Natural strategies for light harvesting in oxygenic photosynthesis: from excess light to
shade
Mascoli, V.
2021
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Mascoli, V. (2021). Natural strategies for light harvesting in oxygenic photosynthesis: from excess light to shade.
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Chapter 5
Photosynthesis with chlorophyll f: trade-off between far-red
absorption and light-use efficiency
Plants and cyanobacteria use the chlorophylls embedded in their photosystems to absorb
photons and perform charge separation, the first step of solar to chemical energy
conversion. While oxygenic photosynthesis is primarily based on chlorophyll a
photochemistry, which is powered by red light, a few cyanobacterial species can harness
less energetic photons when growing in far-red light. Acclimation to far-red light involves
the incorporation of a small number of red-shifted chlorophylls f in the photosystems,
whereas the most abundant pigment remains chlorophyll a. Due to its different energetics,
chlorophyll f is expected to alter the excited-state dynamics of the photosynthetic units
and, ultimately, their performances. Here we combined time-resolved fluorescence
measurements on intact cells and isolated complexes to show that chlorophyll f insertion
slows down the overall energy trapping in both photosystems. While this marginally
affects the efficiency of photosystem I, it decreases substantially that of photosystem II.
Nevertheless, we show that despite the lower energy output, the insertion of red-shifted
chlorophylls in the photosystems remains advantageous in environments that are enriched
in far-red light and therefore represents a viable strategy for extending the photosynthetic
active spectrum in other organisms, including plants. However, careful design of the new
photosynthetic units will be required to preserve their efficiency.
4
This chapter is based on the following publication:
Mascoli, V., Bersanini, L., and Croce, R. (2020). Far-red absorption and light-use efficiency trade-offs in chlorophyll f photosynthesis. Nat. Plants 6, 1044–1053
152
Introduction
Cyanobacteria are organisms that convert visible photons into chemical energy via
oxygenic photosynthesis
337. Most of them use Chlorophyll a (Chl a) as photochemically
active pigment and, for a long time, the absorption wavelength of Chl a (680 nm in
Photosystem II, PSII, and 700 nm in Photosystem I, PSI) was assumed to be the “red
limit”, i.e. the longest wavelength able to power photosynthesis
35. This was a central
dogma in photosynthesis until it was found that the cyanobacterium Acaryochloris Marina
(AM) performs photochemistry using Chlorophyll d (Chl d)
269,270,338, which absorbs in the
near IR (above 720 nm). More recently, new species containing an even more red-shifted
pigment, Chlorophyll f (Chl f), have been discovered
2,24. Like “conventional”
cyanobacteria, these strains use Chl a to drive photosynthesis when grown under visible
light
251,339. However, they are able to undergo a chromatic acclimation that allows them to
perform oxygenic photosynthesis when only far-red light (FRL, 700 nm < 𝜆 < 800 nm) is
available
2,251,253. This acclimation, referred as FaRLiP (Far-Red Light Photoacclimation),
occurs via extensive remodeling of their photosynthetic apparatus, involving the
expression of PSI and PSII paralogs (hereafter referred as photosystems).
FRL-photosystems incorporate the newly synthesized Chl f (and smaller amounts of Chl d)
2,248,
whose absorption wavelength can extend up to 750-800 nm
2,251,253. Even though Chl a
remains their most abundant pigment, Chl f (about 10% of total Chl
251–253) was found to be
located both in FRL-PSI
253–255and FRL-PSII
253,258, resulting in a marked red-shift of their
emission. The acclimation also affects the phycobilisomes (PBS, the major cyanobacterial
antenna complexes), as paralogous allophycocyanin subunits are expressed forming
bicylindrical cores emitting at 720-730 nm
259,260. Furthermore, in some FaRLiP strains, the
FRL-adapted cells can partly retain the photosynthetic complexes present in visible
light
258. Chl f was initially believed to perform only a light-harvesting function
340.
However, later studies located this pigment not only in the antenna, but also in the
reaction centers (RCs), at least in that of PSII, demonstrating that it is photochemically
active
253,256,257,341,342.
Due to their recent discovery, only a few time-resolved spectroscopic studies have been
performed so far on FaRLiP systems. Some of these studies have focused on the energy
equilibration within and between photosynthetic units
258,343–345in vivo, mostly at cryogenic
temperatures, whereas kinetic studies focused on charge separation in isolated
photosystems have been restricted to FRL-PSI only
341,346. A detailed and comprehensive
investigation of the effects of FaRLiP on the energy equilibration and trapping dynamics
in FRL-photosystems is still missing, especially under physiological conditions. This
knowledge is highly relevant for assessing the performance of oxygenic photosynthesis in
the presence of Chl f, which might differ markedly from that attained in Chl a-only
photosystems. Indeed, the insertion of a relatively small number of Chls f in the antenna
of each photosystem can potentially lead to the formation of new energy traps competing
with charge separation at the RCs. In addition, the incorporation of Chl f in the RCs is
expected to change their energetics, thereby affecting the kinetics and thermodynamics of
153
charge separation. Here we address these questions by measuring the excited-state kinetics
of FRL-photosystems via time-resolved fluorescence (TRF) at room temperature. We
performed experiments in vitro on the isolated photosystems and used the results of these
measurements to interpret our in vivo measurements, in which we also compared the
performances of cells adapted to either white light (WL) or FRL. In the following, we will
focus on a specific FaRLiP strain, Chlorogloeopsis fritschii PCC 6912 (CF). However,
since acclimation responses to FRL exhibit a certain degree of variability in FaRLiP
cyanobacteria
2,255,259,260,347, we also studied another organism, Chroococcidiopsis thermalis
PCC 7203 (CT). Experimental data from the latter species are shown extensively in the
Supplementary Information. These results support all the main findings presented in the
main text, but also highlight some differences in the acclimation of the two strains to FRL.
Materials and methods
Cell cultures. The strains CF and CT were obtained from the Pasteur Culture Collection
(Institut Pasteur, Paris, France) and grown at 30 ºC in BG11
348medium supplemented
with 20 mM HEPES-NaOH (pH = 8.0). Acaryochloris marina MBIC 11017 (NBRC
102967, AM) was obtained from Biological Resource Center Collection (NBRC, NITE,
Chiba, Japan) and grown at 30 ºC in IMK medium (Daigo IMK medium, product of
Nihon Pharmaceutical Co., Ltd.). AM, CF and CT were grown under white light (WL) of
30 µmol photons m
-2s
-1starting from OD
750
= 0.1 cm
-1and collecting cells for
experiments at OD
750= 0.8-1.0 cm
-1. CF and CT cells were grown as well under far red
light (FRL, 738 nm; Jazz) of 45 µmol photons m
-2s
-1for 2.5 to 3 weeks prior to
experiments, starting from OD
750= 0.3-0.4 cm
-1, with medium being refreshed every week
keeping an OD
750= 1.0-1.5 cm
-1. The cultures were cultivated in Erlenmeyer flasks
shaking at 100 rpm.
Isolation of photosystems. Membrane fractions of CF and CT were isolated as previously
described for Synechocystis PCC 6803
349,350. Re-suspended thylakoids were solubilized
for 1 h in a buffer containing a final concentration of 1.5% n-dodecyl-b-maltoside
(b-DM), 25 mM BisTris at pH 7.0 and 10 mM MgCl
2(final Chl concentration of ~ 0.5
mg/ml). Un-solubilized material was eliminated by centrifugation (14000 rpm for 10 min
at 4 °C). Solubilized thylakoids were loaded on a sucrose density gradient made by
freezing and thawing 0.5 M sucrose, 50 mM MES-NaOH at pH 6.5, 5 mM CaCl
2, 10 mM
MgCl
2and 0.03% b-DM buffer and separated by ultra-centrifugation in a SW41 rotor at
35000 rpm for 14 h at 4 °C. PSI trimer band and PSII dimer band were subsequently
harvested (Figure S24). The PSII dimer fraction underwent a second round of sucrose
density gradient and ultracentrifugation as specified above. Purity was assessed by
absorption and fluorescence measurements.
Steady-State and Time-Resolved Fluorescence. Room-temperature (RT) absorption
spectra were acquired on a Varian Cary 4000 UV–Vis-spectrophotometer. The
spectrophotometer was equipped with an integrating diffuse reflectance sphere
(DRA-CA-154
50, Labsphere) when measuring on intact cells, in order to correct for light scattering. RT
fluorescence spectra were acquired at an OD < 0.05 cm
-1at Q
y
maximum on a HORIBA
Jobin-Yvon FluoroLog-3 spectrofluorometer.
Time-resolved fluorescence (TRF) measurements were performed with two methods.
Time-Correlated Single Photon Counting (TCSPC) data were acquired with a FluoTime
200 from PicoQuant. The excitation wavelength was 438 nm for all measurements, with a
repetition rate of 10 MHz (unless differently specified). A time window of 20 ns and a
time bin of 4 ps were used. Samples were measured at an OD < 0.1 cm
-1in a magnetically
stirred 1 cm × 1 cm cuvette kept at constant temperature (the sample volume was typically
1 mL). The detection polarization was set to magic angle with respect to the excitation
polarization.
Higher time-resolution fluorescence data were acquired with a Hamamatsu C5680
synchro scan streak camera, combined with a Chromex 250IS spectrograph. A grating of
50 grooves/mm and a blazed wavelength of 600 nm were used for all measurements,
covering a spectral window from 590 to 850 nm. The excitation wavelength was 400 nm
and the laser repetition rate 250 kHz. The excitation beam was vertically polarized and the
detection polarization was set to magic angle when specified. Two time windows were
typically used, one of 1.5 ns and one of 400 ps (the latter with a higher time-resolution,
see following text). Samples were measured at RT (unless differently specified) in a
magnetically stirred 1 cm × 1 cm cuvette. To keep the cells in open state, a sample volume
of 2 mL was used, stirring at 1250 rpm. Alternatively, measurements on cells in closed
state were performed with 1 mL of sample stirring at 600 rpm (after addition of
3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU, see following text). In order to avoid
significant reabsorption in the measurements, the excitation beam was focused in the
sample close to the cuvette wall and emission was collected at right angle close to the
entry point of the laser beam into the cuvette. The measuring time varied from 15 minutes
to 2 hours of CCD exposure. The averaged images were corrected for background and
shading, and then sliced into traces of ∼1.5-nm width prior to analysis.
All measurements with PSII in closed state (both with TCSPC and with the streak camera)
were preceded by addition of 50 µM DCMU and pre-illumination with white light for one
minute to fully close PSII RCs. More details about each type of experiment can be found
in the supplementary information.
Data analysis. Fluorescence traces were globally analyzed using a number of parallel
kinetic components. The total dataset can be described by the fitting function 𝐹(𝑡, 𝜆):
𝐹(𝑡, 𝜆) = { 𝐷𝐴𝑆
•(𝜆) ⋅ exp N−
𝑡
𝜏
•P ⊗ 𝐼𝑅𝐹(𝑡, 𝜆)
ƒ •„q
where each decay-associated spectrum (𝐷𝐴𝑆
•) is the amplitude factor associated with a
decay component k having a decay lifetime
𝜏
•. The instrument response function
155
(lifetime of around 6 ps)
312for TCSPC measurements (FWHM
∼ 90 ps) and from the
fitting in the case of streak camera measurements (FWHM
∼ 20 ps for the 1.5-ns time
window and ∼ 8 ps for the 400-ps time window).
For TCSPC data, the fitting quality was assessed by the χ
2value and by residual
inspection. More details about the global analysis methods can be found in van Stokkum
et al.
293Figure 1. Spectroscopic data of Photosystem I isolated from CF. a) 2D color map representing TRF data
of PSI from WL-adapted CF excited at 400 nm and detected in the Chl Qy region with a streak camera setup (see caption of Figure S1 for experimental details). b) DAS from global analysis of the TRF data in (a). The inset shows a magnification of the small long-lived component. c) Absorption spectra (normalized
156
at Qy maximum) of WL- and FRL-PSI particles isolated from CF. d) Time-integrated (steady-state) fluorescence spectra of PSI particles isolated from WL- (black lines) and FRL- (red lines) adapted cells of CF. The spectra of WL-PSI are obtained from the DAS in (b) (excluding the small long-lived component representing unconnected chlorophylls), those of FRL-PSI are calculated from the DAS in (f) (again excluding the minor long-lived component). e) 2D color maps representing TRF data of PSI from FRL-adapted CF. f) DAS from global analysis of the TRF data in (e). The inset shows a magnification of the small long-lived component. See Figure S2 for the raw/fitted traces. For each sample, the data are representative of two different preparations yielding similar results.
Results
Isolated Photosystem I. TRF experiments were performed on PSI from WL- and
FRL-adapted CF (Figure 1) and CT (Figure S1) to probe the effects of FaRLiP on its energy
landscape and dynamics. The excite-state kinetics of PSI from WL-adapted strains are
very similar to those of PSI from previously studied cyanobacteria growing in WL
108,351.
Energy transfer from blue to red Chls a (peaking around 715-720 nm, which is typical of
cyanobacterial PSI
108,352) takes place in around 10 ps, followed by trapping in about 40 ps
(Figure 1a-b). Upon FaRLiP, however, FRL-PSI shows a new absorption band above 700
nm (Figure 1c) and its emission spectrum drastically red-shifts due to Chl f insertion
(Figure 1d). The steady-state emission spectrum contains two main peaks, the largest at
about 750 nm, the other around 800 nm, suggesting that different Chl f spectral forms
coexist in the complex. The spectra and dynamics of these species are elucidated by
global analysis of TRF data (Figure 1e-f). The 400-nm laser excites mostly Chl a, which
is the most abundant pigment (Table S1). Following excitation, three downhill energy
transfer steps can be observed, shifting energy increasingly towards red. The first step
transfers excitations from bulk Chl a to Chl f and, possibly, red Chl a (black DAS). The
second step (red DAS) mainly involves transfer from Chls at 720 nm (red Chls a and the
blue-most Chls f) to Chl f at 750 nm, while the third and slowest component represents
equilibration between bulk Chl f and strongly red-shifted Chls f emitting at 790-800 nm
(blue DAS). Energy trapping occurs in 140 ps (green DAS), which is about 3.5 times
longer than in the PSI from the WL-adapted strains. The spectra and timescales are nearly
identical in the two FRL-adapted strains.
Isolated Photosystem II. Also PSII incorporates Chl f upon FaRLiP (see absorption
spectra in Figure 2a and Figure S3 and pigment contents in Table S1) and, consequently, a
drastic red-shift of its emission is observed. While PSII of cyanobacteria growing in WL
is known to emit at 680 nm at RT
89,90, FRL-PSII has a fluorescence peak at about 740 nm
(Figure 2b and Figure S3). TRF of FRL-PSII particles was measured with a TCSPC setup,
whose higher signal-to-noise ratio allowed using very low excitation powers. These low
powers are imperative to keep the RCs open (Figure S4). Similar to Chl a-only PSII
89,90,
the fluorescence kinetics of FRL-PSII of CF with open RCs is multi-exponential (Figure
2c): the two major components have lifetimes of approximately 200 ps and 1 ns (red and
blue DAS, respectively). A small > 3 ns component (magenta dashed DAS) can be
ascribed to a minor fraction of FRL-PSII particles with closed RCs.
157
Figure 2. Spectroscopic data of FRL-Photosystem II isolated from CF. a) Absorption spectrum of PSII
particles isolated from adapted CF. b) Normalized fluorescence spectra of PSII particles from FRL-adapted CF excited at different wavelengths. c,d) DAS from global analysis of TRF data from TCSPC measurements (438-nm excitation) of PSII particles isolated from FRL-adapted CF with nearly open (c) and closed (d) RCs. Details on the experimental conditions are given in the caption of Figure S3, and an overlay of the correspondent experimental and fitted TRF traces can be found in Figure S5. The small long-lived component in (c) is due to a minor fraction of particles with closed RCs. The black DAS in (d) is multiplied by 0.5 for a better comparison to the other DAS. e) Normalized TRF traces of FRL-PSII from CF with open (black) and closed (red) RCs. The instrumental response function (IRF) detected at 740 nm is shown in grey. f) Average PSII fluorescence lifetime as a function of wavelength (based on the DAS in (c,d). The black 10-ps energy transfer component in (d) was not included in the calculation of the average lifetime in closed state. Data in (a,b) are representative of two different preparations. Data in (c-e) are representative of 2 technical replicas. All experiments yielded similar results.
158
Upon addition of DCMU and increase of the excitation power, the RCs close and the
excited-state kinetics detected at 740 nm becomes substantially slower (Figure 2e). The
measured fluorescence kinetics of closed PSII is still multi-exponential (as observed in
several WL strains)
89,353: the fastest DAS (Figure 2d, in black) represents downhill energy
transfer from Chl a to Chl f taking place in about 10 ps (see also Figure S6). The
excited-state decay involves three lifetime components of 0.14, 1.2 and 4.2 ns (red, blue and
magenta DAS, respectively), the latter carrying the largest amplitude and approaching the
lifetime of the Chls in organic solvents
34. In both open and closed state, all DAS have
similar shape and peak near 740 nm and, as a consequence, the average lifetime of
PSII particles of CF is essentially wavelength-independent (Figure 2f). The average
FRL-PSII lifetime rises from about 0.6 ns in open state (which is at least 6 times longer than
that measured on Chl a-only PSII with open RCs)
89,90to nearly 2.4 ns in closed state.
Therefore, the fluorescence in closed state (F
m) is nearly 4 times that in open state (F
o), as
shown in Figure S4. This ratio is significantly lower than the F
m/F
otypically measured for
PSII isolated from WL-grown cyanobacteria (which can reach values up to 10 or 20)
89,90,
indicating that charge separation in FRL-PSII is substantially less efficient (the quantum
efficiency of PSII is estimated as: Φ
PSII= 1 - F
o/F
m).
The FRL-PSII of CT is, in many aspects, spectroscopically similar to that of CF (Figure
S3). The main differences between the FRL-PSII of these two strains are: (i) the FRL-PSII
of CT is further red-shifted with respect to that of CF (the same holds for their FRL-PSI,
see Figure S7) and (ii) the PSII particles isolated from FRL-adapted CT are more
heterogeneous due to the presence of some WL-PSII and FRL-PBS (whose bicylindrical
core could remain attached to PSII)
253,258in the preparation. The F
m
/F
oratio for the
FRL-PSII of CT (slightly above 2, see Figure S4) is even lower than that of CF, possibly due to
the presence of FRL-PBS in the preparation.
Measurements on intact cells. FaRLiP induces the appearance of new red-shifted
absorption bands in cyanobacterial cells (Figures 3a and Figure S8) due to the
incorporation of Chl f in their photosystems, as well as the synthesis of red-shifted
allophycocyanin
2. A relative absorbance decrease in the 600-nm region, where the
typically WL-expressed PBS absorb, is also observed. Due to the presence of Chl f, the
emission spectra of FRL-adapted cells significantly red-shift in comparison to those of
WL-adapted cells (Figures 3b and 3c and Figure S8). For FRL cells, the major emission
band in the 720-760 nm region contains contributions from PSI, PSII and
FRL-allophycocyanin. A second band, peaking at about 660 nm, which is sensitized by the
580-nm excitation, stems from PBS (possibly decoupled from FRL-photosystems). The
580-nm excitation also sensitizes emission at 720 nm (which is even more evident in CT,
see Figure S8), as FRL-allophycocyanin is expected to emit mostly around this
wavelength
258,259. At variance with CF, FRL-adapted cells of CT also retain a significant
amount of WL-PSII, as witnessed by the additional 683-nm peak in the emission spectra
of Figure S8.
159
Figure 3. Absorption and emission spectra of intact cells of CF. a) Absorption spectra of intact cells of
CF adapted to WL (black lines) and to FRL (red lines). The contribution of Chl f to the absorption spectrum of FRL-adapted cells can be observed at wavelengths above 700 nm. b) Emission spectra of intact cells of CF adapted to WL excited at different wavelengths. The 440-nm excitation is selective for Chls, whereas 580-nm excitation is selective for PBS. 400-nm light mostly excites Chls, with some small PBS contribution. c) Emission spectra of intact cells of CF adapted to FRL excited at different wavelengths. Data from WL-cells are representative of 2 biological replicas and those from FRL-cells of 3 biological replicas, all yielding similar results.
TRF measurements with a streak camera setup were performed on intact cells to probe the
chlorophyll excited-state kinetics of both PSI and PSII in vivo (Figures 4a and 4b, and
160
Figure S9). The WL-adapted strains display fluorescence kinetics very similar to those of
previously studied WL-grown cyanobacteria
354, both at low power (where most PSII RCs
are open, Figure 4a) and in closed state (Figure 4b). WL-PSI trapping takes place in about
30 ps (red DAS’s in Figures 4a and 4b). In both strains, a 150-ps component (blue DAS’s)
is observed, which is rather independent on the state of PSII and stems prevalently from
PBS, with smaller contributions from the photosystems
354,355. In all these measurements,
the longest-lived component (green DAS’s), whose lifetime markedly increases (from 380
ps to 1.0 ns) in closed state, is clearly related to WL-PSII.
Figure 4. TRF measurements of intact cells of WL- and FRL- adapted CF. a,b) DAS from global
analysis of TRF data of WL-adapted CF measured with a streak camera setup upon 400-nm excitation at low power, when most PSII RCs are open (a) or with closed PSII (b). c,d) DAS from global analysis of TRF data of FRL cells of CF at low powers (c) and with closed PSII (d). The vanishingly small and flat orange component observed at low powers (and magnified in the insets), whose lifetime is much longer than the experimental time window (and therefore designated as infinite, “inf”, in the legends), is needed for baseline correction. More details on the experimental conditions, as well as color maps representing the raw data, can be found in Figures S10 and S12 and their captions. See Figures S11 and S13 for an overlay of experimental/fitted TRF traces and Figure S14 for the time zero spectrum (i.e. immediately after laser excitation) reconstructed from the DAS in (d). Data from WL-cells are representative of 2 biological replicas and those from FRL-cells of 3 biological replicas, all yielding similar results.
161
DAS from TRF measurements on FRL-adapted cells of CF are shown in Figures 4c and
4d and Figure S9. In both conditions (open and closed PSII), two subsequent energy
equilibration steps are observed: a faster transfer from Chl a to Chl f (black DAS), which
is attributed to both FRL-PSI and FRL-PSII based on the in vitro data, and a slower
transfer from bulk Chl f (and possibly red-shifted Chl a) to Chls f red-shifted to 790-800
nm (red DAS), which can be assigned to FRL-PSI. The blue component, with a lifetime of
around 150 ps, a peak around 750 nm and a pronounced shoulder at longer wavelengths,
can be ascribed mostly to FRL-PSI trapping. This is confirmed by the fact that the
spectrum and lifetime of this component is barely affected when PSII RCs close. This
component also includes some contribution from PBS (which are excited to a lesser extent
at 400 nm) at shorter wavelengths. A small dip at 720 nm is observed in the 150-ps DAS
(more clearly when cells are in closed state, Figure 4d), which suggests that the
red-shifted pigments in the FRL-bicylindrical cores accept excitations on this timescale. The
longest-lived component (1.1 ns) observed when PSII RCs are mostly open (green DAS in
Figure 4c) can be assigned to FRL-PSII and is consistent with the measurements on
purified FRL-PSII particles and with previous reports on the other FaRLiP strain
Halomicronema hongdechloris
345. In comparison to the emission spectrum of isolated
FRL-PSII, however, the in vivo FRL-PSII DAS is blue-shifted (Figure S15) due to
contributions from FRL-allophycocyanin (possibly connected to FRL-PSII in vivo). In
closed state, two long-lived PSII-related components can be separated (green and orange
DAS in Figure 4d). The shortest one (green DAS) is blue-shifted and, therefore, arises
mostly from FRL-allophycocyanin, whereas the longest one (orange DAS) has an
enhanced contribution from FRL-PSII. The longest lifetime that can be assigned to closed
PSII in vivo (about 2 ns) is shorter than that observed in vitro (about 4 ns; this shortening
of the closed PSII lifetime in vivo was also observed in Chl a-only PSII of other
strains)
353.
The TRF data presented so far, recorded with a streak camera setup, are essential for
recognizing the spectral signatures of FRL-photosystems in vivo based on our previous
observations in vitro. The time resolution of the streak camera also allows the detection of
ultrafast energy transfer processes between different Chl pools. The relatively high laser
powers required by this technique, however, prevent from keeping PSII RCs fully open
during the in vivo measurements, which is a key requisite for estimating Φ
PSII. In order to
determine the average excited-state lifetime of WL- and FRL-cells in fully open and
closed state, TRF in vivo data were also recorded using the more sensitive TCSPC setup
(Figure 5). The average fluorescence lifetime of WL-cells in closed state at 680 nm
(where PSII emission is the highest) is about 4.6 and 3.3 times that in open state for
WL-CF and CT, respectively. This ratio implies a PSII quantum efficiency of 70-80%, which
is most likely underestimated, as the contribution of PSI and PBS fluorescence at 680 nm
is still significant. As a consequence of the slower charge separation displayed by
FRL-PSII (Figure 5a), the difference in the excited-state kinetics of FRL-cells in open and
closed state is markedly reduced in comparison to what observed for WL-cells (cfr
162
Figures 5b and 5c). F
m/F
ois only about 1.6 for both FRL-CF and CT, pointing to a
remarkably lower efficiency of FRL-PSII (below 40%) with respect to WL-PSII.
Figure 5. TRF measurements on intact cells of different organisms. a) Overlay of normalized TRF
traces detected upon 438-nm excitation of cells of WL-adapted CF CF, black), WL-adapted CT (WL-CT, blue), Acaryochloris marina (AM, green), FRL-adapted CF (FRL-CF, red) and FRL-adapted CT (FRL-CT, orange). The detection wavelength was set to enhance the contribution of PSII in each sample. Details on each experiment are given in the captions of Figures S16, S18 and S23. b) Overlay of TRF traces (excited at 438 nm) of cells of WL-CF with PSII RCs in open (black) and closed (red) state. The detection wavelength is 680 nm to enhance the contribution of PSII. The average excited-state lifetime in open and closed state is shown next to each trace. See Figures S16 and S17 for details on the exponential fittings. c) Overlay of TRF traces (excited at 438 nm) of cells of FRL-adapted CF with PSII RCs in open (black) and closed (red) state. The detection wavelength is 735 nm to enhance the contribution of PSII. The average
163
excited-state lifetime in open and closed state is shown next to each trace. See Figures S18 and S20 for details on the exponential fittings and Figure S19 for the corresponding data of CT. Data are representative of two technical replicas and are fully consistent with correspondent measurements with the streak camera setup (Figure 4 and Figures S9 and S22).
Discussion
The reduced efficiency of FRL-photosystems is explained by their altered energetics.
The two investigated FaRLiP strains show qualitatively similar PSI and PSII kinetics and
the spectroscopic signatures observed for isolated supercomplexes can be recognized in
vivo. The main difference between the two FRL-acclimated strains is in the relative
amount of PSII, which is clearly higher in CT. The reason for retaining some
WL-PSII supercomplexes in an environment where Chl a is barely excited is not clear yet, but
they might be needed to maintain some flexibility for harvesting visible light in case of
sudden availability
258. On the other hand, the FRL-photosystems of both strains display a
rather similar functional architecture. The Chl f molecules incorporated by FRL-PSI and
FRL-PSII are effectively connected with the surrounding Chl a molecules and transfer
their excitations within the first 20 ps. However, the insertion of Chl f has remarkable
consequences on the overall charge separation rates and efficiencies.
Similar to what happens in Chl a-only photosystems, FRL-PSI emission is also red-shifted
with respect to FRL-PSII (Figure S7). In WL-photosystems, this spectral difference is due
to the presence in PSI of the so-called red Chls a
101,102. These red Chls a seem to be
retained, at least partially, in the FRL-PSI, as confirmed by structural data
256. In FRL-PSI,
however, they quickly transfer excitations to the even more red-shifted Chls f. The Chls f
are spectrally heterogeneous and a Chl f pool could be detected emitting at 800 nm. Such
a pool, which is plausibly red-shifted with respect to the RC
253,256,257, can be seen as an
energetic and kinetic analogue of the red Chls a in WL-PSI. Due to its spectral red-shift,
however, the 800-nm Chl f pool might represent an even stronger energy sink, especially
in the case where FRL-PSI RCs only contain Chl a, as suggested by recent structural
data
256,257. This would explain why the overall charge separation is slowed down from
about 40 ps in WL-PSI to 140 ps in FRL-PSI. Nevertheless, the photochemical efficiency
of FRL-PSI, remains above 95%, confirming that PSI remains a strong energy trap also
when incorporating such red-shifted Chls.
Conversely, incorporation of Chl f has a substantial impact on the performances of PSII.
Indeed, the lifetime of FRL-PSII is markedly longer than that of WL-PSII: the longest
WL-PSII related component is just above 200 ps when RCs are open, whereas the average
lifetime of FRL-PSII with open RCs is over 500 ps, with the longest FRL PSII-related
component being at least 1 ns, both in vitro and in vivo. Why does Chl f insertion in PSII
slow down its overall charge separation so dramatically? As a first approximation, the
PSII average fluorescence lifetime is the sum of two contributions: (i) the energy
migration from the outer antenna pigments to the RCs and (ii) the trapping at the RCs
301.
164
primary donor in FRL-PSII (Chl
D1)
253. As the excited-state energy of Chl f is more than
100 mV lower than that of Chl a, Chl
D1can be expected to be a weaker electron donor
towards pheophytin and plastoquinone, leading to a slower and more reversible charge
separation
90. Such a prediction is consistent with the massive increase in
thermoluminescence observed in FRL-PSII (meaning that charge recombination from
pheophytin to Chl f is indeed much more favorable)
253. At the same time, as there are few
Chls f per PSII monomer (more than 3 for the FRL-PSII of CF, see Table S1, and at least
4 for that of CT
253,258), some Chls f must be found in the surrounding antenna (CP43/47
paralogs). After Chl-a excitation, the energy is trapped by these few Chls f in about 10 ps.
If the latter Chls f are not optimally connected to Chl
D1, they could represent kinetic
bottlenecks in the energy migration towards the RCs, resulting in a slower overall
trapping.
To clarify the origin of the slower charge separation attained in FRL-PSII (whether
trap-limited or migration-trap-limited), we compared the excited-state kinetics of FRL-PSII and
WL-PSII of the FaRLiP strains to those of PSII from Acaryochloris Marina (AM). In this
cyanobacterium, the Chl content is largely dominated by the red-shifted Chl d (Figures
S21-23), with only minor amounts of Chl a (< 10%) being present
268,356. Furthermore,
Chl
D1in AM-PSII is a Chl d absorbing nearly at the same wavelength as in FRL-PSII of
CT (∼727 nm)
253,270,357. Interestingly, the average fluorescence lifetime of AM-PSII is also
significantly longer than that of WL-PSII (Figure 5a). Since the Chls d in the AM-PSII are
nearly isoenergetic (compare absorption and fluorescence spectra in Figure S21), similar
to the Chls a in WL-PSII, we can exclude the possibility that the observed difference is
due to energy migration. The slower trapping must therefore be explained by the
substitution of Chl a with Chl d at the RCs. Since in FRL-PSII the primary donor absorbs
at the same wavelength as in AM
253(at least in CT), similarly slower kinetics can be
expected. Charge separation in FRL-PSII is, however, clearly slower (Figure 5a). This
opens up two possibilities, which are not necessarily mutually exclusive: (i) other
differences subsist between AM-PSII and FRL-PSII in terms of their RC energetics (for
example, the redox potential of the primary acceptor pheophytin or that of P
680). This
would account for the yield of thermoluminescence being much higher in FRL-PSII than
in AM-PSII
253,358. (ii) FRL-PSII charge separation is significantly migration-limited. This
could happen if the Chls f in the antenna (which trap the excitations from the surrounding
Chls a nearly irreversibly) are poorly connected to the primary donor. Indeed, the Chls in
the PSII antenna display rather weak electronic couplings with Chl
D1(only few cm
-1or
less), which could potentially lead to energy transfer rates of hundreds of picoseconds (or
longer) between them (see Table S2 for details). The possibility of a migration-limited
trapping in FRL-PSII indicates that the specific location of the newly inserted red-shifted
Chls might play a crucial role in determining the efficiency of the entire unit. The
presence of weekly coupled Chls f in the antenna could also justify the need for a
red-shifted Chl in the PSII-RC, which is known to represent a very shallow trap due to the
165
antenna could represent a stronger energy sink than a Chl a-only RC, making charge
separation more unlikely. Therefore, even if the presence of Chl f in the RC reasonably
reduces the driving force and rate of the primary electron transfer steps, it might be
necessary to enable FRL-PSII to process the low-energy photons harvested in the antenna.
Figure 6. Light-absorption and energy output of photosynthetic units under a dense plant canopy. a)
Absorption spectra of PSII of Synechococcus elongatus from Kuhl et al.359 (designated as WL-PSII, in black) and FRL-PSII of CF (red) normalized to the total Qy absorbance area (λ = 600-800 nm) and relative spectral photon irradiance of unshaded sunlight (daylight) and light filtered by a dense leaf canopy (canopy) from Gan et al.4 b) Wavelength distribution of photons absorbed under a dense plant canopy by the WL- and FRL-PSII complexes (in thick blue/red lines), obtained by multiplying the absorption spectra of the complexes by the spectral irradiance under the canopy shown in (a) at each wavelength. The colored areas represent the wavelength-dependent energy output of each PSII obtained by multiplying the wavelength distribution of absorbed photons by the PSII photochemical yield calculated from the above-discussed in vivo Fm/Fo ratios as ΦWL-PSII = 1 - 1/4.6 = 78% and ΦFRL-PSII = 1 – 1/1.6 = 37%. The total light-absorption is estimated as the area below the thick blue/red lines, while the total output is the total absorption times the photochemical yield. c) Absorption spectra of WL- and FRL-PSI of CF (as in Figure 1c) normalized to the total Qy absorbance area (λ = 600-800 nm). The spectra of unshaded and shaded light as in (a) are also shown for comparison. d) Wavelength distribution of photons absorbed under a dense plant canopy by the WL- and FRL-PSI complexes (in thick blue/red lines) and total energy output (blue/red colored areas) obtained as in (b). The PSI yield are calculated as ΦWL-PSI = 1 – 0.04/4 = 99% and ΦFRL-PSI = 1 – 0.14/4 = 96.5%, where 0.04 and 0.14 (in ns) are the trapping lifetimes of WL-/FRL-PSI and 4 ns is the lifetime of unquenched Chl. The absorption spectra of isolated PSI and PSII are representative of two different preparations for each sample, yielding very similar results.
166
The advantage of absorbing far-red photons under a canopy. A slower charge
separation can be expected to reduce Φ
PSII, as this parameter depends on the ratio between
the average excited-state lifetime of PSII in open and closed state (increasing F
m/F
oincreases the yield of PSII photochemistry). The F
m/F
ovalues of 4.6 and 3.3 measured for
WL-adapted CF and CT in vivo, respectively, imply a Φ
PSIIof about 70-80% for Chl
a-only PSII. The efficiency of FRL-PSII measured in vivo is, by contrast, significantly
lower, as Φ
PSIIdrops below 40% (i.e. nearly half of what found for WL-PSII). All these
pieces of evidence indicate that the altered energetics due to Chl f insertion in PSII
involve a dramatic loss in the efficiency of charge separation, not to mention the higher
chance of incurring into photodamage due to the higher yield of charge recombination
253.
This is probably the reason why oxygenic photosynthesis using red-shifted Chls is
restricted to deep-shade environments that are highly enriched in FRL.
It is worth noticing that the presence of Chl f in the RCs might be responsible for the
slower charge separation observed also in FRL-PSI. In this case, however, it is hard to
establish whether the slower trapping is due to a reduced driving force of charge
separation or to the presence of a strongly red-shifted Chl f pool (emitting at 800 nm) in
the antenna, which could make uphill energy transfer to the RC less favorable, thus
slowing down the overall rate of photochemistry.
Despite the lower yield of charge separation achieved by FRL-photosystems, the insertion
of Chl f in both PSI and PSII remains beneficial for FaRLiP strains in deep-shade
environments, where most available photons have
𝜆 > 700 nm. Even though Chl f
represents only a minor fraction of all Chls, it provides FRL-photosystems with a
significant enhancement in the total amount of absorbed FRL. To quantify the gain in
photosynthetic capacity achieved upon FaRLiP, we calculated the wavelength distribution
of the photons absorbed by the WL-and FRL-units at the bottom of a dense plant canopy
4.
The gain in absorption is particularly evident for PSII (Figures 6a and 6b), as FRL-PSII
harvests about 3.6 times more photons than its WL- counterpart (as Chl a-only PSII does
not absorb above 700 nm). Since the efficiency of Chl f-containing PSII in vivo is roughly
halved with respect to WL-PSII (based on the fluorescence lifetime data), the total energy
output of FRL-PSII (calculated as the product of total light absorption and photochemical
yield) is still 70% larger than for WL-PSII in this specific environment. In the case of PSI,
both the total absorption and total energy output are approximately doubled when Chl f is
inserted (Figures 6c and 6d). Interestingly, the absorption-weighted productivity of both
FRL-photosystems in a FRL-enriched environment is about twice as much as for their
WL-counterparts.
Conclusions
This work shows that Chl f insertion in the photosystems upon FaRLiP involves not only
an increased far-red absorption, but also significant alterations in their excited-state
dynamics, in terms of both energy equilibration and trapping.
167
FRL-PSI displays a charge separation that is about four times slower than in WL-PSI but
remains relatively fast and, therefore, highly efficient. FRL-PSI also contains a Chl f pool
red-shifted to 800 nm that, similar to the “red forms” of WL-PSI, is likely to represent a
bottleneck in the energy migration towards the RCs. FRL-PSI is also more “red” than
FRL-PSII, which appears to be a recurrent motif in photosynthetic linear electron
transport.
Charge separation in FRL-PSII takes place over several hundred ps even when RCs are
fully open. This is primarily a consequence of the primary donor being a Chl f instead of a
Chl a: while this replacement is probably imperative for the RC to harvest excitations
from the other Chls f in the antenna, it irremediably reduces the driving force of the initial
electron transfer steps. In addition, the rate of charge separation might be further limited
by a slower energy migration from the antenna to the RCs. Consequently, FRL-PSII
photochemistry is markedly less efficient than that of WL-PSII, which would explain why
FaRLiP is only used in FRL-enriched niches. Our results suggest that Chl f incorporation
represents a viable strategy for introducing the capacity to utilize far-red light in new
organisms, including plants, but the specific location of this new pigment in the
engineered photosystems may be crucial in determining their photosynthetic efficiency.
Competing interests
The authors declare no competing interests.
Acknowledgments
We thank Judith Schaefers (Vrije Universiteit Amsterdam) for helping with cell growth
and pigment content determination and Martijn Tros for insightful discussions. This
project was supported by the European Union's Horizon 2020 research and innovation
program under the Marie Skłodowska-Curie grant agreement No 675006 and by the
Netherlands Organization for Scientific Research (NWO) via a Top grant to R.C., and by
the EMBO long-term fellowship (EMBO ALTF 292-2017) to L.B..
169
Chapter 5
Supplementary Information
Figure S1. Spectroscopic data of isolated Photosystem I from CT.
A) 2D color map representing TRF data of PSI from WL-adapted CT excited at 400 nm and detected in the Chl Qy region with a Streak Camera setup (with a repetition rate of 250 kHz, an excitation power of 150 µW, the emission polarization set at magic angle with respect to the excitation, the temperature set to 10°C and a 400-ps time window).
650 700 750 800 -200 -100 0 100 200 300 680 720 0 2 4 DAS l (nm) 7 ps 41 ps > 5 ns 400 500 600 700 800 0.0 0.5 1.0 1.5 Abso rbance (a.u.) l (nm) WL-PSI FRL-PSI 680 720 760 800 840 0.0 0.2 0.4 0.6 0.8 1.0 Norm. Fluo rescence l (nm) WL-PSI FRL-PSI 5ps 17ps 51ps 135ps 3.3ns 700 750 800 850 -400 0 400 800 700 750 800 0 20 40 DAS l (nm)
FRL-PSI
679.5 nmWL-PSI
A)
B)
C)
D)
E)
F)
170
B) DAS from global analysis of TRF data in (A). The inset shows a magnification of the small long-lived component.
C) Absorption spectra (normalized at Qy maximum) of WL- and FRL-PSI of CT purified via sucrose gradient as described in the Materials and Methods section.
D) Time-integrated fluorescence spectra of PSI particles isolated from WL- (black lines) and FRL- (red lines) adapted cells of CT. The time-integrated spectra of WL PSI are obtained from the DAS in (B) (excluding the small long-lived component representing unconnected chlorophylls). The time-integrated spectra of FRL-PSI are calculated from the DAS in (F) (again excluding the minor long-lived component). E) 2D color maps representing TRF data of PSI from FRL-adapted CT (same experimental conditions as in (A)).
F) DAS from global analysis of TRF data in (E). The first three components (black, red and blue DAS) describe energy transfer events following Chl excitation, whereas the fourth component (green DAS) describes trapping at FRL-PSI reaction centers. The small long-lived DAS (which is magnified in the inset) is due to small fractions of energetically-uncoupled pigments (and, possibly, PSII). See Figure S2 for an overlay of the raw data with the fitted traces. The same experimental conditions were used for the correspondent measurements on PSI of CF shown in Figure 1.
171
Figure S2. Time-resolved fluorescence (TRF) measurements of PSI isolated from WL- and FRL- adapted strains. A,B) Overlay of raw and globally fitted fluorescence time traces at selected emission
wavelengths from TRF data of WL-PSI of CF (see also Figure 1A-B) and WL-PSI of CT (see also Figure S1A-B). The selected emission wavelengths highlight the contributions from the two main spectral forms, i.e. bulk Chls a (690 nm) and the red Chls a (720 nm). C,D) Overlay of raw and fitted fluorescence time traces at selected emission wavelengths from TRF data of PSI of CF (see also Figure 1E-F) and FRL-PSI of CT (see also Figure S1E-F). The traces show how the initially excited Chl a (represented by the black fluorescence trace at 685 nm) quickly decays due to energy transfer to Chl f, whose rise can be observed at 745-750 nm (black and red DAS’s in Figure 1). The signal at 790 nm due to red-shifted Chl f builds up on a longer timescale due to slower equilibration with the other Chls f at 745-750 nm (blue DAS’s in Figure 1F and Figure S1F). For each sample, the data are representative of two different preparations yielding similar results.
0 100 200 300 0 400 800 1200 Fluo rescence coun ts Time (ps) 690 fit 690 720 fit 720
CF WL-PSI
0 100 200 300 0 50 100 150 200 250 300 Fluo rescence coun ts Time (ps) 690 fit 690 720 fit 720CT WL-PSI
0 100 200 300 0 200 400 600 800 1000 Fluo rescence coun ts Time (ps) 685 685 fit 745 745 fit 790 790 fitCF FRL-PSI
0 100 200 300 0 200 400 600 800 1000 Fluo rescence coun ts Time (ps) 685 685 fit 750 750 fit 790 790 fitCT FRL-PSI
A)
B)
C)
D)
172
Figure S3. Fluorescence data of isolated FRL-Photosystem II from CT.
A) Absorption spectrum of PSII particles isolated from FRL-adapted CT.
B) Normalized fluorescence spectra of PSII particles from FRL-adapted CT excited at different wavelengths. Beside the major emission band peaking at 746 nm and stemming from FRL-PSII, a second band (peaking at 683 nm) in the Chl a region can be observed, which results from the presence of WL-PSII in the preparation. This suggests that this strain retains some WL-PSII after adaptation to FRL, as recently observed also for another FaRLiP strain258. Upon 580-nm excitation, the emission band in the far-red blue-shifts due to an increased contribution in the 720-nm region. This observation can be explained by the presence of FRL-APC forming bicylindrical cores (FRL-BCs) that might stick to PSII in the preparation253,258. 400 500 600 700 800 0.0 0.5 1.0 1.5 Abso rbance (a.u.) l (nm) FRL-PSII 640 680 720 760 800 840 0.0 0.2 0.4 0.6 0.8 1.0 Norm. Fluo rescence l (nm) 400 nm 440 nm 500 nm 580 nm 0.06ns 0.41ns 1.47ns 3.11ns 680 700 720 740 760 0 1 2 3 l (nm) DAS (a.u. )
Open PSII
0.04 ns 0.16 ns 0.57 ns 1.54 ns 4.00 ns 680 700 720 740 760 0.0 0.5 1.0 1.5 DAS (a.u. ) l (nm)Closed PSII
0 2 4 6 8 0.0 0.2 0.4 0.6 0.8 1.0 Norm. Fluo rescence Time (ns) IRF Open Closed740 nm emission
680 700 720 740 760 0.0 0.5 1.0 1.5 2.0 2.5 l (nm) open closed Average Lifetime (ns) 674 nmA)
B)
C)
D)
E)
F)
173
C,D) DAS from global analysis of TRF data on PSII particles isolated from FRL-adapted CT with nearly open (C) and closed (D) reaction centers. The data were recorded with a TCSPC setup exciting at 438 nm. FRL-PSII with open RCs was measured at 0.2 µW (2.5 MHz repetition rate) and upon addition of ferricyanide 0.4 mM, (for each wavelength, the fluorescence trace was recorded for 10 minutes) whilst FRL-PSII with closed RCs was measured at 50 µW (10 MHz repetition rate) after addition of 50 µM DCMU (each trace recorded until reaching 10000 counts at peak maximum). An oxygen scavenging mixture consisting of catalase (50 mg/ml), glucose oxidase (100 mg/ml) and glucose (5 mM) was used to increase sample stability. The temperature was set to 8 °C. After Chl excitation at low powers (where most RCs are open), the excited-state kinetics from PSII particles of CT can be described by four components (C). The fastest (< 100 ps) DAS shows significant amplitude in the Chl a region, with a peak at 680 nm, compatible with the assignment to WL-PSII89,90. The 400-ps component, instead, most likely incorporates contributions from WL-PSII (in the visible region), FRL-BCs (around 720 nm) and FRL-PSII. The 1.5-ns DAS is entirely located in the FR region. The small long-lived component in (C) is due to a minor fraction of particles with closed RCs (see Figure S4 for details). Notably, the 400-ps and 1.5-ns DAS have different shapes in the FR, that of the 400-ps component displaying a larger contribution at 720 nm, which is likely explained by the presence of FRL-BCs. On the other hand, in CF, where FRL-BCs are virtually absent in the FRL-PSII preparation (see emission spectra in Figure 2A), the 200-ps and the 1.0 ns DAS (Figure 2C) have very similar shape. Upon closure of the RCs (D), the excited-state kinetics becomes extremely complex due to the sample heterogeneity. The average lifetime, however, increases both at 680 nm (where the amplitude of the longer-lived component increases with respect to the case of open RCs (C)), and in the FR (see also (E)). FRL-PSII of CT with closed RCs displays, as already observed for the case of CF, a strong 4-ns component. An overlay of the correspondent experimental and fitted TRF traces can be found in Figure S5.
E) Normalized TRF traces of FRL-PSII particles from CT with open (black) and closed (red) RCs. Measurements were performed with a TCSPC setup upon 438-nm excitation. The instrumental response function (IRF) detected at 740 nm is shown in grey.
F) Average fluorescence lifetime of PSII from FRL-adapted CT as a function of wavelength (based on the DAS in (C,D). Due to the sample heterogeneity (WL-PSII, FRL-BCs and FRL-PSII coexist in the preparation), the average lifetime increases at longer wavelengths and peaks at 750 nm. This trend is maintained when PSII particles are measured in closed state.
Data are representative of two different preparations yielding similar results, the only difference being in the relative amount of FRL-BCs.
174
Figure S4. TRF of PSII particles isolated from FRL-adapted strains. A,B) TRF traces recorded at 740
nm (upon 438-nm excitation) at different excitation powers for both strains. The data were recorded with a TCSPC setup at 2.5 MHz repetition rate, unless at 50 µW power, where the repetition rate was 10 MHz. The temperature was set to 8 °C and ferricyanide 0.4 mM was used to keep RCs as much open as possible. The traces are nearly power independent below 0.5 µW and become increasingly longer-lived at higher powers. The instrumental response function (IRF) detected at 680 nm is shown in grey. C,D) Results from a simultaneous multi-exponential fit of the above traces. Three lifetime components were needed to satisfactorily fit the data and the depicted bars represent the fractional amplitude of each component at different excitation powers. In both strains, the increasing excitation pressure involves an increase of the long-lived (> 3 ns) component at the expense of the shorter ones. The > 3 ns component is therefore
0 2 4 6 0.0 0.2 0.4 0.6 0.8 1.0 Normalize d fluorescence Time (ns) 0.1 uW 0.2 uW 0.5 uW 1 uW 5 uW 50 uW IRF
CF
0 2 4 6 0.0 0.2 0.4 0.6 0.8 1.0 Normalize d fluorescence Time (ns) 0.1 uW 0.2 uW 0.5 uW 1 uW 5 uW 50 uW IRFCT
0.1 uW 0.2 uW 0.5 uW 1 uW 5 uW 50 uW 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Ampli tude Fraction 0.24 ns 1.10 ns 3.33 ns 0.1 uW 0.2 uW 0.5 uW 1 uW 5 uW 50 uW 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.20 ns 1.33 ns 3.69 ns 720 740 760 3.2 3.4 3.6 3.8 4.0 F m / F 0 l (nm) 680 700 720 740 760 1.8 2.0 2.2 2.4 F m / F 0 l (nm)C)
D)
E)
F)
A)
B)
175
assigned to PSII particles with closed RCs and its amount is vanishingly small below 0.5 µW, confirming that PSII with open RCs can be only measured at very low powers.
E,F) Ratio between the fluorescence lifetime measured in closed (Fm) and open state (Fo) conditions at different wavelengths for CF (E) and CT (F). Fm/Fo is calculated as the ratio between the average lifetimes measured in closed state and open state, plotted in Figure 2F (for CF) and Figure S3F (for CT).
The power dependency in (A,B,C,D) was investigated once for each strain.
Figure S5. TRF of PSII particles isolated from FRL-adapted strains. Overlay of raw and globally fitted
TRF traces of PSII particles from FRL-acclimated CF and CT (measured with a TCSPC setup upon 438-nm excitation). For each line (representing a different dataset), three experimental time-traces are shown in grey corresponding to three selected emission wavelengths (specified in the legends). For each dataset, all
0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Fluorescenc e (a.u.) Time (ns) 720 720 fit Residuals c2 = 1.04 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Time (ns) 740 740 fit Residuals CF FRL-PSII open c2 = 1.04 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Time (ns) 760 760 fit Residuals c2 = 1.04 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Fluorescenc e (a.u.) Time (ns) 720 720 fit Residuals c2 = 1.02 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Time (ns) 740 740 fit Residuals c2 = 1.02 CF FRL-PSII closed 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Time (ns) 760 760 fit Residuals c2 = 1.02 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Fluorescenc e (a.u.) Time (ns) 680 680 fit Residuals c2 = 0.98 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Time (ns) 720 720 fit Residuals c2 = 0.98 CT FRL-PSII open 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Time (ns) 740 740 fit Residuals c2 = 0.98 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Fluorescenc e (a.u.) Time (ns) 680 680 fit Residuals c2 = 1.03 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Time (ns) 720 720 fit Residuals c2 = 1.03 CT FRL-PSII closed 0 2 4 0.0 0.2 0.4 0.6 0.8 1.0 Time (ns) 740 740 fit Residuals c2 = 1.03
176
traces detected at different wavelengths are globally analyzed as explained in the methods section obtaining the fitted traces (shown in black) and the global χ2 shown at each line. The corresponding residuals (i.e. the difference between experimental and fitted traces) are shown in blue. The DAS obtained from globally analyzing these data can be found in Figures 2C-D (for PSII of FRL-CF) and Figures S3C-D (for PSII of FRL-CT). For FRL-PSII of CF, the traces are representative of 2 technical replicas. For FRL-PSII of CT, the traces are representative of 2 different preparations.
Figure S6. TRF measurements of PSII particles isolated from FRL-adapted strains. DAS from TRF
data of PSII particles from FRL-adapted CF measured with a Streak Camera setup with closed RCs (400-nm excitation, 50 µW power and 250 kHz repetition rate + 50 µM DCMU). The experimental time window for this experiment was 1.5 ns. The temperature was set to 8 °C. The black DAS represents Chl a to Chl f downhill energy transfer, whereas the red and blue DAS represent Chl excited-state decay in presence of closed RCs. These data demonstrate that the amount of excitations retained by Chls a after few tens of ps is negligible. The data were measured once on each strain (data for PSII of CT not shown), with similar results and fully consistent with correspondent measurements with TCSPC (Figures 2 and S3).
Figure S7. Fluorescence spectra of FRL-Photosystems. Normalized steady-state fluorescence spectra of
FRL-PSI and PSII from CF and CT. The emission spectrum of FRL-PSI of CT (peaking at 752 nm) is 6.5 nm red-shifted in comparison to that of FRL-PSI of CF (peaking at 745.5 nm). The emission spectrum of FRL-PSII of CT (peaking at 746.5 nm) is 8 nm red-shifted in comparison to that of CF (peaking at 738.5 nm). The spectra are representative of 2 different preparations with similar results on each strain/sample.
680 720 760 800 -1000 0 1000 2000 DAS (a.u. ) l (nm) 14 ps 310 ps 3.5 ns 680 720 760 800 840 0.0 0.2 0.4 0.6 0.8 1.0 Normalize d fluorescence l (nm) CF FRL-PSI CT FRL-PSI CF FRL-PSII CT FRL-PSII
177
Figure S8. Absorption and emission spectra of intact cells of CT. A) Absorption spectra of intact cells
of CT adapted to WL (black lines) and to FRL (red lines). The Chl f contribution to the absorption spectrum of FRL-adapted cells can be observed at wavelengths > 700 nm. B) Emission spectra of intact cells of CT adapted to WL excited at different wavelengths. 440-nm excitation is selective for Chls, whereas 580-nm excitation is selective for the PBS. 400-nm light mostly excites Chls, with some smaller PBS contribution. C) Emission spectra of intact cells of CT adapted to FRL excited at different wavelengths. Data from WL-cells are representative of 2 biological replicas, those from FRL-cells of 3 biological replicas, all yielding similar results.
400 500 600 700 800 0.0 0.5 1.0 1.5 Abso rbance (a.u.) l (nm) WL FRL 640 680 720 760 800 840 0.0 0.2 0.4 0.6 0.8 1.0 Norm. Fluo rescence l (nm) 400 nm 440 nm 580 nm
CT WL
640 680 720 760 800 840 0.0 0.2 0.4 0.6 0.8 1.0CT FRL
Norm. Fluo rescence l (nm) 400 nm 440 nm 580 nmB)
C)
A)
178
Figure S9. TRF measurements of intact cells of WL- and FRL-adapted CT.
The vanishingly small and flat orange component observed at low powers (and magnified in the insets), whose lifetime is much longer than the experimental time window, is needed for baseline correction. More details on the experimental conditions, as well as color maps representing the raw data, can be found in Figures S10 and S12 and their captions. See Figures S11 and S13 for an overlay of experimental/fitted TRF traces.
A,B) DAS from global analysis of TRF data of WL cells of CT measured with a Streak Camera setup upon 400-nm excitation at low power, were most PSII RCs are open (A), and with closed PSII (B). Under both conditions (open and closed PSII), the two fastest components (black and red DAS) are mostly related to PSI energy equilibration and trapping (which are insensitive to excitation pressure). In both (A) and (B), the blue DAS mostly stems from the PBS, with some smaller contributions from the photosystems, whereas the longer-lived green component, whose lifetime increases from 380 ps at low powers to 840 ps in closed state, can be ascribed to PSII.
C,D) DAS from global analysis of TRF data of FRL cells of CT at low powers (C) and with closed PSII (D). In both conditions (open and closed PSII), the black and red DAS represent subsequent energy equilibration steps: a faster transfer from Chl a to Chl f (black DAS), and a slower transfer from the Chl f pool (and possibly red-shifted Chl a) to a Chl f species red-shifted to 790-800 nm (red DAS), which for its timescale and spectrum can be assigned to FRL-PSI only (Figure S1F). The blue component, whose lifetime (about 150 ps) and spectrum are not much sensitive to the state of PSII RCs, can be ascribed mostly to FRL-PSI (see blue DAS in Figure S1F). This component also includes some contribution from WL-PSII and WL-PBS (the latter are excited to a lesser extent at 400 nm) at shorter wavelengths. At the same time, a small dip at 720 nm is also observed in the 150-ps DAS (more visible when PSII RCs are closed, (D)), which suggests that the red-shifted pigments in the FRL-BCs accept excitations on this
600 650 700 750 -100 0 100 200 650 700 750-0,2 -0,1 0,0 DAS (a.u. ) l (nm) 8 ps 33 ps 160 ps 380 ps inf
