Plant Physiology Preview. Published on December 23, 2009, as DOI:10.1104/pp.109.148213
Plant Physiology revised manuscript
18/12/09
Running title: Kinetic and spectral resolution of NPQ components
Corresponding author: Alfred R. Holzwarth
Max-Planck-Institut für Bioanorganische Chemie
Stiftstr. 34-36, 45470 Mülheim a.d. Ruhr
E-mail: [email protected]
Tel: +49-208-306-3571
Fax: +49-208-306-3951
Research field: Bioenergetics and Photosynthesis
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Copyright 2009 by the American Society of Plant Biologists
KINETIC AND SPECTRAL RESOLUTION OF MULTIPLE
NON-PHOTOCHEMICAL QUENCHING COMPONENTS
IN ARABIDOPSIS LEAVES
Petar H. Lambrev, Manuela Nilkens, Yuliya Miloslavina, Peter Jahns, and Alfred R.
Holzwarth
Max-Planck-Institut für Bioanorganische Chemie, Stiftstr. 34-36, 45470 Mülheim a.d. Ruhr,
Germany; and Institut für Biochemie der Pflanzen, Heinrich-Heine Universität Düsseldorf,
40225 Düsseldorf, Germany
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Financial source:
Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 663
Author affiliations:
Petar H. Lambrev, Max-Planck-Institut für Bioanorganische Chemie
Manuela Nilkens, Heinrich-Heine Universität Düsseldorf
Yuliya Miloslavina, Max-Planck-Institut für Bioanorganische Chemie
Peter Jahns, Heinrich-Heine Universität Düsseldorf
Alfred R. Holzwarth, Max-Planck-Institut für Bioanorganische Chemie *)
*)
Corresponding author, E-mail: [email protected]
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Abstract
Using novel specially-designed instrumentation, fluorescence emission spectra were recorded
from Arabidopsis thaliana leaves during the induction period of dark to high-light (HL)
adaptation in order to follow the spectral changes associated with the formation of nonphotochemical quenching. In addition to overall decrease of PSII fluorescence (quenching)
across the entire spectrum, HL induced two specific relative changes in the spectra – i) a
decrease of the main emission band at 682 nm relative to the far-red (FR, 750-760 nm) part of
the spectrum (ΔF682) and ii) an increase at 720-730 nm (ΔF720) relative to 750-760 nm. The
kinetics of the two relative spectral changes and their dependence on various mutants revealed
that they do not originate from the same process but rather from at least two independent
processes. The
ΔF720
change is specifically associated with the rapidly-reversible energy-
dependent quenching qE. Comparison of the wild type Arabidopsis with mutants unable to
produce or overexpressing PsbS showed that PsbS was a necessary component for ΔF720. The
spectral change ΔF682 is induced both by qE and by PsbS-independent mechanism(s). A third
novel quenching process, independent from both PsbS and zeaxanthin, is activated by a high
turnover-rate of PSII. Its induction and relaxation occur on a time scale of a few minutes.
Analysis of the spectral inhomogeneity of NPQ allows extracting mechanistically valuable
information from the fluorescence induction kinetics when registered in a spectrally-resolved
fashion.
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One of the most important photoprotective mechanisms against high-light (HL) stress in
photosynthetic organisms is the non-photochemical quenching (NPQ) of excitation energy,
which is mostly due to thermal deactivation of pigment excited states in the antenna of
Photosystem II (PSII). There exist a number of literature reviews on the subject (DemmigAdams and Adams, 1992; Horton et al., 1996; Horton and Ruban, 1999; Niyogi, 1999;
Niyogi, 2000; Müller et al., 2001; Krause and Jahns, 2004; Golan et al., 2004; Horton and
Ruban, 2005). Chlorophyll fluorescence, and in particular pulse-amplitude modulated
fluorometry as introduced by Schreiber (1986) has become by far the dominant technique to
measure NPQ in leaves, chloroplasts, and intact microorganisms (Krause and Weis, 1991;
Govindjee, 1995; Maxwell and Johnson, 2000; Krause and Jahns, 2003; Schreiber, 2004),
more recently often combined with specific NPQ mutant studies (Golan et al., 2004; Kalituho
et al., 2006; Dall'Osto et al., 2007; Kalituho et al., 2007). In this technique periodic saturating
light pulses are applied - superimposed on the continuous actinic irradiation applied to induce
NPQ - in order to transiently close the PSII RCs. Since the photochemistry contribution
(photochemical quenching, qP) is thus brought to zero, the method allows to follow the
dynamics of the NPQ development and relaxation by fluorescence in a relatively simple
manner (Krause and Jahns, 2003; Krause and Jahns, 2004).
Mostly based on its relaxation kinetics, NPQ has been divided technically into the three
kinetic components qE, qT and qI, i.e. the rapid, middle and slow phases of relaxation
(Horton and Hague, 1988), initially attributed to energy-dependent quenching, state
transitions, and photoinhibitory quenching (Quick and Stitt, 1989). The rapidly forming and
reversible part of NPQ, qE, is the most thoroughly studied. It is well established that this type
of quenching is a finely regulated process in which the main governing factors are the proton
gradient across the chloroplast thylakoid membrane,
ΔpH
(Wraight and Crofts, 1970;
Briantais et al., 1979), the xanthophyll cycle, i.e. conversion of violaxanthin (Vx) to
antheraxanthin and zeaxanthin (Zx) (Demmig et al., 1987; Demmig-Adams, 1990; DemmigAdams and Adams, 1992), and the action of the PsbS protein (Funk et al., 1995; Li et al.,
2000; Li et al., 2004; Niyogi et al., 2005). The actual molecular mechanism is still unknown,
although there is no shortage of raised hypotheses and proposed quencher candidates: energy
transfer from chlorophyll (Chl) to Zx in the major light-harvesting complex (LHCII) (Frank et
al., 2000), electron transfer from a carotenoid to Chl forming a Zx-Chl or Lut-Chl chargetransfer (CT) state (Holt et al., 2005; Avenson et al., 2009), direct or indirect quenching by the
PsbS protein (Li et al., 2000; Niyogi et al., 2005), energy transfer from Chl to lutein in LHCII
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(Horton et al., 1991; Ruban et al., 2007) linked to the aggregation of or a conformational
change in LHCII, and last not least a far-red (FR) emitting quenched Chl-Chl CT state formed
by the aggregation of LHCII (Miloslavina et al., 2008). Quenching in the PSII reaction centre
(RC) has also been proposed (Weis and Berry, 1987; Finazzi et al., 2004; Huner et al., 2005;
Ivanov et al., 2008) as an additional type of Zx-independent quenching. Alternatively, it has
been suggested that quenching by lutein can complement the Zx-dependent quenching
(Niyogi et al., 2001; Li et al. 2009). Johnson et al. (2009) have recently given support to the
notion that both Zx-dependent and Zx-independent quenching originate from the same PsbSdependent mechanism which is modulated by Zx (Crouchman et al., 2006).
While the rapidly relaxing phase qE is now well characterized in its dependence on the
various factors, the much slower qT and qI phases are still controversial and each of them
may have contributions from more than one mechanism. The qI component has been
traditionally attributed to photoinhibition of PSII (Somersalo and Krause, 1988), associated
with coordinated degradation and repair of the photosystem (Powles and Björkman, 1982;
Kyle, 1987; Krause, 1988; Aro et al., 1993; Long et al., 1994; Murata et al., 2007). Lately
though, it is more widely accepted that under most conditions the photoinhibition is low and
qI is, like qE, a result of thermal deactivation of excited states. Different hypotheses have
been put forward to account for its seeming irreversibility – persistent transmembrane ΔpH
(Gilmore and Yamamoto, 1992), stable protonation of proteins (Horton et al., 1994),
accumulation of inactive PSII reaction centres (Briantais et al., 1992; Schansker and van
Rensen, 1999), or stable binding of Zx to CP29 (Färber et al., 1997). The connection of the qT
phase with state transitions has been doubted as well, and in fact it is now thought that the
fraction of energy redistributed from PSI to PSII under high light conditions is negligible
(Walters and Horton, 1991; Walters and Horton, 1993) and the qT must have a different
origin or that it has erroneously been ascribed as non-photochemical quenching (Schansker et
al., 2006).
Along with the large amount of contradictory evidence on the nature and location of the NPQ
quenching site(s), the question whether the light-induced reversible NPQ represents one
single mechanism of deexcitation located in a single site brought about by the combined
action of PsbS and Zx (Johnson et al., 2009) or whether it comprises several parallel and
largely independent mechanisms acting on different parts of the PSII antenna, has not been
finally answered. One way to answer this question might be to carefully examine the spectral
properties of NPQ-related fluorescence changes. Quenching in different locations of the PSII
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antenna or with different mechanisms might give rise to a differential quenching in various
parts of the PSII antenna that might affect the PSII fluorescence spectra in different ways.
This appears possible since the various pigment-protein complexes of the photosynthetic
apparatus have slightly different absorption and emission spectra (Holzwarth, 1991;
Holzwarth and Roelofs, 1992). However in the vast majority of modulated chlorophyll
fluorescence instrumentation – including the most widely used PAM fluorometer (Schreiber
et al., 1986) - the signal is integrated over a broad wavelength range, usually covering the
whole range ≥ 710 nm. This integration over the long-wave part of the spectrum has several
undesirable consequences and is associated with the unnecessary loss of available
information. For example, the fluorescence of PSII peaks in the region of 680-685 nm,
whereas beyond 700 nm the PSII fluorescence intensity drops to less than 20% of its peak
intensity. In contrast, the fluorescence of intact PS I complexes is dominant in the region
above 710 nm (Haehnel et al., 1982; Karukstis and Sauer, 1983; Holzwarth et al., 1985;
Holzwarth, 1986; Slavov et al., 2008). Thus the widely used instrumentation measures the
NPQ parameters in a region with reduced PSII contribution and relatively high PS I
contribution to total fluorescence, despite the fact that NPQ is generally considered to be
primarily a PSII phenomenon. Only in a few studies the fluorescence in the red and the FR
region has been separated in order to evaluate the contribution of Photosystem I (PSI) and its
influence on the NPQ parameters (Genty et al., 1990; Peterson et al., 2001). NPQ might also
shift the fluorescence properties of the PSII antenna complexes or give rise to entirely new
fluorescing components (Miloslavina et al., 2008). This would remain undetected if the NPQ
fluorescence changes are not resolved in the spectral domain. It follows from these
considerations that a great deal of insight into the NPQ mechanisms and locations may be
gained if the spectral dimension is added to the NPQ fluorescence characterization. Among
the many advantages of such an approach, one would then be able e.g. to distinguish whether
NPQ simply leads to a uniform decrease of PSII fluorescence across the emission range, or
whether this decrease is non-uniform, localized in specific pigment protein complexes, and/or
whether new fluorescing species are actually being produced in the NPQ process.
The high-light (HL) induced NPQ effects on the leaf fluorescence spectra have often been
studied also at low temperature, where the differentiation between pigment sites is better
(Krause et al., 1983; Demmig and Björkman, 1987; Ruban and Horton, 1994). However the
possibility to resolve the kinetics of NPQ development and relaxation is largely lost when
performing the measurements at low temperatures. The 77 K spectra of leaves and thylakoid
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membranes are characterised by three main peaks, F685, F695, and F730, believed to originate
predominantly from Chl a in CP47 of PSII, a specific Chl in CP43 of PSII, and from PSI,
respectively (Satoh and Butler, 1978; van Dorssen et al., 1987; Andrizhiyevskaya et al., 2005;
Komura et al., 2007). Fluorescence from the major LHCII peaks at 680 nm (Rijgersberg and
Amesz, 1978) and from the PSII reaction centre chlorophylls at 683 nm (Roelofs et al., 1993;
Andrizhiyevskaya et al., 2005). Low temperature studies on the effects of HL irradiation are
confined to the changes in the far-red (FR) to red (R) fluorescence ratio, which are the result
of the quenching of PSII fluorescence or energy redistribution between the photosystems
(state transitions). Ruban and Horton have shown that photochemical quenching in Guzmania
is maximal at 688 nm whereas non-photochemical processes quench preferentially at 683 and
698 nm (Ruban and Horton, 1994).
In this study we undertook a detailed investigation of the NPQ-associated spectral changes in
the fluorescence spectra of Arabidopsis thaliana measured at room temperature (RT) and at
77 K. It follows from the above discussion that deeper insight into the mechanisms of NPQ
processes may be gained by combining the kinetic and the spectral information of the
fluorescence changes occurring in NPQ. For this purpose we developed a multi-wavelength
spectrometer with parallel detection allowing to follow the entire time-dependent fluorescence
spectra of leaves during the induction and relaxation phases of NPQ with high sensitivity.
Specific questions to be addressed in this study are the following: Are there more than one
NPQ process(es) and NPQ sites? Are these processes occurring in a linked fashion or are they
independent? How do they depend on the various cofactors known to affect NPQ, in
particular regarding the roles of PsbS and Zx. Using this novel approach of adding the
spectral information to the NPQ fluorescence changes we discovered specific spectral changes
associated with different NPQ components. By comparing the effects measured on various
NPQ mutants of Arabidopsis it is possible to assign these NPQ components to specific
quenching processes. The results provide evidence that the total NPQ is a combination of
several parallel and largely independent processes, likely occurring at different locations in
the photosynthetic apparatus.
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Results
77 K fluorescence spectra
Fluorescence emission spectra measured at 77K from dark-adapted wild-type (w.t.)
Arabidopsis thaliana leaves and from leaves irradiated for 30 min with 600 μmol photons m-2
s-1 red light (620 nm) are shown in Fig. 1. To facilitate detailed comparison of their shape, all
spectra were normalized. Since NPQ is supposed to alter the properties of PSII rather than
PSI, we wanted to use PSI fluorescence as a reference for normalization to better visualize the
changes in PSII. According to the notion that at shorter wavelengths the emission originates
from PSII and at longer wavelengths from PSI, we normalized the spectra at 760 nm – near
the FR tail of the spectra, where the contribution from PSII is minimal and from PSI maximal.
More precisely, the spectra were normalized to the averaged intensity between 755 and 765
nm in order to further reduce the already low noise in the data (the signal to noise ratio at 760
nm is about 300:1) and thus avoid inaccurate normalization. The 77 K fluorescence emission
spectra of w.t. leaves show peaks at 683 nm, 691 nm, and 730 nm. The relative ratio between
them has been reported to be strongly dependent on the leaf anatomy, particularly the
thickness and the chlorophyll content, which determine its optical properties, and also on the
angle of excitation and detection (Weis, 1985). However, for leaves at similar physiological
state at a fixed orientation we found the spectral differences to be insignificant. Following HL
treatment of the w.t. leaves the spectra revealed characteristic and reproducible differences – a
decrease of fluorescence in the PSII wavelength range (< 700 nm) and an increase in the FR
range (> 710 nm). The light-minus-dark difference spectra (Fig. 1B) show two negative peaks
– at 683 and 691 nm, matching the peaks of the emission spectra – and a positive band
appearing around 727 nm, i.e. somewhat blue-shifted to the major PSI emission peak (730
nm). In some cases, a negative difference band at 715 nm was also observed (only a shoulder
in Fig. 1B).
Whereas the negative difference bands are undoubtedly associated with quenching of the PSII
antenna fluorescence – F685 and F695, the origin of the positive 730 nm band is less clear. In
order to check whether its appearance corresponds to the kinetics of NPQ, leaves were rapidly
frozen in the light at different times of HL irradiation and 77 K fluorescence spectra were
recorded. Furthermore, spectra were taken after keeping pre-illuminated leaves for different
times in darkness. The resulting time-dependent changes of the ratio F730/F760 are shown in
Fig. 2. The ratio rose within the first several minutes of irradiation towards a maximum value
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and declined back after a few minutes of darkness. This is a typical behaviour for energydependent quenching, qE. However because these measurements have to be carried out on
different leaves, the level of accuracy is not high enough to quantitatively compare the
kinetics of the 77 K fluorescence changes at different wavelengths.
Room-temperature fluorescence spectra
RT measurements allow for a far better quantification of the relative changes in the
fluorescence spectra before and after illumination when they are registered from the same leaf
without changing its orientation in the optical path. To exploit this possibility we constructed
a special dual-LED instrument, based on an Ocean Optics USB2000 CCD spectrometer (See
Materials and Methods). Since the spectrometer is able to register a full emission spectrum
with high signal-to-noise ratio in less than 10 ms, the instrument can follow fast light-induced
spectral changes with remarkable sensitivity. The dual-LED mode enables probing of the
fluorescence spectra and kinetic development from closed PSII RCs either under variable
actinic light or in the absence of it. This allows us, on the one hand, to correlate the spectral
changes with kinetic components of NPQ and, on the other hand, to also discriminate the
processes using different excitation wavelengths if necessary.
The NPQ kinetics, measured with a single actinic/excitation source, is presented in Fig. 3 as
the time and wavelength dependence of the NPQ parameter, calculated as NPQ = F(t0) / F(t) –
1 (Briantais et al., 1979; Bilger and Björkman, 1990). Along the time axis the figure shows
the known kinetics of NPQ formation in the wild type Arabidopsis and the mutants npq4 and
npq1. The PsbS-deficient npq4 plants generate much less NPQ as compared to the wild type
and the kinetics lacks the fast NPQ phase, whereas in npq1, which can not form Zx, most of
the NPQ is formed in the fast initial phase, attributed to the action of PsbS. Along the
wavelength axis the figure reveals the significant non-homogeneity of the NPQ parameter
with respect to the detection wavelength. A distinctive ‘valley’ in the surface plot between
700 and 730 nm is observed for w.t. leaves but is lacking in the npq4 mutant and is smaller in
npq1.
The normalized (to the average intensity at 745-755 nm) emission spectra measured in dualLED mode from a w.t. leaf in the dark-adapted state, after 30 min of irradiation with 600
μmol photons
m-2 s-1 red light, and after 5 min re-darkening are shown in Fig. 4. The RT
spectra are characterised by a major PSII band with a single maximum at 685 nm and a lower10
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intensity broad band in the 710-740 nm range, which corresponds to the vibrational tails of
the two photosystems and to the ‘red’ chlorophylls of PSI. The most pronounced HL-induced
effect on the spectral shape is, as expected, the decrease of the main PSII band. The lightminus-dark difference spectra have a negative peak at 682 nm. In the FR region, the spectra of
light-adapted leaves (dashed line in Fig. 3A) show a well-resolved increase above the darkadapted spectra. In the light-minus-dark difference spectra this is observed as a positive band
with a maximum at 720 nm. Five minutes after switching off the actinic light, the main
fluorescence band recovered part of its intensity (dotted line in Fig. 4A). Interestingly, at the
same time the observed increase around 720 nm disappeared completely. This is a first
indication that the two light-induced effects, i.e. the decrease of F682 and the increase of F720
are not always matched. The effect is clearly demonstrated by the difference spectra in Fig.
4B. The light-minus-re-dark difference spectrum (dashed) shows that F720 is a fast-relaxing
component and the re-dark-minus-dark spectrum (dotted) shows the complete absence of the
F720 difference band after five minutes of dark, the only difference remaining in the F682 band.
We compared the described light-induced spectral changes in several Arabidopsis NPQ
mutants – npq4, which lacks the PsbS protein (Li et al., 2000), L17, which overexpresses
PsbS (Li et al., 2002), npq1, which is unable to convert violaxanthin to zeaxanthin, npq2,
which lacks violaxanthin and neoxanthin but accumulates Zx (Niyogi et al., 1998), and stn7 –
a mutant lacking the Stn7 protein kinase, thus being unable to phosphorylate LHCII and to
undergo state 1 – state 2 transitions (Bellafiore et al., 2005). Figure 5 shows the light-minusdark difference spectra for the different mutants, irradiated for 30 min with 600 µmol photons
m-2 s-1 red light. In all six genotypes the magnitude of the relative F682 decrease seemed
comparable, however there were marked dissimilarities in the F720 difference band. Most
notably, this band was absent in the npq4 mutant and conversely, was significantly enhanced
in the PsbS overexpressor L17, compared to the w.t.
The kinetics of induction and relaxation of NPQ, recorded in dual-LED mode with saturating
pulses every 60 s, are shown in Fig. 6 for the different mutants. Note that the scaling on the
vertical axis is different because the NPQ values largely differed between specific mutants,
e.g. L17 typically shows stronger NPQ than w.t., whereas in the npq1 and npq4 mutants NPQ
is weak. In all mutants during the slow phase of NPQ induction the increase of the NPQ
parameter was significantly smaller in the FR region than at 682 nm. This behaviour is wellknown and attributed to PSI fluorescence in the FR region, which is not quenched (Genty et
al., 1990; Pfündel, 1998). Furthermore, in all mutants but npq4, during the first minutes of
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irradiation the two curves representing the NPQ parameter at 720 and 750 diverge, forming a
gap between each other, which however remained constant with further increase of time under
illumination but quickly disappeared after turning off the actinic light. This is in line with the
already shown rapidly reversible light-induced increase of F720. In npq4 there are no
detectable differences in the NPQ parameter at 720 and 750 at any time and in npq1 the
difference is very small.
As a quantitative measure of the spectral changes described above, we use the fluorescence
ratios F682/F750 and F720/F750. Since the shape of the detected spectrum can slightly vary from
leaf to leaf due to anatomical and hence light scattering and re-absorption differences, we
compare only the HL-induced changes in these ratios, i.e. the light-minus-dark differences, –
ΔF682
= –[(F682/F750)light–(F682/F750)dark]·100 and
ΔF720
= [(F720/F750)light–(F720/F750)dark]·100.
The time courses of these changes are shown in Fig. 7 for the different Arabidopsis
genotypes. The most important conclusion from these plots is that
ΔF682
and
ΔF720
have
substantially different time courses. The ratio increases rapidly in the first few minutes of
irradiation and then continues to grow at a slower rate. Clearly F682 is quenched even in the
absence of PsbS and without operation of the violaxanthin cycle. After switching the actinic
light off, ΔF682 quickly drops to a transient minimum but does not relax completely and even
continues to increase in the absence of background actinic illumination. This transient drop is
negligible in npq4 and npq1 and stronger in the L17 mutant.
Unlike ΔF682, the time course of the ΔF720 spectral change lacks the slow phase of increase in
ΔF720 relaxes almost completely within several minutes
after switching the continuous light off. The lifetime of the relaxation of ΔF720 in w.t. was
about 2 min. The magnitude of ΔF720 was very sensitive to the various NPQ-related
mutations. Lack of the PsbS protein completely inhibited the light-induced ΔF720 changes. The
HL. Furthermore, the light-induced
effect was reversed in the PsbS overexpressor, where the ratio increased to a value higher than
in the w.t. The Zx-deficient mutant npq1 and the Zx-accumulator npq2 both exhibited the HLinduced ΔF720 but in npq1 it was significantly reduced and appeared to have a more complex
kinetics. In npq2 the fluorescence spectral change was enhanced but in the the dark it
recovered more slowly than in the w.t., which is characteristic for the kinetics of NPQ
recovery of this mutant (Niyogi et al., 1998). The absence of the Stn7 kinase did not result in
substantial changes in any of the observed parameters. More importantly, the inability to
undergo state transitions was not accompanied by absent or smaller ΔF720 change.
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The values of the parameters
ΔF682
and
ΔF720
obtained after 30 min of irradiation are
summarized in Table 1 for all measured plant genotypes, together with the technical SternVolmer type parameters NPQ, qE, and qI (Krause and Jahns, 2003), detected at 682 nm. The
most striking result is the very strong correlation between ΔF720 and the rapidly forming and
relaxing quenching component qE, whose contribution is defined as the part of total
quenching which relaxes after 10 min of darkness. The studied mutants have a largely
different capacity for qE, ranging from 0 (in npq4) to about double the w.t. value in L17. In
all mutants the light-induced ΔF720 remarkably followed the qE. In contrast, all mutants were
able to produce qI, i.e. the part of quenching which did not relax in 5 min, and the variations
in this parameter and in
ΔF682 were rather small. It is important to stress that while qE and
(especially) the ΔF720 fluorescence change can be attributed to a particular (PsbS-dependent)
photochemical/physical mechanism of quenching, the qI parameter cannot be associated with
a single mechanism and is purely a technical term.
The behaviour of the F682/F750 ratio was strongly dependent on the measurement protocol, and
particularly the frequency of the saturating pulses. When saturating pulses were applied at 3
min interval (Figure 8A), the changes in the F682/F750 were significantly smaller as compared
to the changes measured with 1 min pulse interval. The slow phase and particularly the slow
increase after switching the actinic light off were basically abolished. The NPQ parameter and
the magnitude of ΔF682 and ΔF720 are shown in Figure 8B for 1 min, 3 min and 6 min pulse
intervals. The pulse repetition rate has impact on the NPQ and on the ΔF682 change but the
ΔF720 change is independent from it.
Discussion
Spectral changes detected at 77 K
The normalized fluorescence emission spectra of HL-treated leaves showed the wellestablished decrease of fluorescence in the PSII region compared to the PSI region, shown by
many authors (Kyle et al., 1983; Krause et al., 1983; McTavish, 1988). In addition to this
change, a characteristic light-induced change, not previously reported, was found in the FR
region – the increase of F730 relative to F760. There are two possible explanations to interpret
this effect – 1) the negative and the positive relative difference bands have the same common
origin, i.e. quenching of PSII fluorescence; 2) there are different mechanisms or sites which
are responsible for the different positive/negative light-induced spectral changes. The latter
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hypothesis brings two further possibilities: 1) the relative increase of F730 is a consequence of
the quenching of fluorescence components with maxima outside the difference band, and 2)
F730 is a genuine new fluorescence emission component resulting from the HL-induced
formation of a new FR emitting excited-state species. The 77 K fluorescence data alone did
not provide sufficient evidence to distinguish between these possibilities. However, the results
cannot be explained within the usual concept that at low temperature the fluorescence in the
FR region originates from PSI alone. The observed increase of F730 relative to F760, if regarded
as a consequence of PSII quenching, would only be possible if PSII fluorescence had a
broader vibrational tail extending more to the FR than PSI, i.e. the ratio F760/F730 of the pure
PSII spectrum were higher than that of the pure PSI spectrum. A state 1 – state 2 transition
also cannot explain the observed effect. Both the quenching of PSII and a potential state
transition could explain the negative F683 and F691 difference band but not the positive F730
band demonstrated in the normalized spectra.
Different mechanisms are responsible for the HL-induced spectral changes
The hypothesis that the positive difference band in the FR region discovered under NPQ
conditions has a different mechanism of origin than the quenching of the main PSII
fluorescence band was tested by a series of RT experiments in which the time course of lightinduced formation and dark relaxation is followed and compared for each fluorescence
difference band. The reasoning is that in case these difference bands have a common origin
and location in the PSII antenna, they should have the same kinetics and the same spectral
characteristics, and, conversely, differences in the kinetics of formation or relaxation would
be an indication that the underlying mechanisms or sites of action are different for these
bands. Although at RT the spectral changes in the FR region are smaller than at 77 K, the
positive difference band at 720 nm can be resolved clearly and with high precision in our
experiments to reveal that it shows drastically different kinetics of induction and recovery, as
compared to the quenching of PSII detected at 682 nm.
The red (682 nm) and FR (720 nm) spectral changes not only differ in their time course
during irradiation and subsequent re-darkening but are also affected in a distinctly different
manner by mutations. For example the presence or absence of PsbS has a strong impact on the
FR change only but no statistically significant effect on the 682 nm changes. The 682 nm
changes depend significantly on the applied frequency of saturating light pulses, yet the 720
nm changes are insensitive to it. Therefore we have to conclude that there are at least two
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independent processes responsible for the fluorescence time changes. One only leads to a
relative decrease of the main PSII fluorescence band at 682 nm. The other quenches the PSII
fluorescence as well but concomitantly develops a specific new fluorescence in the 720-730
nm range.
The FR fluorescence change is related to qE
We attempted to investigate the particular biological process underlying the newly found
spectroscopic feature of NPQ, ΔF720. Although the increase of FR (>700 nm) relative to red
fluorescence (680-690 nm) has been commonly interpreted as a result of a state transition
(Butler and Kitajima, 1975; Mawson and Cummins, 1986; McTavish, 1988; Walters and
Horton, 1991), this cannot explain the
ΔF720
change for two reasons: first, the relative
increase of F720 to F750 tail of PSI fluorescence has no obvious explanation and, second, the
amount of energy redistribution from PSII to PSI under HL conditions used in our
experiments is generally believed to be very low, if present at all (Walters and Horton, 1991).
Without relying on any such assumptions however we can completely exclude a state
transition hypothesis since in the stn7 mutant of Arabidopsis which is unable to undergo state
transitions (Bellafiore et al., 2005), the ΔF720 band is unaffected. Therefore the ΔF720 spectral
change is definitely not associated with a state 1 – state 2 transition.
The time course of the HL-induced ΔF720 spectral change, which reaches a stationary state in
a few minutes of irradiation and reverses completely within a few minutes in darkness, clearly
associates it with the energy-dependent type of quenching, qE, and distinguishes it from the
so-called ‘sustained’ quenching qI. The magnitude of the relative difference ΔF720 is strongly
correlated with the value of the qE parameter in all examined mutants. The ΔF720 band is thus
a spectral marker for the mechanism underlying qE, and as such it opens new possibilities to
probe qE specifically and independently from other quenching components, as e.g.
photoinhibition or others.
The most striking feature of the HL-induced FR fluorescence change is its complete absence
in the PsbS-deficient mutant npq4 and its enhancement in the PsbS overexpressor. This
demonstrates the direct role of the PsbS protein and further confirms the association of PsbS
with qE but not with qI. Since the ΔF682 change was not inhibited by the lack of PsbS (on the
contrary, it seems to be slightly enhanced), it becomes clear that Arabidopsis plants have at
least two different mechanisms for the rapidly-inducible NPQ – a PsbS-dependent one and a
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
PsbS-independent one. Furthermore it follows that the function of PsbS can be exerted
independently from the xanthophyll cycle since the rapid light-induced ΔF720 is detected also
in the npq1 and npq2 mutants. However when Zx cannot be formed the amplitude of the
spectral change is significantly smaller, this is consistent with the lower ability of the npq1
mutant to generate both qE and ΔF720. It must be also noted that double mutants containing
the npq4 mutation – like e.g. the npq1npq4 and the npq2npq4 mutants – also did not have any
ΔF720 (data not shown), confirming the compulsory role
relation of ΔF720 with qE.
detectable
of PsbS for qE and the
Could the observed FR fluorescence change have a different and more trivial origin? The
measured fluorescence time changes could be in principle influenced by changes in leaf
absorbance and reflectance. But such changes would affect the whole fluorescence intensity
and would not primarily give rise to distinct spectral changes of the fluorescence. These
effects would thus be compensated by the normalization procedure. Changes in the effective
absorption might also lead to different re-absorption and thus affect the spectral shape of the
fluorescence emission. Clearly leaves have a strong re-absorption of fluorescence in the range
below 695 nm. However, there is little or no absorption in the FR region and thus such
artefacts can be excluded. Another source of error could be light-scattering changes, e.g.
associated with ΔpH-induced thylakoid swelling. However, light scattering primarily affects
the measurement of absorbance but has a much smaller - and almost independent from the
optical arrangement - effect on fluorescence. Furthermore, such light-scattering changes could
hardly give rise to a distinct spectral change of the fluorescence in a relatively narrow range.
More importantly though, it is extremely unlikely that effects such as ΔpH-induced thylakoid
swelling and others, that in general might give rise to such scattering changes, would follow
the well-known qE dependence on PsbS content, as does the observed
ΔF720
fluorescence
change. Therefore, while we cannot totally exclude some disturbances due to possible optical
artefacts, it is extremely unlikely that they could in fact explain the majority of the observed
ΔF720
spectral fluorescence change. Rather the dependence of this change on the different
PsbS mutants strongly suggests that it is indeed qE-related.
Photophysical origin of the FR spectral change
The actual photophysical origin of the HL-induced changes in the FR region of the
fluorescence spectrum cannot be determined from the present steady-state fluorescence
measurements alone. However, recent ultrafast fluorescence data on isolated LHCII
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aggregates and intact leaves of Arabidopsis acquired in our lab (Miloslavina et al., 2008;
Holzwarth et al., 2009) indicate the likely origin of this NPQ-related emission component.
Time-resolved fluorescence spectra of HL-treated leaves at RT revealed a new PsbSdependent antenna fluorescence component, not present in the dark-adapted leaves
(Holzwarth et al., 2009). Remarkably, the emission spectrum of this fluorescence decay
component showed a strong enhancement in the FR region, in the range 700-730 nm as
compared to normal PS II fluorescence. This component was absent in PsbS-deficient plants
and its amplitude was enhanced in the PsbS-overexpressor L17. It can be imagined that both
the HL-induced FR decay component and the ΔF720 change detected here in the steady-state
spectra presented originate from the same mechanism.
Interestingly, when LHCII forms aggregates, or higher-order oligomers, in vitro, it exhibits
enhanced fluorescence in the FR region, particularly strong at low temperatures (Ruban and
Horton, 1992; Mullineaux et al., 1993; Vasil'ev et al., 1997) but also at RT (Miloslavina et
al., 2008). Because oligomerization of LHCII leads to strong fluorescence quenching (Ide et
al., 1987; Ruban and Horton, 1992; Mullineaux et al., 1993), it has actually been proposed as
a mechanism for NPQ in vivo (Horton et al., 1991; Horton et al., 2005; Ruban et al., 2007).
An essential finding in this direction is that the spectrum and lifetime of the detached antenna
component measured in intact quenched leaves under NPQ conditions closely match the FR
fluorescence of LHCII aggregates in vitro (Miloslavina et al., 2008). Furthermore, when these
LHCII aggregates are cooled to 77K a new FR fluorescence located in the range 700-730 nm
appears (Ruban and Horton, 1992; Mullineaux et al., 1993; Miloslavina et al., 2008). Thus the
FR fluorescence at low temperature in LHCII aggregates also parallels the
ΔF720
band
observed here. We thus suggest that the qE-associated PsbS-dependent FR spectral change
has the same origin as the FR emission of quenched LHCII aggregates.
Flash-induced quenching component
Whereas the ΔF720 band can be ascribed to a specific mechanism of quenching, i.e. the PsbSdependent qE, this does not hold true for the
ΔF682 band, which reflects any processes that
quench PSII fluorescence and will depend both on the qE and qI components of NPQ. The
F682/F750 ratio can be then expected to decrease gradually with the slower formation of Zx and
not to reverse immediately after turning off the actinic light because of the remaining qI.
However this cannot explain two important observed effects: 1) the slow changes in the
F682/F750 ratio in the npq1 and npq2 mutants in which the Zx content does not change; and 2)
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the rise of the ratio observed after switching off the actinic light. These changes are obviously
induced by the short saturating flashes only and accumulate with each flash provided that the
time interval between flashes is short. The total irradiation dose from the flashes in our
measurements is negligibly small compared to the background actinic light – only 0.4% of the
total number of incident photons at a flash interval of 60 s. This rules out the possibility that
the fluorescence changes are simply an additive effect of the measuring and actinic light.
Therefore the flash-induced changes reflect a specific process and appear to be triggered by
the high turnover rate of PSII during the saturating flash. A key observation is that the flashinduced
ΔF682
depends on the flash repetition rate. This suggests that these flash-induced
changes are reversible on a time scale of a few minutes. At shorter intervals between flashes
the time is not sufficient for these changes to relax and the effects are cumulative. This
behaviour shows that there is a reversible component of NPQ, independent from both PsbS
and Zx, which can be induced by a short pulse of strong light and relaxes on a time scale of
several minutes. This then represents an additional independent mechanism of NPQ. It might
be responsible for the quenching observed in mutants having neither PsbS nor xanthophyll
cycle activity such as the npq1npq4 and npq2npq4 double mutants. Based on our data we
cannot tell at present what the actual underlying quenching mechanism might be or whether it
takes place in the antenna or the RC of PSII. We hypothesize however that this quenching
might be located in the RC of PSII, thus relating it to RC-quenching that has been proposed in
the literature (Finazzi et al., 2004; Ivanov et al., 2008)
It must be stressed that the flash-induced quenching component severely distorts the measured
NPQ relaxation kinetics when measuring flashes are spaced at short intervals (i.e. shorter than
5 min.) and, more importantly, it contributes significantly to qI, interpreted usually as
irreversible or slowly-reversible quenching, whereas in fact it is also a quenching component
relaxing on a relatively short time scale of about 5-10 min. Thus, in our measurements by far
the largest part of the slow induction and relaxation phases, generally called the qI phase in
the literature, are genuine reversible NPQ quenching components and are not related to
damage or photoinhibition. In fact, our experiments strongly suggest that true photoinhibition
is minor under the conditions applied here even for HL-irradiation up to 1 h on intact leaves.
Conclusion
We have been able with the help of a newly-designed multi-wavelength fluorometer for
registering spectrally-resolved fluorescence and NPQ induction kinetics to dissect and
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distinguish three contributions to NPQ. We also discovered spectral features specific to the
different components of NPQ. We demonstrate three separate and independent mechanisms or
sites of action of NPQ. The first one is the rapidly inducible and rapidly relaxing PsbSdependent qE mechanism, which gives rise to a relative FR fluorescence increase around 720
nm. This spectral signature of qE suggests that a new emitting species is produced together
with the qE quenching induction. The second slower NPQ quenching process occurs
essentially independently from PsbS but can be correlated to the formation of Zx. It does not
give rise to new FR fluorescing species but only decreases PSII fluorescence uniformly across
the spectrum. Finally, a third quenching process, independent from both PsbS and Zx, was
detected, which appears to be reversible in the dark on a ca. 5 min time scale but is
completely unrelated to the mechanism of qE and is triggered by a high excitation or turnover
rate in PSII. All of these three NPQ mechanisms are reversible over the time scales and
excitation intensities used in our experiments. Any non-relaxing quenching contributions
appear to be minor.
Materials and Methods
Plant material
Arabidopsis thaliana (ecotype Columbia 0) wild-type and mutant plants were grown in soil at
a light intensity of 150 µmol photons m-2 s-1 and a constant temperature of 20°C under longday conditions (14 h light / 10 h dark). Leaves from 5-6 week-old plants were used for all
experiments. The following mutants have been used: npq1, defective in the Vx de-epoxidase,
(Niyogi et al., 1998); npq2 (also termed aba1-6), defective in the Zx epoxidase (Niyogi et al.,
1998); npq4, PsbS deficient (Li et al., 2000); L17, PsbS overexpressing (Li et al., 2002); and
stn7, defective in the LHCII kinase (Bonardi et al., 2005).
77K fluorescence measurements
In all experiments plants were dark-adapted for at least one-two hours. Prior to the
measurement leaves were detached, moisted and placed between two glass plates where they
were irradiated at room temperature for certain time using a high-power red (620 nm) LED
(Philips Lumileds Lighting Co., USA) at intensity of 600-1000 μmol m-2 s-1. The leaves were
then immediately frozen in liquid nitrogen and placed in an optical cryostat (Oxford
Instruments, UK). Fluorescence spectra were recorded in a home-made system based on an
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USB2000 CCD spectrometer (Ocean Optics Inc., USA) using a blue (465 nm) LED as an
excitation source, filtered with a 550 nm long-pass glass filter.
Dual-LED fluorescence spectrometer for RT measurements
For measurements of the NPQ generation and relaxation at room temperature, a special Dual
LED fluorescence setup was devised, schematically illustrated in Fig. 9A. The optical setup is
assembled in a dark casing. Two high-power LEDs (Luxeon Star-O, Philips Lumileds
Lighting Co., USA), one red (620 nm) and one blue (460 nm), are focused with lenses onto
the leaf holder. Short-pass 630 nm filters (Thorlabs Inc., USA) are placed in front of the LEDs
to remove long-wavelength emission. The fluorescence is collected via another lens at an
angle of 45°C with the leaf surface. Scattered light is filtered through a custom-made 660 nm
long-pass interference filter (LayrTech, USA). The fluorescence is focused onto a fibre-optics
guide, connected to a CCD spectrometer (USB2000, Ocean Optics Inc., USA). The dataacquisition protocol is programmed in Microsoft Visual Studio using an interface library from
Ocean Optics. Data analysis is done in MATLAB. In our experiments the red LED was used
as continuous actinic light for the induction of NPQ and the blue LED provided periodic short
pulses used at the same time to close the reaction centres and as excitation (measuring)
source. For this purpose, a fast-response DC power supply with TTL input was built, ensuring
pulse rise/decay time of less than 50 µs. The triggering signal for the pulses is supplied by the
spectrometer and programmatically synchronized with the fluorescence detection.
Fluorescence spectra are recorded with each given saturating pulse as depicted by the diagram
in Fig. 9B. Before the pulse, a background spectrum is recorded. The LED is triggered and
after a pre-defined delay, the actual pulse-excited fluorescence spectrum is recorded, the LED
is then switched off and the background signal is subtracted by software. This way the output
signal represents only the fluorescence excited by the saturating pulses but not by the actinic
light. After switching off the actinic light, the relaxation of NPQ can be monitored provided a
sufficient time separation between the measuring pulses to prevent quenching induction.
The actual measurement conditions used in the reported dual-light experiments were as
following: Blue measuring/saturating pulses of 200 ms duration and approximate intensity
1500 μmol photons m-2 s-1 were given at every 60 s. Fluorescence detection was started 30 ms
after the pulse onset. After the first pulse (to acquire Fm) the red actinic light with intensity of
600 μmol photons m-2 s-1 was switched on.
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The setup can also be used in a single-LED mode where the same LED is used as actinic and
excitation source. This allows for following faster induction changes registering a full
spectrum every few milliseconds. Thus, in effect, the described setup provides most of the
functionality of both conventional pulse-amplitude-modulated (PAM) and direct detection
fluorometers, however with full spectral resolution. The time-dependent spectra shown in Fig.
3 were acquired in single-LED mode using red light (1000 μmol photons m-2 s-1) and a 2 s
recording interval.
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Table 1. NPQ parameters and fluorescence ratios in Arabidopsis leaves.
Genotype
NPQ6821
qE682
qI682
−ΔF682
ΔF720
w.t.
2.9 ± 0.1
2.1 ± 0.4
0.9 ± 0.2
30 ± 2
4.4 ± 0.2
stn7
3.4 ± 0.1
2.8 ± 0.1
0.6 ± 0.0
27 ± 3
5.4 ± 0.3
npq4
1.4 ± 0.1
0.0 ± 0.0
1.3 ± 0.1
29 ± 4
0.3 ± 0.2
L17
6.8 ± 0.5
5.6 ± 0.5
1.3 ± 0.2
35 ± 3
10.4 ± 0.5
npq1
1.5 ± 0.1
0.5 ± 0.1
1.1 ± 0.1
27 ± 2
1.9 ± 0.3
npq2
2.3 ± 0.1
1.6 ± 0.2
0.8 ± 0.2
31 ± 5
6.7 ± 0.4
1
NPQ = Fdark / Flight – 1, qE = Fdark / Flight – Fdark/Fre-dark, qI = Fdark/Fre-dark – 1,
[(F682/F750)light – (F682/F750)dark]·100,
ΔF682
=
ΔF720 = [(F720/F750)light – (F720/F750)dark]·100, where Fdark,
Flight and Fre-dark correspond to the fluorescence of dark-adapted leaves, after 30 min HL
irradiation, and after 5 min subsequent re-darkening, respectively. Average values ± SEM (n
= 6-9).
29
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Figure Legends
Figure 1. A. 77 K fluorescence emission spectra, normalized at 760 nm, of dark-adapted
Arabidopsis w.t. leaves (solid line) and leaves pre-illuminated for 30 min with red light (PFD
600 μmol m-2 s-1). The curves represent average from 4-6 measurements on different leaves.
The standard error is shown for selected wavelengths. B. Difference spectrum (light minus
dark).
Figure 2. Dependence of the fluorescence ratio F730/F760 on the duration of irradiation with
600 μmol m-2 s-1 red light (A) or on the time during re-darkening (B) of w.t. Arabidopsis
leaves preilluminated for 15 min. The parameter is calculated from fluorescence emission
spectra registered at 77 K after rapid freezing of the leaves. Each value represents an average
of 2-6 leaves, error bars represent SEM.
Figure 3. Three-dimensional plot of the NPQ parameter (NPQ = F(t0)/F(t)–1) of leaves of
Arabidopsis w.t. (A), npq4 (B), and npq1 (C) as a function of emission wavelength and
irradiation time. Actinic/excitation light – 620 nm, PFD 1000 μmol m-2 s-1. Fluorescence
spectra are recorded every 2 seconds using the actinic light as excitation source.
Figure 4. A. Room-temperature fluorescence emission spectra, normalized at 750 nm, of w.t.
Arabidopsis leaves in dark-adapted state, after 30 min illumination (actinic light 620 nm, PFD
600 μmol m-2 s-1), and after 5 min re-darkening. Additional pulses of blue light (0.2 s, PFD
1500 μmol m-2 s-1) are applied every 60 s to detect the fluorescence spectra from closed PSII
reaction centres. B. Relative difference spectra (in % relative to the emission at 750 nm):
light-minus-dark, light-minus-re-dark, and re-dark-minus-dark. The plots show results of a
single leaf measurement. Error bars on panel B represent the SEM at 682 and 720 nm for 9
measured leaves.
Figure 5. Comparison of light-minus-dark fluorescence difference spectra (30 min HL),
obtained as in Fig. 4, for Arabidopsis w.t., npq4 and L17 (A) and stn7, npq1, and npq2
mutants (B). Error bars represent the largest standard errors at 682 and 720 nm corresponding
to 6-9 leaves measured individually for each mutant.
Figure 6. Time-courses of the light-induced formation and subsequent dark relaxation of the
NPQ parameter (NPQ = F(t0)/F(t)–1) calculated at three wavelengths in Arabidopsis w.t. (A)
and npq4 mutant (B) irradiated at 600 μmol m-2 s-1 PFD red light for 30 min (white bars). The
30
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fluorescence is recorded every 60 s by applying a 0.2 s pulse (460 nm, PFD 1500 μmol m-2 s1
).
Figure 7. Time-dependent changes in the relative fluorescence differences −ΔF682 (white
circles) and
ΔF720 (black circles) during illumination (620 nm,
PFD 600 μmol m-2 s-1) and
subsequent re-darkening of leaves of Arabidopsis w.t. and the mutants L17 (PsbSoverexpressor), npq4 (PsbS-less), npq1 (Zx-deficient), npq2 (Zx-accumulator), and stn7
(Stn7-less). Fluorescence is excited by saturating pulses applied every 60 s. The parameters
are calculated as the light-minus-dark difference in the ratios F682/F750 and F720/F750
respectively (ΔF720 is multiplied by 5 for clarity). The curves represent the results from a
single leaf measurement. Standard errors corresponding to 6-9 measured leaves are shown as
bars.
Figure 8. A. Time-dependent changes in the relative fluorescence difference −ΔF682 during
illumination and re-darkening of leaves of w.t., L17 and npq4. Conditions are as in Fig. 7,
except that saturating pulses are applied at 3 min interval. B. NPQ parameter at 682 nm ,
−ΔF682, and
ΔF720
obtained after 30 min of HL irradiation of w.t. leaves with additional
saturating pulses every 1, 3, or 6 min. Error bars represent SEM (n = 3-9).
Figure. 9. A. Schematic representation of the dual-LED fluorescence setup for registering
fluorescence emission spectra of leaves during formation and relaxation of NPQ. Solid black
lines represent electrical connections, dotted lines - the optical path. 1. Regulated DC power
supply; 2. Excitation LED; 3. Actinic LED; 4. Short-pass filter (630 nm); 5. Lens; 6. Leaf
holder; 7. Long-pass filter (660 nm); 8. Fibre optics; 9. Casing; 10. USB2000 spectrometer;
11. Triggering signal; 12. Computer interface. B. Timing diagram of a typical measurement
cycle showing the recording of the background fluorescence, turning on the saturating pulse
and recording of the fluorescence spectrum.
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