Spectral diversity of photosystem I from flowering plants

Abstract

Photosystem I and II (PSI and PSII) work together to convert solar energy into chemical energy. Whilst a lot of research has been done to unravel variability of PSII fluorescence in response to biotic and abiotic factors, the contribution of PSI to in vivo fluorescence measurements has often been neglected or considered to be constant. Furthermore, little is known about how the absorption and emission properties of PSI from different plant species differ. In this study, we have isolated PSI from five plant species and compared their characteristics using a combination of optical and biochemical techniques. Differences have been identified in the fluorescence emission spectra and at the protein level, whereas the absorption spectra were virtually the same in all cases. In addition, the emission spectrum of PSI depends on temperature over a physiologically relevant range from 280 to 298 K. Combined, our data show a critical comparison of the absorption and emission properties of PSI from various plant species.

Full text

Vol.:(0123456789)
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Photosynthesis Research (2023) 155:35–47
https://doi.org/10.1007/s11120-022-00971-2
ORIGINAL ARTICLE
Spectral diversity ofphotosystem I fromflowering plants
PeterR.Bos1· ChristoSchiphorst1· IanKercher1· SiekaBuis1· DjanickdeJong1· IgorVunderink1·
EmilieWientjes1
Received: 8 June 2022 / Accepted: 30 September 2022 / Published online: 19 October 2022
© The Author(s) 2022
Abstract
Photosystem I and II (PSI and PSII) work together to convert solar energy into chemical energy. Whilst a lot of research
has been done to unravel variability of PSII fluorescence in response to biotic and abiotic factors, the contribution of PSI
to invivo fluorescence measurements has often been neglected or considered to be constant. Furthermore, little is known
about how the absorption and emission properties of PSI from different plant species differ. In this study, we have isolated
PSI from five plant species and compared their characteristics using a combination of optical and biochemical techniques.
Differences have been identified in the fluorescence emission spectra and at the protein level, whereas the absorption spectra
were virtually the same in all cases. In addition, the emission spectrum of PSI depends on temperature over a physiologically
relevant range from 280 to 298K. Combined, our data show a critical comparison of the absorption and emission properties
of PSI from various plant species.
Keywords Light harvesting· Photosystem I· Fluorescence· Absorption· Spectroscopy
Introduction
Photosynthesis is driven by light absorbed by photosystem
I (PSI) and photosystem II (PSII). Both photosystems are
located in the thylakoid membrane of oxygenic photosyn-
thetic organisms (Blankenship 2021). The supramolecular
PSI complex oxidises plastocyanin and photoreduces ferre-
doxins in the photosynthetic electron transport chain (Gobets
and van Grondelle 2001). In plants PSI is composed of multi-
ple proteins that can be divided in two moieties: (i) the core,
harbouring the reaction centre, all electron transport cofac-
tors, 102 chlorophyll (Chl) a molecules and ~ 22 β-carotenes
and (ii) an outer light-harvesting complex (LHCI) composed
of four gene products (Lhca1-4), which coordinate Chl a,
Chl b, lutein, violaxanthin and β-carotene (Ben-Shem etal.
2003; Amunts etal. 2007; Wientjes and Croce 2011). The
PSI core is much conserved over the course of evolution and
can be traced back to cyanobacteria, the oldest known clade
of oxygenic photosynthetic organisms (Amunts and Nel-
son 2008; Cardona 2018, Sánchez‐Baracaldo and Cardona
2020). Due to the low mutation rate of PSI core proteins
the structure and pigment organisation of the PSI core from
plants, algae and cyanobacteria is almost identical (Jordan
etal. 2001; Galka etal. 2012; Qin etal. 2015; Mazor etal.
2017; Pan etal. 2018; Steinbeck etal. 2018).
Contrarily, LHCI has emerged later in evolution in
green algae and higher plants and is found to be more vari-
able between species than the PSI core complex (Green
2003; Croce and van Amerongen 2020; Pan etal. 2020).
LHCI of Chlamydomonas reinhardtii consists of nine gene
products (Lhca1-9) that form two parallel concentric half
rings, whilst the LHCI complex of higher plants only
forms a single half-moon shaped belt around PSI, organ-
ised as an Lhca1/4 and Lhca2/3 dimer (Amunts etal. 2007;
Drop etal. 2011; Wientjes and Croce 2011; Su etal. 2019;
Suga etal. 2019). In plants a fifth protein Lhca5 can be
present in low concentrations and can substitute for Lhca4
(Klimmek etal. 2006; Wientjes etal. 2009). Recently it
was reported that the optical properties of LHCI from dif-
ferent higher plant species can differ (Chukhutsina etal.
2020). A distinct feature of LHCI of higher plants is the
absorption of photons with a wavelength > 700nm by
Chls which are called red forms. These red forms slow
down excitation energy trapping but increase the range of
wavelengths plants can use for photosynthesis (Croce etal.
* Emilie Wientjes
emilie.wientjes@wur.nl
1 Laboratory ofBiophysics, Wageningen University, P.O.
Box8128, 6700ETWageningen, TheNetherlands
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36 Photosynthesis Research (2023) 155:35–47
1 3
2000; Le Quiniou etal. 2015). Red forms can arise when
two Chls with transition dipole moments that are parallel
and in line have strong electronic interaction (Van Amer-
ongen and Van Grondelle 2000). The effect can become
stronger when the Chls experience different polar envi-
ronments, leading to the mixing of excitonic states with
charge transfer states (Gobets etal. 1994; Romero etal.
2009; Novoderezhkin etal. 2016). Lhca3 and Lhca4 har-
bour such coupled Chls and therefore give rise to the most
red-shifted absorption and emission bands of PSI (Morosi-
notto etal. 2003; Croce etal. 2007). The organisation of
these low-energy red forms in Lhca3 and Lhca4 is very
similar (Wientjes etal. 2011a, b). Excitations residing on
the red-shifted Chls must overcome their energy deficit
with environmental heat for transfer to neighbouring Chls.
Decreasing the temperature leads to an increase of the time
the excitation resides on the red forms and, as such, the
fluorescence of the low-energy Chls becomes more pro-
found. This has been demonstrated over the temperature
range from 17 to 280K (Croce etal. 1998; Jelezko etal.
2000). The effect of temperature, on the PSI emission
spectrum and intensity, in the biological relevant range
has so far not been investigated.
Regardless of slower trapping due to red-shifted Chls, PSI
is arguably the most efficient nanomachine in nature, using
almost every absorbed photon for charge separation (Amunts
and Nelson 2008). Since PSII has a higher fluorescence yield
than PSI, most leaf Chl fluorescence is emitted from Chls
affiliated with PSII. PSII emission has been well studied
and is known to vary due to differences in light conditions
and plant stresses (Dau 1994; Goltsev etal. 2016). Contrast-
ingly, less is known about the fluorescence emission proper-
ties of PSI. First of all, because its weak emission makes it
difficult to obtain samples which are pure enough to study
the properties of PSI and not that of PSII contaminations.
Furthermore, the emission intensity of PSI is very similar
for open and closed reaction centres and not affected by non-
photochemical quenching processes (Itoh and Sugiura 2004;
Wientjes and Croce 2012; Porcar-Castell etal. 2014). How-
ever, PSI fluorescence can contribute significantly to leaf
fluorescence depending on the detection wavelength and the
level of (non)-photochemical quenching of PSII emission.
At 680nm, the fluorescence signal is dominated by PSII,
but with detection above 720nm, the PSI contribution can
reach up to 40% of the fluorescence signal from whole leaves
(Agati etal. 2000; Franck etal. 2002). Therefore, knowing
the emission spectra of both photosystems is essential for
a correct interpretation of invivo fluorescence data. Espe-
cially pulse amplitude modulation (PAM) measurements
and remote sensing applications, techniques that commonly
use detection wavelengths of invivo fluorescence > 700nm,
would benefit from an accurate correction with a PSI fluo-
rescence spectrum (Porcar-Castell etal. 2014).
The PSI trapping kinetics have been studied with time-
resolved fluorescence measurements before (Mukerji
and Sauer 1993; Pålsson etal. 1995; Croce etal. 2000;
Ihalainen etal. 2002, 2005; Van Oort etal. 2008; Wientjes
etal. 2011a, b; Jennings etal. 2013; Akhtar and Lambrev
2020). However, a wide range of average trapping times
ranging from 40 to 64ps at room temperature or even 99ps
at 280K have been reported. This difference might be due
to variations in the plant species that were investigated,
but could also be due to isolation methods or measurement
techniques. Recently it has been shown that the PSI emis-
sion spectrum and trapping kinetics varies from species to
species (Chukhutsina etal. 2020). These results indicate
that the PSI spectrum is not as invariable as thought. To
disentangle the contribution of PSI and PSII of total leaf
fluorescence we need to have accurate knowledge of the
PSI spectrum, how it is affected by temperature and how
it potentially differs between plant species.
Here, we used an array of biochemical and optical
techniques to study the biological variability of PSI. To
this end, we isolated PSI-LHCI supercomplexes from 5
different angiosperms (flowering plants), namely the sun-
tolerant eudicot species Arabidopsis thaliana and Spinacia
oleracea and the monocot species Zea mays, Spathiphyl-
lum wallisii and Calathea roseopicta [in lesser used offi-
cial nomenclature Goeppertia roseopicta (Borchsenius
etal. 2012)]. The latter two are houseplants introduced
from the South American tropical forests and are adapted
a shade environment (Schott and Endlicher 1832; Van
Huylenbroeck etal. 2018). Zea mays is also cultivated
from South America, but is a sun-tolerant species (Benz
2001). The diversification of monocots and eudicots is
hypothesised to have occurred around 130 million years
ago which is therefore the maximum time between the
most-recent common ancestor of two species in this study
(Moore etal. 2007).
Since canopy shade light is enriched in far-red light
(Rivadossi etal. 1999; Johnson and Wientjes 2020),
plants that adapted to a niche in a shaded environment
could benefit from using a broader spectrum of light, espe-
cially in the far-red part. Therefore, we hypothesise that
the plants from the tropical rainforest, S. wallisii and C.
roseopicta will have red-shifted red forms compared to
the crop/pioneer species A. thaliana, S. oleracea and Z.
mays. For sun-tolerant species the extended lifetime and
the accompanied risk of damage to the photosystems could
have suppressed adaptation to more red-shifted Chls. We
found, based on multiple technical and biological repli-
cas, significant differences in the emission properties of
PSI of the five studied plant species. Moreover, we show
that the PSI emission spectrum and intensity is affected
by changes in temperature within a biologically relevant
range (280–298K).
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37Photosynthesis Research (2023) 155:35–47
1 3
Materials andmethods
Plant material
Arabidopsis thaliana and Zea mays were grown in a plant
cabinet at 60% humidity, 125µmol/m/2/s, and 8hday.
Plants from both species were harvested after 6–8weeks.
Spinacia oleracea was purchased at the local supermarket.
Spathiphyllum wallisii and Goeppertia/Calathea roseop-
icta were bought at the local gardening shop and grown
in a living room without direct sunlight. The species were
identified with the Pl@ntnet app (Identify, explore and
share your observations of wild plants. Pl@ntNet. (n.d.).
Retrieved January 25, 2022, from https:// ident ify. plant net.
org/).
Protein sequence alignment
The protein sequences of Lhca1-4 of A. thaliana were
acquired from the Arabidopsis Information Resource
(TAIR) (Berardini etal. 2015). Orthologs in Z. mays and
S. oleracea were detected using the “plant orthologs”
section at the TAIR webpage of the specified protein. In
case of multiple orthologs per species, all were aligned to
the A. thaliana protein sequence and the sequences with
the highest degree of cover were chosen. A deep learning
approach was used to detect and remove chloroplast tran-
sit peptides from the sequences (Armenteros etal. 2019).
Chloroplast transit peptides were detected with high prob-
ability (> 0.77). Sequences were aligned with the web-
based tool multiple sequences alignment Clustal Omega
(Sievers etal. 2011; McWilliam etal. 2013) from which
percent identity was obtained.
PSI‑LHCI isolation
Thylakoids were harvested according to a protocol adapted
from Caffari etal. (Caffarri etal. 2009). In short, leaves
were homogenised quickly in buffer 1 (400-mM sorbitol,
5-mM EDTA, 10-mM NaHCO3, 5-mM MgCl2, 20-mM
tricine and 10-mM NaF) in a blender. The solution was
filtered through a 400-µm and 100-µm mesh sized filter
and centrifuged for 3min at 2000g. The pellet was care-
fully resuspended in buffer 2 (300-mM sorbitol, 5-mM
MgCl2, 20-mM tricine, 2.5-mM EDTA, 10-mM NaF and
10-mM NaHCO3) and the chloroplasts were again pelleted
for 3min at 2000g. The pellet was resuspended in buffer
3 (5-mM MgCl2, 2.5-mM EDTA, 10-mM NaF and 20-mM
Hepes) and centrifuged for 10min at 10,000g. The centri-
fuge was cooled to 4°C and the material was kept on ice
as much as possible. The pellet was resuspended in buffer
1 and stored at −80°C until further use.
PSI-LHCI complexes were isolated according to a slightly
modified protocol of Wientjes etal. from a second sucrose
gradient (Wientjes etal. 2009). Solubilisation happened with
0.6% α-DM or β-DM and sucrose gradients were centrifuged
for 16–20h at 40.000rpm in a Beckman SW-41Ti rotor
(1.97 × 105g). The lowest green band contained PSI-LHCI
and was isolated with a syringe, concentrated and loaded
on a second sucrose gradient. The lowest band was isolated
with a syringe, concentrated and stored at −80°C. The one
but lowest band on the first sucrose gradient contained the
PSII core and was isolated and stored at −80°C.
Pigment analysis
Pigments were extracted in 80% acetone and absorption
spectra were recorded from 350 to 750nm. Spectra were
fitted with the spectra of individual pigments as described
by Chazaux etal. to determine Chl concentration and Chl
a/b ratio (Chazaux etal. 2022).
Gel electrophoresis
The SDS-PAGE electrophoresis protocol was adapted from
Laemmli (1970). A ratio of 32:1 acrylamide/bisacrylamide
was used to a concentration of 15% in the running gel and
5% in the stacking gel. 2M urea was incorporated in the gel.
Samples were diluted to loading concentration (~ 150µg/
mL Chl) in the presence of DTT and were heated to 70°C
for 10min. Around 2µg Chl was loaded per lane. The gel
was stained with Coomassie R and imaged with a Bio-rad
Universal Hood II.
Spectroscopy
Samples were diluted to 10µg/mL in buffer 1. Absorption
spectra were recorded with a Cary-4000 UV–VIS spectro-
photometer (Agilent Technologies, Inc., Santa Clara, USA)
from 350 to 800nm. Absorption at 750nm was set to zero
and spectra were corrected for scattering by subtracting a
linear line with the slope set to the average slope of the spec-
trum between 750 and 800nm. Spectra were normalised
by the area between 650 and 750nm. Technical replicates
were averaged and significant differences between biologi-
cal replicates were determined with a Tukey test. Biological
replicates were averaged and plotted.
Fluorescence spectra in a 1-cm cuvette in a Fluorolog 3.22
spectrofluorometer (HORIBA Jobin Yvon, Longjumeau,
France) with 435-nm excitation (10-nm slit width) and
600–800-nm emission were recorded. The temperature was
regulated with a water bath and measured with a temperature
sensor. The temperature was increased stepwise with 4–5
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38 Photosynthesis Research (2023) 155:35–47
1 3
degrees steps from 280 to 298K whilst cooling the sample
in between to minimise damage. Damage was checked by
taking a second spectrum at 280K after the 298-K measure-
ment. Samples were recorded three times with an integration
time of 0.4s and averaged. To quantify the change in the
red-form emission, the minimal fluorescence value was set
to zero and the data were normalised to the maximum value
between 675 and 690nm to correct for the damage to the
photosystem that resulted in a lower fluorescence. The sum
of intensity between 700 and 800nm was calculated and
the resulting value from the measurement at 293K was set
to 1. Where applicable, data were interpolated and values at
298K and 286K were averaged.
Steady-state 77-K fluorescence measurements were per-
formed in a glass Pasteur pipette (pathlength ~ 1mm) in a
glass Dewar filled with liquid nitrogen with excitation wave-
length 435nm and emission recorded from 600 to 800nm
with a 1-nm step-size and an integration time of 0.4s. Spec-
tra were recorded three times and averaged.
Room temperature and 77-K spectra were normalised to
the maximum fluorescence value > 700nm, smoothed with
the Savitzky–Golay filter, 20 points of window, 2nd poly-
nomial order and normalised again using Origin (Origin,
Version 2020b. OriginLab Corporation, Northampton, MA,
USA). Where applicable, technical replicates were averaged
and significance was determined by a Tukey test (p < 0.05)
between different species. Averages were taken per species
and plotted. Visualisation in boxplots was achieved with Ori-
gin. Temperature-controlled spectra between 280 and 298K
were also smoothed with the Savitzky–Golay filter, 20 points
of window, and 2nd polynomial order with Origin.
Time‑resolved spectroscopy
Time-resolved fluorescence measurements were performed
with a streak-camera setup as described earlier (Van Oort
etal. 2009). Excitation wavelength was 400nm and time
window was set to 800ps. Samples were diluted to 10µg/
mL Chl in a 1 × 1-cm cuvette and stirred continuously. Tem-
perature of the sample was regulated with a Peltier-based
thermostat connected to a copper sample holder and cooled
with a water bath.
The collected images were corrected for background sig-
nal and spatial variations in detector sensitivity. Corrected
images were averaged over 5nm and globally analysed
with the specialised software Glotaran to construct decay-
associated spectra (DAS) (Mullen and Van Stokkum 2007;
Snellenburg etal. 2012).
4 DAS fitted the PSI streak images best. Spectra were
normalised to the total area under the graphs and interpo-
lated using Origin (Origin, Version 2020b. OriginLab Cor-
poration, Northampton, MA, USA). DAS1 described excita-
tion energy transfer within PSI. DAS2 and DAS3 described
the decay of PSI, whilst DAS4 described the decay of con-
taminations, like free Chls, LHCII or PSII. To calculate the
average lifetime of PSI, DAS2 and DAS3 were used:

𝜏

=AreaDAS2 ×𝜏2+AreaDAS 3 ×𝜏3∕

AreaDAS2 +AreaDAS 3

, with
AreaDAS#
the area under the DAS, and
𝜏#
the fluorescence
lifetime associated with the DAS. The emission maximum
of DAS3 was determined in the section between 703 and
778nm. Values for lifetime and λmax were averaged between
technical replicates and statistical differences between bio-
logical replicates were determined with a one-way ANOVA
and a Tukey test (p < 0.05) using Origin (Origin, Version
2020b. OriginLab Corporation, Northampton, MA, USA.).
Calculation PSI contribution tototal fluorescence
Fluorescence spectra at various temperatures of PSI from
S. oleracea were used. For the PSII spectrum we recorded
the emission of freshly prepared grana membranes prepared
according to (Barbato etal. 2000). Since no temperature
dependence of PSII was observed, this PSII spectrum was
used for all temperatures. The spectra were normalised to
the total area and multiplied with the transmission spectrum
of the RG9 filter, acquired from Schott by linear interpo-
lation between given transmission data points in the RG9
datasheet (Schott AG, Mainz, Germany). This filter is
common in many PAM systems. Spectra were multiplied
with their lifetime (69ps for PSI as determined with time-
resolved spectroscopy, 224ps for open PSII reaction centres
(Wientjes etal. 2013a, b) and 1.6ns for closed PSII reaction
centres (Roelofs etal. 1992; Matsubara and Chow 2004;
Rizzo etal. 2014)) and added up to get the total fluorescence
spectrum in F0 and FM situation. Equal excitation of the two
photosystems was assumed. The PSI contribution to the total
fluorescence in F0 and FM (PSI/(PSI + PSII)) and Fv/FM (Fv/
FM = 1− F0/FM) was calculated.
Results
Protein composition ofPSI‑LHCI isolates
PSI-LHCI complexes from five plant species were isolated
and purified on sucrose gradients and subjected to an array
of spectroscopic and biochemical techniques. Firstly, the
protein content of the isolates was examined on an SDS-
PAGE gel to identify the composition of PSI-LHCI in the
different species and potential contaminations (Fig.1). In all
lanes, the core-subunits of PSI, PsaA and PsaB, are clearly
visible. Two smaller bands around 60kDa can be seen in
Z. mays and S. oleracea, which are most likely originat-
ing from ATP synthase α and β subunit. Since these pro-
teins do not contain pigments, they do not interfere with
the spectroscopic measurements (Tian etal. 2017). In the
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39Photosynthesis Research (2023) 155:35–47
1 3
lane of A. thaliana, five bands between 26 and 17kDa are
visible, which correspond to Lhca1-4 and PsaD (Ballottari
etal. 2007). The migration behaviour of the Lhca bands of S.
oleracea is very similar to the ones from A. thaliana, but the
bands of the other three species differ. The large variation
in migration behaviour of the Lhcas from the investigated
species indicates that there is a variation in the lengths and/
or amino acid composition of the polypeptide. This variation
is less apparent in the Chl a/b ratios of the samples loaded on
this gel. In addition, a high degree of similarity is observed
in the protein sequences of Z. mays, A. thaliana, and S. oler-
acea, the three species of which the DNA sequences are
known (90–94% between A. thaliana and S. oleracea and
85–89% between A. thaliana and Z. mays, see Supplemen-
tary TableS1). Despite the high similarity, repeated SDS-
PAGE of different PSI-LHCI isolates show consistently a
different migration behaviour of Lhca proteins between the
species. The sizes of the mature proteins of these three spe-
cies differ maximally 0.3kDa. Therefore, the differences in
migration behaviour must be due to the variation in amino
acid composition. Indeed, small variations in the amino acid
composition of membrane proteins can have large effects
on the SDS-PAGE migration behaviour (Rath etal. 2009).
PSI fluorescence varies betweenspecies
At 77K, fluorescence of the red forms of PSI is very pro-
found, since the uphill energy transfer from a red-shifted
Chl to the bulk Chls is severely slowed down due to the
lack of environmental heat. Less environmental heat means
a higher chance of the excitation residing on one of the
low-energy Chls and eventually emitting a photon. Steady-
state-fluorescence measurements on PSI complexes of dif-
ferent plant species at 77K allow us to chart their difference
in fluorescence spectra and especially in the red-emitting
Chls. Moreover, the integrity of the complexes and contami-
nations can be examined at this temperature as mainly PSI
emits around 730–740nm, whilst PSII and free Chls a emit
predominantly in the 670–690-nm region. S. wallisii and
to a lesser extent C. roseopicta are the only two isolates
with notable contamination with non-PSI bound pigments,
as can be seen by a second fluorescence peak around 680nm
(Fig.2 and Supplementary Fig.S1). Most likely these stem
from PSII complexes or free Chls. Since the contamination
in S. wallisii could not be diminished with a second sucrose
gradient and increased substantially after being subjected to
temperature changes, this could also point towards instabil-
ity of the PSI complex for this plant species.
The 77-K emission spectra of the PSI complexes were
compared with the emission of the intact thylakoid mem-
branes to check if PSI isolation affects its emission prop-
erties (Supplementary Fig.S2). Perfect overlap, signifying
no effect of the isolation, was observed for A. thaliana, Z.
mays, and S. oleracea. Instead, the spectra of isolated PSI
from S. wallisii and C. roseopicta show a broader and a few
nm blue-shifted spectrum when compared to the intact thy-
lakoids. This suggests that these PSI complexes are indeed
less stable in detergent.
Variations in the PSI fluorescence spectra at < 700nm
can be explained by contamination with other compounds,
but differences at wavelengths > 700nm are likely due to
Fig. 1 SDS-PAGE of PSI-LHCI complexes from Arabidopsis thali-
ana (At), Zea mays (Zm), Spinacia oleracea (So), Spathiphyllum wal-
lisii (Sw) and Calathea roseopicta (Cr). Also a marker (M) is loaded
and the bands annotated with their molecular weight. 2–2.5µg Chl
was loaded per lane. Chl a/b ratios of the loaded samples are listed
below the lanes
Fig. 2 77-K fluorescence measurements on PSI from five plant spe-
cies. A Average PSI spectra recorded at 77K normalised to the emis-
sion maximum. Excitation at 435 nm. B Boxplot of the emission
maxima. The boxes express 25th and 75th percentile, with median
(line) and mean (open circle) indicated. Whiskers indicate the 5th and
95th percentile. Significant groups as determined by a Tukey test are
indicated with letters (p < 0.05). Number of biological replicates is 4
(N = 4) for all species
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40 Photosynthesis Research (2023) 155:35–47
1 3
diversity in PSI across species. In Fig.2 it can be observed
that the emission maximum of PSI from A. thaliana
(λmax = 731 ± 0.4nm) is blue shifted and the spectrum of C.
roseopicta (λmax = 740 ± 0.8nm) red shifted with regard to
the complexes of the other species. Significant differences
are found when comparing the λmax of the different PSI
complexes (Fig.2). It is interesting to note that C. roseop-
icta showed an even more red-shifted emission maximum
in intact thylakoids, with an emission maximum of 745nm
(Supplementary Fig.S2). The emission maxima of Z. mays
(λmax = 735 ± 0.7nm), S. oleracea (λmax = 736 ± 0.5nm) and
S. wallisii (λmax = 736 ± 1.4nm) cannot be significantly dis-
tinguished from each other. As far as we know these results
show for the first time that PSI complexes from different
plant species are significantly different in their spectral
properties.
PSI absorption spectra are similar
Next, absorption spectra of the PSI isolates from the spe-
cies were recorded, showing minor differences based on
four independent sample preparations per plant species
(Fig.3). A small but significant difference is found between
Z. mays and S. wallisii regarding the amount of absorption
at 465nm, resembling Chl b absorption (p = 0.049 as deter-
mined by one-way ANOVA with Tukey test). Although it
is possible that PSI from different species have a slightly
altered Chl a/b ratio, contamination with PSII complexes can
also explain this deviance. In support for the latter hypoth-
esis, the 77-K spectrum also indicates the PSI samples of S.
wallisii are more contaminated with uncoupled Chls and/or
PSII complexes than the sample of Z. mays (Supplementary
Fig.S1). When studying the far-red absorption of PSI of
the five species, no significant differences are detected. This
points out that although PSI fluorescence at 77K differs
significantly between species, the RT absorption spectra are
hardly affected by this diversity.
Variability ofRT PSI fluorescence
Next, we present the room-temperature steady-state fluores-
cence spectra that can be used in remote sensing applications
as PSI spectrum (Fig.4). Contrarily to the earlier absorp-
tion and 77-K fluorescence spectra, only the spectra with the
least contamination are presented, since impurities of PSII or
free pigments fluoresce more than PSI and tend to dominate
the spectrum. Like the 77-K fluorescence spectra, the RT
fluorescence spectra also display variation in the amount of
fluorescence in the far-red region. C. roseopicta has the most
red-shifted fluorescence spectrum and A. thaliana the most
blue shifted. These differences are apparent both from the
values of λmax, the wavelength at maximum emission, and
the red tail of the spectra. All in all this points again towards
heterogeneity in PSI of different plant species that was also
visible in the 77-K fluorescence spectra.
Excitation energy trapping byPSI
Streak camera time-resolved fluorescence measurements
allow for accurate determination of both spectra and fluo-
rescence decay kinetics of different components within a
sample. In the case of PSI isolates, the data can be fitted
with four decay-associated spectra (DAS); 3 DAS for PSI
and one for impurities (Fig.5A). The short-lived DAS1,
with lifetimes between 6 and 20ps, has both positive and
negative parts and represents transfer from the bulk Chls
to the red-absorbing Chls. DAS 2 and 3, with lifetimes
between 23–46ps and 79–168ps, respectively, correspond
to PSI. DAS2 describes the decay of the bulk Chls, whilst
DAS3 is related to red-Chl decay. The lifetime of DAS3 is
larger than the one of DAS2 since uphill energy transfer
is required for the excitation to reach the reaction cen-
tre. DAS4 belongs to impurities of the sample that can
either be PSII contamination or free pigments. From the
decay-associated spectra of PSI, an average lifetime of the
complex can be calculated by taking the sum of the prod-
ucts of the relative area of a DAS and its lifetime for the
relevant spectra. Average lifetimes for the different PSIs
calculated this way range from 60 to 70ps at 293K, which
is longer than normally reported for PSI-LHCI (Croce
etal. 2000; Ihalainen etal. 2002, 2005; Van Oort etal.
2008; Wientjes etal. 2011a, b; Jennings etal. 2013). The
lifetimes of PSI in this study do not differ significantly
from each other (Fig.5B). The same is true for the aver-
age lifetime and the λ-max of DAS2 (Fig.5C, D). These
Fig. 3 Average absorption spectra of PSI from 5 plant species.
Absorption at 750nm is set to zero and spectra are normalised to the
area between 650 and 750nm. N = 4 for Zm, So, Sw and Cr, N = 5 for
At
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41Photosynthesis Research (2023) 155:35–47
1 3
results illustrate that the bulk Chls in PSI are conserved
between these five species and that their energetic connec-
tions to the core complex are comparable. However, signif-
icant differences are found in the DAS3 emission maxima
and lifetimes (Fig.5E, F). A. thaliana has a blue-shifted
λmax (719 ± 1.8 nm) and shorter lifetime (100 ± 6ps)
compared to C. roseopicta (λmax = 729 ± 1.3 nm, life-
time = 127 ± 2ps). The C. roseopicta DAS3 lifetime is
also longer than the lifetimes form Z. mays (97 ± 2ps) and
S. oleracea (100 ± 3ps). The S. wallisii DAS3 lifetime
(λmax = 727 ± 2.6nm, lifetime = 126 ± 11ps) is distinct
from Z. mays. An increased DAS3 lifetime for a species
correlates with a higher λmax found in the 77-K fluores-
cence measurements (Fig.2).
These combined results illustrate that the fluorescent
properties of PSI from different plant species differ. The
RT, 77K and time-resolved fluorescence measurements
all demonstrate that the red forms of PSI from C. roseop-
icta are red shifted and the ones from A. thaliana are blue
shifted compared to the other species. Little differences
are found between the red forms of Z. mays, S. oleracea
and S. wallisii.
PSI fluorescence spectrum changes atbiologically
relevant temperatures
Temperature is known to influence the shape and intensity
of fluorescence spectra (Croce etal. 1998). Several stud-
ies have presented fluorescence spectra recorded at differ-
ent temperatures invitro for PSI core, LHCI complexes
and PSI (Croce etal. 1998, 2000; Agati etal. 2000). Dif-
ferences in the amount of fluorescence from the red forms
were observed. More specifically, since environmental heat
is required to transport excitation energy from the red forms
to the PSI core, a decrease in red fluorescence is observed
with increasing temperatures. However, the experiments
on LHCI were performed at 80–280K, which makes the
spectra less relevant for natural conditions. Fluorescence
changes have also been observed invivo in whole leaves at
biologically relevant temperatures, but these changes were
attributed to changes in PSII fluorescence (Agati etal. 2000).
Here, we present temperature-dependent changes in the PSI
fluorescence spectrum from S. oleracea between 280 and
298K (Fig.6A). A clear decrease in fluorescence from the
red forms can be recognised upon increasing the tempera-
ture and this fluorescence makes an almost full recovery
Fig. 4 RT steady-state fluorescence spectra of PSI isolates from five plant species at excitation of 435nm. Spectra are normalised to the maxi-
mum fluorescence intensity peak of PSI around 723nm. All spectra are plotted together in the last panel for comparison
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42 Photosynthesis Research (2023) 155:35–47
1 3
Fig. 5 Overview of time-resolved fluorescence spectra, λmax and
lifetimes. A Representative decay-associated spectra of a PSI isolate
from S. oleracea. B Average lifetimes of PSI from five species, cal-
culated from DAS2 and DAS3. C, D λmax and lifetime of DAS2. E, F
λmax and lifetime of DAS3. Where applicable, significant groups are
indicated with letters, based on a Tukey test (p < 0.05). N = 4 for all
species
Fig. 6 Temperature dependence of steady-state PSI fluorescence
spectra with excitation at 435 nm. A S. oleracea PSI fluorescence
spectra recorded at stepwise increased temperatures (280–298 K).
Recovery of the original spectrum was checked by cooling back down
to 280 K (280K-2). B Steady-state modelled temperature-depend-
ent spectrum of PSI-LHCI. Temperature-dependent transfer rates
were calculated via a Boltzmann equilibrium and applied in a model
resembling the one from Schiphorst etal. (2022). A more detailed
description of the construction of the model can be found in the Sup-
plementary information
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43Photosynthesis Research (2023) 155:35–47
1 3
when cooling back to 280K. The total area under the graph
decreases at higher temperature, indicating faster trapping
of excitations by the reaction centre. The peak at ~ 685nm
increases a little upon increasing temperature, but does not
drop back when cooling back down to 280K, most likely
pointing towards a minute increase of free pigments. This is
in accordance with the slightly incomplete recovery of the
red fluorescence peak upon cooling, which points towards a
small amount of damage to the PSI complex by either expo-
sure to light or elevated temperatures. Heating to 307K led
to considerably less recovery of the original fluorescence
data at 280K (Supplementary Fig.S2). The area under
the fluorescence spectrum from 700 to 800nm of PSI of
S. oleracea at 286K increases to 110% (0.3% SE, N = 3
technical replicates) of the area at 293K and at 298K; the
area is only 93% (0.2% SE, N = 4 technical replicates) of
the area at 293K after normalisation to the peak at 685nm
(Fig.6A). To check if the PSII emission intensity was also
affected by temperature the PSII core was isolated and it
appeared that PSII was to a substantially larger extent dam-
aged by temperature than PSI (Supplementary Fig.S3).
More importantly, the shape of the fluorescence spectrum of
PSII is not temperature dependent. This shows that the total
invivo fluorescence does not only vary due to biological
reasons, such as quenching of PSII, but also due to abiotic
variables, such as a temperature effect on PSI. The temper-
ature-dependent change in PSI fluorescence is comparable
to differences found with simple temperature-dependent PSI
spectrum modelling (Fig.6B, Supplementary Fig.S4 and
supplemental text), indicating that the differences arising
with changing temperature can be explained by Boltzmann
equilibrium distribution.
Using the PSI and PSII spectra from S. oleracea, an esti-
mation of the PSI contribution to the total invivo chloro-
phyll fluorescence at different temperatures can be made.
The total fluorescence spectrum (the addition of the PSI and
PSII spectrum multiplied with their lifetimes) was calcu-
lated considering the F0 situation in which all PSII reaction
centres are open and the FM situation with only closed PSII
reaction centres (Fig.7). Considering the use of an RG9
filter as is common in most modern PAM set-ups (Porcar-
Castell etal. 2014; Pfündel 2021), PSI contributes 41% to
F0 fluorescence. At 280K this contribution increases to 43%
but at 298K it is 40%. However, this does not change the
FV/FM ratio (measure for PSII quantum efficiency), which
remains 0.78 in these calculations. In FM conditions, the PSI
contribution is only 9% and is not significantly influenced by
changes in temperature.
Discussion
All higher organisms depend on oxygenic photosynthesis
for their survival. Oxygenic photosynthesis is driven by
PSI and PSII. Whilst multiple studies have investigated PSI
spectral properties and trapping kinetics (Croce etal. 1996,
1998, 2000; Ihalainen etal. 2002, 2005; Wientjes etal.
2011a, b; Jennings etal. 2013; Bos etal. 2017; Molotokaite
etal. 2017; Chukhutsina etal. 2020), little is known about
how these properties differ between plant species. Here we
Fig. 7 PSI and grana spectra from S. oleracea at 293K. A PSI and
PSII spectra multiplied with their lifetime considering open PSI and
PSII reaction centres (69 and 224 ps, respectively), together with
the resulting F0 fluorescence spectrum. In red the PSI contribution
to the fluorescence. B FM situation with closed PSII reaction cen-
tres (1.6ns). A smaller PSI contribution to the total fluorescence is
observed in FM
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44 Photosynthesis Research (2023) 155:35–47
1 3
isolated PSI-LHCI from five different plant species to com-
pare their protein composition and optical properties.
We found significant differences in the red forms of the
various PSI complexes, based on 77K steady-state fluo-
rescence measurements. These differences most likely arise
from differences in Lhca3 and Lhca4, the LHCI subunits
in which the red Chls are located (Morosinotto etal. 2003;
Wientjes and Croce 2011). We hypothesised that plants
adapted to life in the canopy shade (e.g. S. wallisii and C.
roseopicta) are likely to have evolved more red-shifted
Chls than plants adapted to full-sun conditions, such as
crop plants (Z. mays and S. oleracea) or pioneer species (A.
thaliana). The 77K fluorescence spectrum of C. roseopicta
is indeed found to be red shifted and the one of A. thaliana
blue shifted with regard to the other species. The lifetime of
the DAS associated with the red forms and the RT fluores-
cence spectra is in agreement with this result. However, the
absorption spectra of the complexes of the different species
do not differ in the part > 700nm, which indicates that red-
shifted emission is not correlated with a detectable change
in absorption of far-red photons. Our hypothesis that PSI of
species that are adapted to shadow-rich conditions can use
more far-red light for photosynthesis is not confirmed for the
plants investigated in this study.
Since A. thaliana is a model species, its PSI 77K fluores-
cence spectra have been reported several times (Drop etal.
2011; Wientjes etal. 2011a, b; Galka etal. 2012; Benson
etal. 2015; Chukhutsina etal. 2020), with an emission maxi-
mum ranging between 730 and 733nm. Our determined
emission maximum of 731 ± 0.4nm is in agreement with
these studies. Also the emission maximum of S. oleracea
of 736 ± 0.5nm is similar to previously determined values
(Pålsson etal. 1995). However, Chukhutsina etal. found
for Z. mays an emission maximum of 730nm (Chukhut-
sina etal. 2020), which does not fall within our determined
range (λmax = 735 ± 0.7nm). The differences between these
studies are surprising considering the determined emission
maxima of A. thaliana in these studies are similar. Moreover,
only small variations have been found between studies that
recorded the 77K emission maximum of the same species, a
range of 730–733nm for A. thaliana in 5 studies and a range
of 733–736nm for P. sativum based on three studies (Akhtar
and Lambrev 2020; Wang etal. 2021; Yan etal. 2021), indi-
cating that differences in isolation procedure do not lead to
large deviations. It has also been shown that various light
conditions during growth do not lead to a shift in the PSI
fluorescence maximum in A. thaliana (Wientjes etal. 2013a,
b). In our opinion, differences between cultivars of Z. mays
are the most likely explanation for the deviation between
the studies, which makes Z. mays an interesting species to
study further.
In this study, we found that C. roseopicta has the
most red-shifted λmax reported at 77K for flowering
plants (λmax = 740 ± 0.8nm for the isolated complex and
λmax = 745nm in thylakoids). The combined data on PSI
from different angiosperms suggest that emission maxima
of flowering plants fall in a range of about 15nm between
730 and 745nm at 77K. More diversity is found between
the red forms of other species of oxygenic photosynthetic
organisms (Chen etal. 2020; Huang etal. 2021; Yan etal.
2021). The moss P. patens with a similar LHCI build-up
as angiosperms is found to have an emission maximum at
727nm at 77K (Yan etal. 2021). The blue shift of λmax
in comparison to flowering plants is due to the replace-
ment of Lhca4 with Lhca5, which lacks red-shifted Chls
(Wientjes etal. 2009; Yan etal. 2021). As such, the LHCI
of PSI of P. patens is composed of an Lhca1/5 and an
Lhca2/3 dimer. The red forms of Lhca3, in the Lhca2/3
dimer, are responsible for the 727-nm emission (Wientjes
etal. 2011a, b). The 77-K emission of the PSI complex of
C. reinhardtii is about 18nm blue shifted compared to that
of A. thaliana (Drop etal. 2011; Le Quiniou etal. 2015;
Huang etal. 2021). Interestingly, their trapping times are
similar, mainly because LHCI of C. reinhardtii is larger
than the one of A. thaliana, which increases the lifetime
to a similar value (Le Quiniou etal. 2015). Within algae,
variation in antennae size and red shift of red forms is
identified, but no species with 77-K emission maxima as
red shifted as those of angiosperms have been reported
(Swingley etal. 2010; Perez-Boerema et al. 2020).
Cyanobacteria lack LHCI, but in several species (more
red shifted) red forms that arise from oligomerisation of
PSI have been detected, even up to λmax of fluorescence
of 760nm at 6K in Spirulina platensis (Karapetyan etal.
1999, 2014; Gobets etal. 2001).
In PAM measurements on whole leaves, the PSI contribu-
tion to the total fluorescence is usually neglected (Pfündel
etal. 2013; Giovagnetti etal. 2015). However, several stud-
ies have shown that the PSI contribution can reach up to 40%
to the minimal fluorescence level (F0) and up to 10% to the
maximum fluorescence value (FM) at detection wavelengths
of 720nm or larger (Agati etal. 2000; Franck etal. 2002). It
has been determined that PSI can contribute up to 25% in A.
thaliana, 50% in Z. mays (Pfündel etal. 2013), and 45% in
Prunus laurocerasus L. (Pfündel 2021) to the fluorescence
at detection wavelengths > 700nm. Our calculations of the
PSI contribution to F0, based on the PSI and PSII spectra of
S. oleracea at 293K, correspond to these values. We also
show that changes in temperature between 280 and 298K do
not significantly alter this contribution, nor does it change
the FV/FM ratio. Therefore, PSI correction of PAM data is
essential for a correct interpretation of the PSII quantum
yield, but the correction does not have to be carried out with
a temperature-dependent PSI spectrum. However, variations
in PSI contribution in F0 and resulting changes in Fv/FM can
arise between different plant species.
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45Photosynthesis Research (2023) 155:35–47
1 3
Put together, our results show variability of the PSI fluo-
rescence spectrum between different angiosperms. More
specifically, the shadow-tolerant plant C. roseopicta has
the most red-shifted emission maximum for PSI reported
for flowering plants. However, its absorption spectra does
not differ significantly from our other studied plants in the
far-red region which challenges the view that red-shifted
red forms allow plants to use a broader light spectrum. In
addition, we showed that biologically relevant temperature
changes have a substantial effect on the emission spectrum
of PSI.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s11120- 022- 00971-2.
Acknowledgements We thank Herbert van Amerongen for providing
helpful comments that improved the manuscript. This work was sup-
ported by the Dutch Organisation for Scientific Research (NWO) via a
Vidi grant no. VI.Vidi 192.042 (E.W.).
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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