions as previously described: 250 μmol photons m−2 s−1 cool-white 0.5 at the maximum of the Qy absorption band was measured at RT. The
fluorescent light (WL), 38 °C, and sparging with 1% (v/v) CO2 in air sample was magnetically stirred in a cuvette with speed of 750 rpm and
[36]. The ΔPSII::ChlF mutant, obtained by deleting the psbD1 and psbD2 to avoid reabsorption the excitation laser was focused in the sample
genes but expressing the chlF gene from Ft7521 [35], was grown under close to the cuvette walls. The averaged image was corrected for
different light qualities: low-intensity white light (LL: ~10 μmol pho- background and shading, and then sliced into traces of ~2 nm width.
tons m−2 s−1), green light (GL: 11 μmol photons m−2 s−1) and FRL
(25–30 μmol photos m−2 s−1) and the medium was supplemented with 2.6. Global and target data analysis
20 mM glycerol [37]. Differences in light qualities were obtained with
neutral density filters as described previously [22,23]. Data obtained with the streak-camera setup were first globally
analyzed with the R package TIMP-based Glotaran [42]. The metho-
2.2. Purification of trimeric Photosystem I complexes dology of global analysis is described in [43,44]. The datasets were
analyzed with a sequential model, the evolution associated spectra
PSI complexes were purified from WT, WT::ChlF, ΔPSII::ChlF cells (EAS) from the resulting analysis were used to calculate the decay as-
grown at different light conditions by sucrose gradient centrifugation of sociated spectra (DAS). The instrument response function (IRF) was
membrane solubilized proteins, following procedures described pre- fitted with a single Gaussian with a full width at half max (FWHM) of
viously [35,38,39]. Purified PSI complexes were resuspended in MES 5–6 ps. The amplitudes of the resulting DAS are normalized to the first
buffer (pH 6.5) containing 10 mM CaCl2, 10 mM MgCl2, 0.05% (w/v) β- EAS, which is the t0 spectrum. Disregarding the longest lifetime which
D-dodecyl maltoside (DM) and 5% (w/v) glycerol. is attributed to free pigments, the average decay lifetimes τavg could be
calculated from the sum of all lifetimes weighed by the relative am-
2.3. Pigment extraction and analysis plitudes of each of the DASs: τavg = Σ(τn*An) / Σ(An). In which An is the
amplitude calculated as the area under the DAS of component n and τn
Cyanobacterial cells were harvested by centrifugation and washed its respective decay lifetime. This average lifetime, representing the
once in 50 mM HEPES pH 7.2 prior to pigment extraction and analysis. time in which charge separation (CS) occurs, can be used to calculate

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M. Tros, et al. BBA - Bioenergetics 1861 (2020) 148206

Fig. 1. Spectroscopic properties at RT of isolated PSI complexes: Absorption spectra (A), fluorescence emission (λexc = 500 nm) (B) and CD spectra (C). The absorption and fluorescence spectra are normalized to the
the efficiency of the PSI complex. This quantum efficiency of CS, ΦCS, is
calculated with ΦCS = 1–(τavg / τnoCS) where τnoCS is the decay time of
the excitation when no CS occurs. For τnoCS the natural fluorescence
decay rate of free Chl a (0.2 ns−1) is used.
In a target analysis the datasets of all five samples were simulta-
neously fitted with the same kinetic model (Fig. 3A). In this model the
PSI core complexes were described with three compartments for re-
spectively the bulk Chl a, red Chl a and the newly introduced Chl f
pigments. Additionally, two compartments had to be added to account
for the small but variable amounts of free Chl a and free Chl f with ns-
lifetime emission. For the PSI0-Chlf sample, which did not contain Chl f,
the bound and free Chl f compartments were removed in the analysis.
As 400 nm excitation light was used all compartments are populated
from the Soret compartment (see Supplemental Methods S1 for more
details).

3. Results

3.1. Integration of Chl f into the native PSI protein of Synechococcus 7002

Like the majority of cyanobacteria, also Synechococcus 7002 syn-

thesizes only Chl a, which is incorporated into PSI and PSII. The Chl f
synthase gene (ChlF) from Fischerella thermalis PCC 7521 was hetero-
logously expressed in Synechococcus 7002 WT and ΔPSII strains, as
previously described [21,35]. The resulting strains, designated
WT::ChlF and ΔPSII::ChlF, respectively, were grown at different light
intensities and using different light colours (white, green, far-red) [35].
From these strains, the PSI trimeric complexes were isolated by sucrose
density gradients and subsequently analyzed with different techniques.
The properties of these complexes were compared with those of PSI
trimers purified from the WT Synechococcus 7002 strain.
HPLC analysis of PSI pigment extracts showed that Chl f was bound
to the PSI complexes (Table 1). However, the content of Chl f per PSI
varied according to the different growth light conditions, from a
minimum of ~1 Chl f per PSI (WT::ChlF in WL) to a maximum of ~4 Chl
f per PSI (ΔPSII::ChlF in FRL) [12,35].
Absorption spectra of isolated PSI complexes containing Chl f re-
vealed a long-wavelength shoulder when compared to WT PSI (Fig. 1A).
The shoulder increased in intensity as the content of Chl f per PSI in-
creased (Fig. 1A insert). Similarly, at room temperature (RT) the
fluorescence emission at 720 nm (Fig. 1B) increased with the number of
Chl f per complex, and at 77 K the emission band gradually shifted from
714 nm for PSI-0Chlf to 719 nm for PSI-4Chlf (Fig. S1). The CD spectra
of these complexes were not significantly different from those of the WT
(Fig. 1C), which suggested that no major changes in the overall pigment
organization had occurred to accommodate the Chl f molecules. maximum and the CD spectra are normalized to the absorption.

3.2. Excitation energy transfer and trapping in PSI-Chl f-containing

complexes

Time-resolved fluorescence measurements with excitation at

400 nm were performed on the PSI complexes with a streak camera
setup, and the data were globally analyzed. The kinetics of all the PSI
complexes isolated from different strains/growth conditions could be
well-fitted with four components. In Fig. 2 and Table 2 respectively, the

Table 1
Strains, growth conditions and calculated Chl f amounts per PSI monomeric
complex quantified from HPLC analysis.
Name of the strain Growth conditions # of Chl f per PS I Short name

WT White light (WL) 0 (0%) PSI-0Chlf

WT::ChlF White light (WL) 1.1 (1.1%) PSI-1Chlf
ΔPSII::ChlF Low light (LL) 1.6 (1.7%) PSI-1.5Chlf
Green light (GL) 2.6 (2.7%) PSI-2.5Chlf
Far-red light (FRL) 3.8 (4.0%) PSI-4Chlf

3
M. Tros, et al. BBA - Bioenergetics 1861 (2020) 148206

Fig. 2. Decay Associated Spectra (DAS) obtained from time-resolved fluorescence emission measurements performed at RT upon 400 nm excitation on the PSI
samples normalized to the T0 spectrum of each sample.

decay associated spectra (DAS) and corresponding lifetimes of the PSI 0Chlf) has a maximum at 690 nm. However, a broad shoulder is present
samples are displayed. in the far-red region, which can be assigned to red-shifted Chls a
The first component (Fig. 2A) with a lifetime of 4–6 ps represents [11,45]. The insertion of Chl f in PSI results in a significant change of
excitation energy transfer (EET) from bulk Chls a to red Chls a and f DAS3: PSI-1Chlf and PSI-1.5Chlf show a new peak at λ = 721 nm (grey
(when present). The red-shifted bleach in the spectra of PSI-2.5Chlf and and blue traces in Fig. 2C), the relative amplitude of which increases in
PSI-4Chlf compared to the other samples can be assigned to Chl f, the the samples with 2.5 Chl f and 4 Chl f per PSI (green and red traces in
content of which is higher in these complexes. The second component Fig. 2). The DAS3 lifetime increases only slightly at increasing Chl f
(16–19 ps) is dominated by the decay; it accounts for most of the content (from 40 to 60 ps, see Table 2), indicating that the Chls f mo-
trapping and has a similar lifetime in the five complexes. A small but lecules are very well connected to the reaction center (RC) and that
significant broadening of DAS2 is observed in the samples with higher trapping still occurs very efficiently.
Chl f content (Fig. 2B), especially in the spectra of the PSI-2.5Chlf and DAS4 (Fig. 2D) has a very small amplitude and a lifetime of about
PSI-4Chlf samples. Similar to DAS2, DAS3 (Fig. 2C) of WT PSI (PSI- 4 ns for all samples (Table 2). It represents the contribution of

Table 2
Results from global analysis. Each dataset is normalized to the T0 spectrum. The average lifetimes τavg were calculated (disregarding the longest lifetime) with the
formula τavg = Σ(τn*An) / Σ(An). The quantum efficiency of charge separation (CS) ΦCS was calculated with ΦCS = 1–(τavg / τnoCS) where τnoCS is the free Chl a decay
rate of 0.2 ns−1.
Sample A1 (%) τ1 (ps) A2 (%) τ2 (ps) A3 (%) τ3 (ps) A4 (%) τ4 (ns) τavg (ps) ΦCS (%)

PSI-0Chlf 20.3 4.5 58.1 16.5 17.6 35.2 4.0 4.74 17.4 99.7
PSI-1Chlf 18.3 5.7 66.8 18.1 13.6 42.4 1.4 6.09 19.1 99.6
PSI-1.5Chlf 14.8 5.2 68.0 18.9 12.4 47.1 4.8 3.84 20.4 99.6
PSI-2.5Chlf 21.7 5.9 54.8 18.6 19.7 61.0 3.8 4.46 24.4 99.5
PSI-4Chlf 20.7 5.0 54. 17.7 20.9 59.6 3.7 4.91 24.1 99.5

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M. Tros, et al. BBA - Bioenergetics 1861 (2020) 148206

Fig. 3. Model used for the simultaneous target analysis of all datasets from Chl f-containing PSI samples (A). The full model is shown in Supplemental information
(Fig. S3). Estimated SAS of Bulk Chl a, Red Chl a, Chl f and free Chls a and f (B).

disconnected Chls a (max at ~680 nm) and possibly Chl f (max at compartments were assumed to be equal among the samples. Only for
~720 nm). the PSI-4Chlf sample these rates had to be adapted slightly. This as-
The presence of Chl f in the PSI complexes results in an increased sumption is supported by the Chl f emission bands at λ = 721 nm in the
average lifetime τavg (Table 2) in all samples with the exception of PSI- steady-state emission spectra (Fig. 1B) and DAS3 from the global ana-
4Chlf. Despite the increase of τavg, in all samples, the trapping is still lysis (Fig. 2C). The increasing amplitude of this band indicates the in-
very fast in comparison to the intrinsic decay kinetics of the pigments, tegration of a different number of Chl f molecules in the complexes.
and thus the quantum efficiency of CS ΦCS (Table 2) remains high. However, the central wavelength of the band is independent on the
number of integrated Chl f, indicating a similar environment of all in-
serted Chls f. Moreover, this central wavelength is very similar to that of
3.3. Target analysis Chl f in solvent (Pyridine [48]). If a Chl f would replace one of the red
Chls a the strong interaction between Chls is expected to result in a
To understand better how the insertion of Chl f influences excitation significantly more red shifted band (> 720 nm). Although the frac-
energy transfer and trapping kinetics within the new hybrid PSI com- tional binding stoichiometry of PSI-1.5Chlf and PSI-2.5Chlf indicate
plexes, all datasets were simultaneously subjected to a target analysis. that these samples contain heterogeneous populations of complexes
The kinetic model was based on the isolated PSI core model with different amounts of Chl f, they can still be described with a
[41,46,47], consisting of two compartments to describe the bulk and homogeneous kinetic model as the physiochemical environment of the
red Chls a (respectively, black and red boxes in Fig. 3A). An extra integrated Chls f is very similar.
compartment was included in the model to account for a small amount As demonstrated in Fig. S4, this kinetic model fits the datasets very
of disconnected/free Chl a pigments (blue box in Fig. 3A). These three well, and the additional emission attributable to Chl f is well visible.
compartments were used to describe the PSI-0Chlf sample. As in the The estimated SAS resulting from the simultaneous fit of all the samples
global analysis the maximum of DAS3 is at the same position as in DAS2 are displayed in Fig. 3B and the estimated kinetic rates are listed in
and its intensity is relatively small, only one compartment was used for Table S1. The decay rate of the bulk compartment from which CS occurs
the red Chls a. For the PSI complexes containing Chl f, two additional was estimated to be kT = 64 ns−1, which is similar to the trapping rates
compartments were required: one representing Chl f connected to the reported before for isolated PSI [46,47,50,51]. The first SAS (black)
bulk Chl a compartment and a second one representing the small represents the spectrum of the bulk Chl a, peaking at 690 nm. The SAS
amount of free Chl f (respectively, magenta and green boxes in Fig. 3A), for the Red Chl a (red) and Chl f (pink) compartments are well sepa-
observed in the global analysis. Trapping was only allowed to occur rated. The SAS of the Red compartment is centered at 715 nm, which is
from the bulk compartment and, as it was assumed that no Chl f was in agreement with the emission of the most red-forms of Synechococcus
inserted in the RC, the trapping rate kT was set to be equal for all 7002 [52]. The spectrum is also broader than that of the other com-
samples. The decay rates of the PSI red and the free Chl f compartments partments, which is a typical property of the spectra of the far-red forms
were set to be equal to the intrinsic decay rate of free Chl a [47,51,52]. The emission spectrum of the Chl f (pink) compartment
ki = 0.2 ns−1 because the excited state lifetime of Chl f in solvents is peaks at 723 nm, which matches the wavelength of the red band seen in
very similar to that of Chl a [48]. The amplitudes of the Species Asso- the RT emission spectra of PSI-2.5Chlf and PSI-4Chlf (Fig. 1B). The
ciated Spectra (SAS) for the red and the Chl f compartments were set to increasing Chl f content results in an increase in the energy transfer
zero below 685 nm and 690 nm, respectively. Equal area constraints for rates k3 and k4 between the bulk and Chl f compartments (Table S1)
the area under each SAS were applied and the spectrum of free Chl f with the equilibrium shifting to the latter one. The free Chl a SAS (blue)
was assumed to be equal to the SAS of Chl f within the complex. The peaks at 678 nm with a distinct long vibrational band in the red. The
area of the Chl f SAS is expected to be very similar to the one of Chl a, amounts of free Chls a and f in all samples are listed in Table S2 (see
because the extinction coefficients of the two Chls were found to be Supplemental Methods S1 for more details).
almost the same [49]. Although the position of Chl f emission band The time dependence of the concentrations of each compartment for
(721 nm) is very close to that of the pigment in solvent (Pyridine [48]), the different samples is given by the amplitude matrices, which are
we cannot exclude the possibility that the spectrum of Chl f bound to displayed in Table 3. The matrix shows the lifetime and corresponding
the protein differs from that of the pigment in the buffer solution. amplitudes of the population (negative amplitude) and depopulation/
However, as the concentration of free Chl f is very low and its fluor- decay (positive amplitude) of each compartment [41,51,53]. When
escence decay is much longer than the energy trapping in the PSI RC, it focusing on the Red and Chl f compartments, it is seen that in all
will not have a large effect on the calculations. Moreover, the free Chl f samples the first lifetime component of around 7 ps represents energy
signal observed in DAS4 of the global analysis (Fig. 2D) peaks at a transfer from bulk to red Chls and a minor portion of the Chl f. The
comparable position with the bound Chl f (Figs. 2C, 1B). In the fit it was second component of 15–20 ps encompasses two major events, namely
assumed that the insertion of Chl f did not affect the red Chl a forms, i.e. i) trapping of the energy from the Red compartment and ii) population
the energy transfer rates k1 and k2 between the bulk and red

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M. Tros, et al. BBA - Bioenergetics 1861 (2020) 148206

Table 3 trapping rate that is approximately two and a half times lower than the
Amplitude matrices of all samples calculated from results target analysis. trapping from the red Chl a molecules. As a result, the overall trapping
Sample τ (ps) Amplitude compartment Trapped slowly time in the Chl f-containing PSI is longer than in the WT complexes, but
it is still far shorter than that the intrinsic decay processes of the pig-
Red Chl f Bulk Chl ments, assuring a high trapping efficiency (99.5% or greater).
Target analysis supports the view that the presence of Chl f does not
PSI-0Chlf 7.18 −0.204 – 0.375
20.8 0.199 – 0.593 substitute the far-red Chl a forms in any of the samples. In agreement
– – – – 0% with Kurashov et al. [12], our data also suggest that the Chl f pigments
PSI-1Chlf 7.14 −0.210 −0.005 0.396 in these hybrid PSI-Chlf complexes are not associated with the electron
20.1 0.201 −0.031 0.583 transport chain (ETC) in the RC subdomain: if Chl f would be present in
46.4 0.005 0.036 0.020 3.5%
the ETC, no Chl f fluorescence would be emitted, while Chl f emission is
PSI-1.5Chlf 7.07 −0.205 −0.015 0.401
17.1 0.122 −0.095 0.296 observed in all the samples and its intensity increases with the number
26.2 0.078 0.110 0.274 10.8% of Chls f associated with the complexes (Figs. 1B-2C).
PSI-2.5Chlf 6.90 −0.207 −0.041 0.445 Notably, the Chls f bound to the PSI subunits of Synechococcus 7002
16.7 0.177 −0.147 0.417
are not as red-shifted as the Chls f bound to the PSI subunits of FaRLiP
44.2 0.027 0.186 0.115 18.2%
PSI-4Chlf 4.55 −0.211 −0.023 0.329
cyanobacteria expressed under FRL [23], which extend the absorption
15.1 0.174 −0.191 0.523 region to approximately 800 nm [12,25,35]. However, the addition of a
44.7 0.030 0.211 0.128 21.5% small number of Chls f in the Synechococcus 7002 PSI complexes with
WT sequence still permits to extend the photosynthetically active ra-
diation up to 750 nm. In the complexes analyzed in this work the en-
of the Chl f from the bulk. This energy, and the remaining energy from hancement in FRL absorption > 700 nm is limited up to 7% of the total
the red Chls, is trapped in ~45 ps (third component). As expected the QY absorption (λ = 600–750 nm) (Fig. 4A, red). Although this en-
amplitudes of the Chl f compartment increase with the increase in Chl f hancement is rather small, it can have a large effect in FRL-enriched
content. Because the trapping time of ~45 ps is still much faster than environments. To quantify this effect, we calculated the wavelength
the natural fluorescence decay time of Chl (5 ns), the depopulation of distribution of the photons absorbed by the PSI complexes (Awl) at the
PSI excited states almost exclusively occurs by charge separation. The bottom of a dense plant canopy [22,33] (Fig. 4). The effective absorp-
percentages of excitation trapped slowly were calculated from these tion of the PSI complexes in this environment is calculated by i) nor-
depopulation amplitudes and are listed in Table 3. In contrast to the malizing the absorption spectra of Fig. 1A to their respective area (total
global analysis, in this target analysis the contribution of the Red Chls a QY absorption, range λ = 600–750 nm, Fig. 4A, black and red) and (ii)
and Chls f can be resolved and quantified completely. In Table 3, the multiplying these normalized spectra with the irradiance spectrum of
amplitude of the Chl f decay with ≈44 ps differs significantly for PSI- the light under the leaf canopy (Fig. 4A, blue). In the resulting spectra
2.5Chlf and PSI-4Chlf, being 0.186 and 0.211, respectively. (Fig. 4B), showing the light that is absorbed by the PSI complexes under
a dense plant canopy, the absorption in the far-red significantly in-
4. Discussion creases with the number of integrated Chl f. From these spectra, the
share of absorbed far-red photons is calculated by Awl > 700nm =
To address how photosynthetic efficiency is affected by the presence 750 750
700 wl / 600 wl . Similarly, Awl > 700nm was calculated for the com-
A A
of far-red-absorbing chlorophylls in engineered systems, we character- plexes in unshaded sunlight [22,33] (Fig. 4A, purple). The absorption
ized PSI complexes differing in the number of Chl f molecules. In these enhancement due to Chl f is quantified by comparing the absorption of
complexes, a Chl f content up to 4% could be reached in optimized the Chl f-containing complexes with that of PSI-0Chlf
750 750 750
growth conditions (Table 1) [35], which represents 50% of the Chl f (RAbs = ( 600 Awl A (PSI-0Chlf))/ 600 Awl (PSI-0Chlf)). The re-
600 wl
content in the PSI of FaRLiP cyanobacterial strains when they are grown sults are shown in Table 4.
in FRL [24,25]. It should be noted that while in the FaRLiP strains the Under the shade of a dense plant canopy a significant part of charge
Chls f are associated with paralogs of the PSI subunits only expressed in separation events would be caused by far-red photons (Fig. 4B), as
FR light, in the Synechococcus 7002 strains, they are associated with the plants absorb little light > 700 nm [33,54] (Fig. 4A, blue). Here more
WT proteins of this model cyanobacterium. The successful integration than half of the red light absorbed by the 4 Chl f pigments (3.8%) is FRL
of Chl f into the PSI complexes of this cyanobacterium that normally (Fig. 4B, Table 4), leading to a 50% increase in total light energy ab-
contain only Chl a, under different conditions [35], resulted in hybrid sorption compared to PSI-0Chlf (Table 4). Obviously, increasing the
complexes with increased far-red absorption and emission (Fig. 1). number of Chl f molecules by a factor of two and shift their absorption
The results presented here show that the Chl f pigments are func- further to the red to achieve the properties typically observed in FRL-
tionally connected to the other pigments in the complexes. As expected, PSI complexes of FaRLiP cyanobacteria would produce a much greater
the low-energy states of the Chl f pigments function as local traps with a impact on photosynthesis by leaves beneath the canopy.

Fig. 4. A) Absorption spectra of PS-0Chlf (black) and

PSI-4Chlf (red) normalized to the total QY absorption
(λ = 600–750 nm) and relative spectral photon
fluence (irradiance) of unshaded sunlight (daylight)
and light filtered by a dense leaf canopy (canopy)
taken from Gan and Bryant [22]. B) Wavelength
distribution Awl of photons absorbed under a dense
plant canopy by the PSI complexes.

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