Planta (2020) 252:19
https://doi.org/10.1007/s00425-020-03423-0
ORIGINAL ARTICLE
Effects of low temperature on photoinhibition and singlet oxygen
production in four natural accessions of Arabidopsis
Heta Mattila1 · Kumud B. Mishra2 · Iiris Kuusisto1 · Anamika Mishra2 · Kateřina Novotná2 · David Šebela2 ·
Esa Tyystjärvi1
Received: 20 December 2019 / Accepted: 8 July 2020 / Published online: 15 July 2020
© The Author(s) 2020
Abstract
Main conclusions Low temperature decreases PSII damage in vivo, confirming earlier in vitro results. Susceptibility to photoinhibition differs among Arabidopsis accessions and moderately decreases after 2-week cold-treatment.
Flavonols may alleviate photoinhibition.
Abstract The rate of light-induced inactivation of photosystem II (PSII) at 22 and 4 °C was measured from natural accessions
of Arabidopsis thaliana (Rschew, Tenela, Columbia-0, Coimbra) grown under optimal conditions (21 °C), and at 4 °C from
plants shifted to 4 °C for 2 weeks. Measurements were done in the absence and presence of lincomycin (to block repair). PSII
activity was assayed with the chlorophyll a fluorescence parameter Fv/Fm and with light-saturated rate of oxygen evolution
using a quinone acceptor. When grown at 21 °C, Rschew was the most tolerant to photoinhibition and Coimbra the least.
Damage to PSII, judged from fitting the decrease in oxygen evolution or Fv/Fm to a first-order equation, proceeded more
slowly or equally at 4 than at 22 °C. The 2-week cold-treatment decreased photoinhibition at 4 °C consistently in Columbia-0
and Coimbra, whereas in Rschew and Tenela the results depended on the method used to assay photoinhibition. The rate of
singlet oxygen production by isolated thylakoid membranes, measured with histidine, stayed the same or slightly decreased
with decreasing temperature. On the other hand, measurements of singlet oxygen from leaves with Singlet Oxygen Sensor
Green suggest that in vivo more singlet oxygen is produced at 4 °C. Under high light, the PSII electron acceptor Q A was
more reduced at 4 than at 22 °C. Singlet oxygen production, in vitro or in vivo, did not decrease due to the cold-treatment.
Epidermal flavonols increased during the cold-treatment and, in Columbia-0 and Coimbra, the amount correlated with
photoinhibition tolerance.
Keywords Acclimation · Charge recombination · Chilling stress · Cold-hardening · Photodamage · Photoinactivation ·
Reactive oxygen species · SOSG
Communicated by Dorothea Bartels.
Heta Mattila and Kumud B. Mishra contributed equally to this
work.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00425-020-03423-0) contains
supplementary material, which is available to authorized users.
* Esa Tyystjärvi
esatyy@utu.fi
1
Department of Biochemistry, Molecular Plant Biology,
University of Turku, 20014 Turku, Finland
2
Global Change Research Institute of the Czech Academy
of Sciences, Bělidla 986, 4a, Brno 603 00, Czech Republic
Abbreviations
CD
Developed at cold (grown 15 weeks at 4 °C)
CT
Cold-treated (grown 6 weeks at 21 °C and
2 weeks at 4 °C)
DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea
Fv/Fm
Ratio of variable to maximum fluorescence
HL
High light (grown 6–10 weeks under PPFD of
1000 µmol m−2 s−1 at 21 °C)
kPI
Rate constant of photoinhibition of PSII
NT
Non-treated (grown at 21 °C)
NPQ
Non-photochemical quenching (of fluorescence)
ROS
Reactive oxygen species
1
O2
Singlet oxygen
SOSG Singlet Oxygen Sensor Green
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Page 2 of 17
Introduction
Photosystem II (PSII) is constantly damaged by light, and
synthesis of a new D1-protein is needed for the recovery
of the electron transfer activity (for reviews, see Tyystjärvi
2013; Nath et al. 2013). The initial rate of the damage is
directly proportional to the intensity of the illumination
(Tyystjärvi and Aro 1996); if damage occurs faster than
the repair, e.g. under high light, non-functional PSII units
accumulate. In the literature, the term “photoinhibition”
has been used to describe several phenomena; here, we
strictly refer to the light-induced irreversible loss of activity of PSII. The molecular mechanism of the damage to
PSII is still under debate (Tyystjärvi 2013).
Low temperature is a major factor limiting growth and
geographical distribution of plant species. The repair of
PSII slows down if temperature drops (Greer et al. 1986;
Grennan and Ort 2007). Results about the effect of temperature on the rate of the damage itself, however, vary.
A decrease in the rate of damage with a decrease in temperature in pumpkin thylakoids (Tyystjärvi et al. 1994;
Tyystjärvi 2013) and chloramphenicol-treated leaves
(Tyystjärvi 1993), lack of a clear temperature dependence
in the cyanobacterium Synechocystis (Allakhverdiev and
Murata 2004) and an increase in the rate of damage with
decreasing temperature in the leaves of Chenopodium
album (Tsonev and Hikosaka 2003) and Gossypium hirsutum (Kornyeyev et al. 2003) have been reported. Light
absorption and subsequent charge separation in PSII
are almost temperature-independent but, upon a sudden
decrease in temperature, sink capacity decreases because
carbon fixation slows down, leading to increased “excitation pressure” in the chloroplasts (Dietz et al. 1985). High
excitation pressure is thought to cause photo-oxidation
events leading to variegated leaf phenotypes in several
mutants like immutans (Rosso et al. 2009). The “overreduction” of electron transfer chain may increase singlet
oxygen (1O2) production because reduced electron acceptors promote PSII charge recombination reactions. 1O2, in
turn, could damage PSII (see e.g. Vass and Cser 2009).
Therefore, high excitation pressure has been proposed to
increase photoinhibition (Sonoike et al. 1999; Kornyeyev
et al. 2003). However, these considerations do not directly
probe the relationship between excitation pressure and
photoinhibition of PSII.
Growth at a low temperature triggers cold-acclimation
in several plant species (for a review, see Theocharis et al.
2012), causing large changes in gene expression and modifying multiple physiological processes, including synthesis of protective substances, changes in the composition
of membrane lipids, and changes in enzyme activities or
amounts of other substances that protect against reactive
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Planta (2020) 252:19
oxygen species (ROS). Light plays an important role in
the development of full cold-acclimation (Soitamo et al.
2008), and it has been suggested that reduction of the photosynthetic electron transfer chain partly causes the coldinduced responses (Maxwell et al. 1995; Gray et al. 1996;
Ivanov et al. 2006). Indeed, increased excitation pressure
has been shown to trigger cold-acclimation responses,
such as redox potential changes in PSII (see below; Sane
et al. 2003).
Growth at (or long exposure to) cold (Gray et al. 1996;
Krause et al. 1999; Savitch et al. 2000; Venema et al. 2000;
Sane et al. 2003) or over-expression of cold-inducible genes
(Yang et al. 2010) has been reported to attenuate photoinhibition of PSII at low temperatures. In some species, this is
due to increased activity of the repair cycle of PSII (Krause
et al. 1999; Venema et al. 2000; Grennan and Ort 2007;
Rogalski et al. 2008) but also the rate of the damaging
reaction has been reported to diminish due to cold-acclimation (Vonshak and Novoplansky 2008). The protection
has been hypothesized to be based on the ability of coldacclimated plants to keep the QA electron acceptor of PSII
more oxidized in the light even at low temperatures (Öquist
et al. 1993; Gray et al. 1996), thereby decreasing excitation pressure. Cold-acclimation can increase activities of
the enzymes of the Calvin-Benson cycle, which increases
the rate of carbon fixation at low temperatures (Strand et al.
1999). Alternative electron transfer routes (cyclic electron
transfer and electron transfer to the plastid terminal oxidase)
may also function more efficiently after cold-acclimation
(e.g. Ivanov et al. 2012; Mishra et al. 2019). In many plant
species, cold-acclimation also leads to changes in the redox
potentials of the electron transport chain of PSII, possibly
modifying recombination reactions and affecting 1O2 yield
(Janda et al. 2000; Ivanov et al. 2001; Sane et al. 2003).
The amounts of xanthophyll pigments and/or non-photochemical quenching of excitation energy (NPQ) can increase
during cold-acclimation (e.g. Krause et al. 1999; Venema
et al. 2000). Furthermore, cold-acclimation may affect concentrations of anthocyanins and flavonols. Although mainly
in the vacuole, flavonols have been found in chloroplasts of
several species (Saunders and McClure 1976). Flavonols are
preferentially located at the lipid-water interphase (Scheidt
et al. 2004), which allows them to quench 1O2 produced
within membranes. These properties may make flavonols
important scavengers of 1O2, as the lifetime of 1O2 in plant
cells is so short (for reviews, see Mattila et al. 2015; Arellano and Naqvi 2016) that the damage caused by 1O2 is
expected to occur near the site of origin of this ROS. In
Phillyrea latifolia chloroplast-envelope-located flavonols
were reported to quench 1O2 (Agati et al. 2007).
The capacity of plants to cold-acclimate varies greatly,
but cold-tolerance is correlated with the distance of a species or accession from the equator (e.g. Hannah et al. 2006;
Page 3 of 17 19
Planta (2020) 252:19
Mishra et al. 2011). Arabidopsis thaliana grows over a broad
geographic range with varying temperatures, and therefore,
effects of low temperature and cold-acclimation on photoinhibition can be investigated in natural accessions of this
model species. In the present study, we used four accessions
with different freezing tolerances; the LT50 (lethal temperatures at which 50% of tissue damage occurs as measured
by electrolyte leakage) has been reported to be − 5.7 °C,
− 7.7 °C, − 5.2 °C and − 4.6 °C, for Rschew, Tenela, Columbia-0 and Coimbra, respectively (Hannah et al. 2006; Mishra
et al. 2011). A shift to a cold growth temperature causes an
increase in the freezing tolerance; a 2-week cold-treatment
at 4 °C, as used in the present study, lowered the LT50 temperatures by 4.5–5.2 °C in Rschew, Columbia-0 and Tenela
but only by ~ 1.5 °C in Coimbra (Hannah et al. 2006; Mishra
et al. 2011). In addition, stable changes in gene expression
and metabolites are observed after 2 weeks at 4 °C (Hannah
et al. 2006).
Previous investigations on photoinhibition of PSII at low
temperatures have mostly been conducted by illuminating
plants in the absence of a translation inhibitor (Gray et al.
1996; Krause et al. 1999; Sonoike et al. 1999; Venema
et al. 2000; Sane et al. 2003). Thus, it is not clear whether
the observed temperature dependence of photoinhibition
depends on differences in the rate of damage or repair.
Moreover, the effect of cold-acclimation on photoinhibition
of PSII has been mostly studied only by chlorophyll a fluorescence (e.g. Gray et al. 1996; Krause et al. 1999; Venema
et al. 2000; Sane et al. 2003). In the present study, to differentiate between NPQ, repair and damage, we illuminated
leaves in the presence and absence of the chloroplast translation inhibitor, lincomycin, and assayed photoinhibition by
chlorophyll a fluorescence as well as by oxygen evolution.
In addition, 1O2 production was measured to understand the
effects of cold-treatment and this ROS on photoinhibition.
Materials and methods
Plant material and growth conditions
Arabidopsis thaliana accessions, Rschew (from Russia),
Tenela (Finland), Columbia-0 (Central Europe) and Coimbra
(Portugal), obtained as gifts from Prof. Arnd G. Heyer, were
grown in a growth chamber (FytoScope FS-RI 1600, Photon
Systems Instruments, Brno, Czech Republic) for 6 weeks
at day/night temperatures of 21 °C/18 °C (photosynthetic
photon flux density, PPFD, of 100 µmol m−2 s−1) with ~ 60%
humidity (Mishra et al. 2014). After 6 weeks of growth,
half of the plants were shifted for cold-treatment to 4 °C for
2 weeks (CT) while the rest of the plants (NT) were kept at
21 °C/18 °C. In addition, Columbia-0 was shifted to 4 °C
for 15 weeks after the emergency of first real leaves (CD).
For growth at 4 °C and excitation pressure measurements,
Columbia-0 was grown in growth chambers equipped with
Osram Powerstar HQI-BT lamps at 22 °C or with fluorescent tubes at 4 °C, otherwise as previously described. To
test the effect of growth light, plants (Columbia-0) were
shifted under PPFD of 1000 µmol m−2 s−1 (Dyna, Heliospectra, Sweden) for 6–10 weeks after the emergence of
first real leaves (HL). The temperature was around 20 °C. In
all experiments, the day/night light rhythm during growth
was 8 h/16 h, and fully expanded leaves were used for
measurements.
Photoinhibition measurements
After 8 weeks of growth, detached leaves, with petioles
in water, were illuminated for 0–45 min at the PPFD of
2000 µmol m−2 s−1 at 22 °C or 4 °C in a growth chamber (FytoScope FS 130, Photon Systems Instruments). To
block the repair cycle of PSII, leaf petioles were incubated
overnight in lincomycin (0.4 mg/ml) solution, under the
low irradiance of ~ 10 µmol m−2 s−1. Before and after the
illumination treatment, leaves were kept for 30 min in the
dark at 22 °C, after which the chlorophyll a fluorescence
parameter F v/F m (variable to maximum fluorescence)
was measured by Handy Fluorcam FC 1000-H (Photon
System Instruments). Weak measuring flashes (620 nm,
10 µs, ~ 1 µmol m−2 s−1, 19.63 Hz) were used to measure
minimal fluorescence (F0) and an 800-ms saturating pulse
(white light, ~ 2000 µmol m−2 s−1) was fired on the top of the
measuring flashes to measure maximal fluorescence (Fm).
After the fluorescence measurements, thylakoid membranes
from 3 to 6 leaves were isolated as described by Hakala
et al. (2005) and immediately stored at − 80 °C. PSII activity was then measured with a parallel method, by measuring
the light-saturated (PPFD ~ 4000 µmol m−2 s−1 from a slide
projector) rate of oxygen evolution from the thylakoid membranes (10 µg chlorophyll/ml) at 22 °C in a buffer (40 mM
HEPES–KOH (pH 7.6), 1 M betaine monohydrate, 330 mM
sorbitol, 5 mM MgCl2, 5 mM NaCl, 1 mM KH2PO4 and
5 mM NH4Cl) with an oxygen electrode (Hansatech, King’s
Lynn, UK) using 0.5 mM 2,6-dimethylbenzoquinone as an
electron acceptor.
The reduction state of QA during the photoinhibition
treatments was assayed with PAM-2000 fluorometer (Walz,
Effeltrich, Germany). Detached leaves were dark-acclimated
for 30 min at room temperature and F0 and Fm were measured. After that, the temperature was set to either 22 °C
or 4 °C, leaves were placed on wet paper and light was
switched on (PPFD of 2000 µmol m−2 s−1 from a high pressure xenon lamp; Sciencetech Inc.). F (fluorescence intensity
recorded just before the pulse) and Fm′ (fluorescence intensity during the pulse) were measured during illumination
every 15 min by firing an 800-ms saturating pulse (white
13
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light, ~ 4000 µmol m−2 s−1). The quantum yield of PSII in
the light was calculated according to Genty et al. (1989), F0′
(F0 in light), required for calculation of qP and qL, according
to Oxborough and Baker (1997), qP (photochemical quenching) according to Schreiber et al. (1986) and qL (photochemical quenching assuming connected PSII units) according to
Kramer et al. (2004).
Pigments
Chlorophyll and flavonol contents were measured from
intact leaves with a nondestructive handheld device (Cerovic
et al. 2012; Dualex Scientific, Force-A, Paris, France) after
7–8 weeks of growth. At least three individual uniform-sized
NT and CT plants of each accession were selected, from
which three leaves were measured. Chlorophyll concentration of isolated thylakoid membranes was measured according to Porra et al. (1989).
Gas exchange measurements
Net CO2 assimilation rates of individual attached leaves of
NT and CT plants of each accession, after 7–8 weeks of
growth, were measured with a gas exchange measuring system LI-6400-17 (Li-Cor, Biosciences, Linkoln, NE, USA)
using a 6400-15 Arabidopsis chamber with the aperture
diameter of 1 cm (Li-Cor). CO2 concentration in the chamber was set to 385 ppm, air humidity to 60 ± 5%, and temperature to 22 °C. Light-acclimated leaves were illuminated
first for 2 min at the PPFD of 100 µmol m−2 s−1 and then for
45 min at the PPFD of 2000 µmol m−2 s−1.
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Page 4 of 17
O2 and thermoluminescence measurements
The rate of 1O2 production by isolated thylakoid membranes (100 µg chlorophyll/ml) in high light (PPFD
4000 µmol m−2 s−1) at 4 °C or at 20 °C, was estimated by
measuring the consumption of oxygen, occurring due to the
reaction of 1O2 with 20 mM L-histidine (Sigma-Aldrich,
Saint Louis, MO, USA; Rehman et al. 2013) with the oxygen electrode (Hansatech). Oxygen concentration decreases
linearly from 5 to 45 s after switching on the high light, during which time the rate of 1O2 production was calculated.
Consumption of oxygen by the thylakoids without added
histidine was subtracted from the final results.
1
O2 production by NT, CT and HL Columbia-0 leaves
was measured with Singlet Oxygen Sensor Green (SOSG;
Invitrogen™). Leaf disks (diameter 6 mm) were vacuuminfiltrated and subsequently incubated overnight in a
solution containing 200 µM SOSG and rinsed before the
measurements. Leaf disks were illuminated with red light
(> 650 nm; PPFD 2000 µmol m−2 s−1), to avoid 1O2 production by SOSG itself (Ragás et al. 2009), at 4 °C or at 22 °C.
13
For 1O2 assays, illumination with red light was interrupted,
and SOSG fluorescence was excited by illuminating the leaf
with 500 nm light, obtained through a 10 nm bandpass filter
(Corion, Newport Corporation), and fluorescence from 535
to 555 nm was recorded with QE Pro spectrometer (Ocean
Insights).
Temperature dependencies of the reactions of 1O 2
with SOSG or histidine were tested by illuminating (red
light of PPFD 2000 µmol m−2 s−1 or white light of PPFD
4000 µmol m−2 s−1, for SOSG and histidine, respectively)
methylene blue solution (absorbance at 665 nm = 0.32 or
0.08, for SOSG and histidine, respectively) with 200 µM
SOSG or 20 mM histidine at 4 °C or at 22 °C. SOSG fluorescence and oxygen consumption were recorded as above
described.
Thermoluminescence bands were recorded with a luminometer from isolated thylakoid membranes (500 µg chlorophyll/ml) as described earlier (Tyystjärvi et al. 2009) in
the presence or absence of 20 µM 3-(3,4-dichlorophenyl)1,1-dimethylurea (DCMU). The thylakoid membranes for
1
O2 and thermoluminescence measurements were isolated
as described above from NT and CT plants taken directly
from growth chambers.
Statistical tests and figures
Significances of differences were tested by calculating Student’s t test (two-tailed, unequal variances; for calculations
and original data, see Online Resource 1). Whenever lincomycin was used, the rate constant of photoinhibition (kPI)
was calculated by fitting the decrease in Fv/Fm or in the rate
of oxygen evolution, as indicated, to the first order reaction equation in SigmaPlot (Systat Software Inc, San Jose,
CA, USA). All figures were prepared in SigmaPlot (Systat
Software Inc).
Results
Assaying photoinhibition of PSII, in the presence
of repair, by chlorophyll a fluorescence.
Detached leaves from A. thaliana accessions (Rschew,
Tenela, Columbia-0 and Coimbra) grown at 21 °C for
8 weeks (non-treated; NT) were illuminated with high light
(PPFD 2000 µmol m−2 s−1) either at their growth temperature (at 22 °C) or at 4 °C. Maximum quantum yield of PSII
photochemistry (Fv/Fm; after 30 min of dark-incubation) was
measured after 0 min, 15 min, 30 min and 45 min in the
high light (Fig. 1a, b). Illumination was conducted in the
absence of lincomycin to allow the repair of the D1-protein
to proceed simultaneously with photoinhibitory damage
to PSII. In addition, leaves from plants cold-treated (CT)
Planta (2020) 252:19
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photoinhibition of PSII, respectively (Fig. 1). In NT plants,
on the average, Fv/Fm values declined faster at 4 °C than at
22 °C (Fig. 1a, b). The difference was statistically significant
for Tenela after 15 min (P = 0.03) and 30 min (P = 0.01)
and for Rschew after 45 min (P = 0.01) of the light treatment. However, the rate of photoinhibition at 4 °C did not
differ significantly between NT and CT plants in any of the
accessions.
Assaying photoinhibition of PSII, in the absence
of repair, by chlorophyll a fluorescence
Fig. 1 Photoinhibition at 22 °C (a) or at 4 °C (b, c) in the absence
of lincomycin, quantified by the chlorophyll a fluorescence parameter Fv/Fm. Fv/Fm was measured from detached leaves of four noncold-treated (NT; a, b) or cold-treated (CT; c) A. thaliana accessions, at different time points during the 45-min illumination (PPFD
2000 µmol m−2 s−1), after subsequent 30-min dark incubation. The
error bars show standard deviations (SD) from at least three biological replicates. Statistically significant differences at any time-point
between the indicated accessions are marked with **P < 0.05 or
***P < 0.01. The control values of Fv/Fm (± SD) were 0.82 (0.03),
0.80 (0.03), 0.82 (0.03) and 0.76 (0.05) for Rschew (R), Tenela (T),
Columbia-0 (Col) and Coimbra (Co), respectively, in a, 0.82 (0.03),
0.82 (0.03), 0.81 (0.04) and 0.79 (0.06) in b, and 0.82 (0.03), 0.79
(0.04), 0.77 (0.08) and 0.79 (0.06) in c
for 2 weeks at 4 °C were illuminated under the same high
light at 4 °C (Fig. 1c). Of the four accessions, Coimbra
and Rschew seemed the most susceptible and tolerant to
To determine if the observed differences in photoinhibition
were due to differences in the rate of damage to PSII or
the rate of repair, we repeated the experiments in the presence of lincomycin. When the repair cycle is blocked with
lincomycin, decline in PSII activity can be fitted to the first
order reaction equation for calculation of the rate constant
of photoinhibition of PSII (kPI). A good fit was obtained for
NT Rschew, Tenela and Columbia-0 at both 22 and 4 °C, but
for NT and CT Coimbra and CT Tenela, the fit was of low
quality (standard errors of the fits are presented in Online
Resource 1).
Similarly to what was observed without lincomycin
(Fig. 1), we found that NT Coimbra and Rschew were,
respectively, the least and the most tolerant to photoinhibition, both at 22 °C and at 4 °C (Fig. 2a, b, see Table 1
for the kPI values). After the cold-treatment, the differences
between accessions were no longer statistically significant
(Fig. 2c, Table 1). However, in contrast to the experiments
in which the repair was allowed to function, Fv/Fm values
declined more rapidly at 22 °C than at 4 °C in 21 °C-grown
Columbia-0 (P = 0.02) and Coimbra (P = 0.05) (Fig. 2a, b).
In Rschew and Tenela the differences in kPI values were
not statistically significant between the temperatures. In all
four accessions, photoinhibition at 4 °C proceeded more
slowly in CT plants than in NT plants (Fig. 2b, c). Accordingly, the kPI values of CT Rschew, Tenela and Coimbra
were circa 77% and of CT Columbia-0 84% of those of NT
plants (Table 1). The difference was significant in the case
of Rschew (P = 0.0002) and Tenela (P = 0.01).
Assaying photoinhibition of PSII, in the absence
of repair, by oxygen evolution
To test whether the results are universal or specific to a
particular method of quantification of photoinhibition,
we assayed photoinhibition of PSII also by measuring the
light-saturated rate of oxygen evolution in the presence of
an artificial electron acceptor from thylakoid membranes
isolated from the illuminated leaves. Good first-order fits
were obtained for NT plants at 22 °C and for NT Rschew,
Tenela and Columbia-0 at 4 °C; the fit for NT Coimbra at
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Photoinhibition appeared faster when measured with
oxygen evolution, in comparison to data obtained with the
fluorescence parameter Fv/Fm (c.f. Figures 2 and 3; Table 1).
However, similarly to the fluorescence data, we found that
Rschew was the most tolerant accession and Coimbra the
least, both at 4 °C and 22 °C (Table 1). Furthermore, photoinhibition proceeded more slowly at 4 °C than at 22 °C
in all accessions (Fig. 3a, b), although the differences in
the kPI values (Table 1) were significant (P < 0.05) only for
Rschew and Columbia-0. Two-weeks of cold-treatment alleviated photoinhibition at 4 °C statistically significantly only
in Coimbra (Fig. 3b, c).
Physiological parameters of cold‑treated
and non‑treated plants
Fig. 2 Photoinhibition at 22 °C (a) or at 4 °C (b, c) in the presence
of lincomycin, quantified by the chlorophyll a fluorescence parameter Fv/Fm. Fv/Fm was measured from detached leaves of four noncold-treated (NT; a, b) or cold-treated (CT; c) A. thaliana accessions, at different time points during the 45-min illumination (PPFD
2000 µmol m−2 s−1), after subsequent 30-min dark incubation. The
error bars show SD from at least three biological replicates. The lines
show a fit to the first order reaction equation. The control values of
Fv/Fm (± SD) were 0.82 (0.03), 0.79 (0.05), 0.83 (0.01) and 0.78
(0.04) for Rschew, Tenela, Columbia-0 and Coimbra, respectively, in
a, 0.80 (0.01), 0.77 (0.02), 0.81 (0.03) and 0.79 (0.03) in b and 0.82
(0.03), 0.77 (0.07), 0.79 (0.06) and 0.79 (0.06) in c
4 °C was of low quality, and in all accessions, the first data
point measured from CT plants after 15 min of light treatment deviated from first order. The standard errors of the fits
are presented in Online Resource 1.
13
To find the factors affecting the different photoinhibition
tolerances of the accessions, we measured chlorophyll content, epidermal flavonols and the rate of CO2 assimilation
non-invasively from intact leaves of NT and CT plants.
Chlorophyll contents, measured per leaf area with an optical method, were only little affected by temperature, age or
accession. In NT Rschew the chlorophyll content slightly
increased during the 14-day time-frame of the experiment
(Fig. 4a). The shift of the plants to 4 °C caused a slight
decrease in the chlorophyll content after 7 days only in
Columbia-0 (Fig. 4a), and even in this accession, no statistically significant difference was anymore observed
after 14 days of the cold-treatment. The relative differences in the amounts of chlorophyll between the accessions
(Rschew > Columbia-0 > Tenela > Coimbra) stayed the same
in all the treatment groups (Fig. 4a).
Contrary to chlorophylls, epidermal flavonols increased
significantly during the cold-treatment, both after 7 and
14 days, in all four accessions (Fig. 4b). In Tenela and Coimbra the cold-induced increase in epidermal flavonols was
smaller than in Rschew and Columbia-0, and the difference
was more obvious after 14 days of cold-treatment (Fig. 4b).
In Tenela and Coimbra, flavonols also increased at 21 °C
when the NT plants grew older (Fig. 4b).
To probe the effect of the high-light-illumination on the
whole photosynthetic electron transport chain (up to carbon
fixation), the rate of net CO2 assimilation, during illumination, was measured. Even though PSII activity declined
by about 20% during the 45-min high light illumination
at 22 °C in the absence of lincomycin (Fig. 1a), a similar
illumination treatment did not cause a decline in the rate
of net CO2 assimilation, measured per leaf area (Fig. 5).
Maximum assimilation rates varied between the accessions
(Rschew > Columbia-0 > Tenela ≈ Coimbra), and the values
of net CO2 assimilation (Fig. 5) and the chlorophyll content
of the leaves (Fig. 4a) showed similar order between the
accessions, especially in NT plants. A 2-week cold-treatment
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Table 1 The rate constants of photoinhibition (kPI) in min−1 calculated by fitting the decline in Fv/Fm (Fig. 2) or in PSII-dependent oxygen evolution (Fig. 3), during the 45-min illumination at 22 °C or at
4 °C, to the first order reaction equation in non-cold-treated (NT) and
cold-treated (CT) A. thaliana accessions
kPI, min−1 (SD)
Non-treated, at 22 °C
Rschew
Tenela
Columbia-0
Coimbra
Non-treated, at 4 °C
Cold-treated, at 4 °C
Fluorescence
Oxygen evolution
Fluorescence
Oxygen evolution
Fluorescence
Oxygen evolution
0.0086a,A (0.0011)
0.0105b,A (0.0012)
0.0128c,A (0.0014)
0.0160d,A (0.0011)
0.0194a,A (0.0044)
0.0210ab,A (0.0024)
0.0247b,A (0.0036)
0.0241ab,A (0.0051)
0.0093a,A (0.0008)
0.0115bc,A (0.0013)
0.0111b,B (0.0027)
0.0135c,B (0.0027)
0.0124a,B (0.0044)
0.0192b,A (0.0047)
0.0179ab,B (0.0059)
0.0226b,A (0.0080)
0.0072a,B (0.0016)
0.0088a,B (0.0034)
0.0093a,B (0.0042)
0.0105a,B (0.0053)
0.0155ab,B (0.0048)
0.0220a,A (0.0048)
0.0128b,B (0.0044)
0.0124b,B (0.0044)
SD values (in parentheses) were calculated from at least three biological replicates. Statistically significant differences (P < 0.05) between accessions are indicated with lower-case letters and between treatment groups (between NT 22 °C and NT 4 °C, or between NT 4 °C and CT 4 °C)
with upper-case letters. Significances of the differences between different accessions are shown only within the same treatment group. Significances of the differences between fluorescence and oxygen evolution data were not calculated
caused a decrease in the maximum rate of CO2 assimilation
in high light at 22 °C in all four accessions (Fig. 5). The
maximum CO2 assimilation rate after switching on the high
light, however, was reached faster in the CT plants (Fig. 5).
1
O2 production in thylakoid membranes and leaves
To investigate if the physiological differences of the accessions led to differences in the 1O2 production capacity of
PSII, we measured the rate of 1O2 production by isolated thylakoid membranes of NT and CT plants, at 20 °C and at 4 °C
in high light (Fig. 6a). Undamaged thylakoids were obtained
by isolating them directly from growth conditions (21 °C or
4 °C). 1O2 production was measured with a histidine-based
method; the method measures short-term (1–2 min) 1O2 production upon switching light on. Thylakoids isolated from
CT plants produced more 1O2 at 20 °C than those isolated
from NT plants, but the difference was statistically significant only in Coimbra (Fig. 6a). In both NT and CT thylakoids, production of 1O2 was slightly slower at 4 °C than at
20 °C, but a statistically significant difference was observed
only in CT Coimbra. The reaction of histidine with 1O2,
probed by producing 1O2 with methylene blue, proceeded
slightly faster at 20 °C than at 4 °C (Fig. S1), indicating that
1
O2 production by methylene blue is weakly if at all dependent on temperature in the physiological range. Interestingly,
the production of 1O2 by CT thylakoids at 4 °C did not significantly differ from those of NT thylakoids (Fig. 6a). Even
thylakoids of Columbia-0 leaves that had grown and developed at 4 °C (CD) did not produce less 1O2 than thylakoids
of NT or CT Columbia-0 (Fig. 6a).
To see if 1O2 production in vivo would differ from the
observed in vitro results, 1O 2 production in high light
(PPFD 2000 µmol m−2 s−1 of red light) was measured from
leaf disks of Columbia-0 with SOSG (Fig. 6b–d). The
reaction of SOSG with 1O2, again probed by methylene
blue, proceeded over two times as fast at 22 °C than at
4 °C (Fig. 6b). During the illumination of leaves, SOSG
fluorescence increased more rapidly at 22 °C than at 4 °C
(Fig. 6c). The 2-week cold-treatment did not seem to affect
1
O2 production, as SOSG fluorescence increased similarly
in NT and CT leaves (Fig. 6c). To test the effect of growth
light intensity, 1O2 production at 4 and 22 °C was measured
also from high light (PPFD 1000 µmol m−2 s −1) grown
(HL) Columbia-0 (Fig. 6d) at PPFD 1000 µmol m −2 s−1.
Expectedly, when illuminated with the lower PPFD of
1000 µmol m−2 s−1, NT plants produced less 1O2 than at
PPFD 2000 µmol m−2 s−1. However, SOSG fluorescence
increased similarly in NT and HL plants (Fig. 6d). At
this PPFD, SOSG fluorescence increased at similar rates
at 4 °C and at 22 °C (Fig. 6d). Considering the strong
temperature dependence of the reaction between SOSG
and 1O2 (Fig. 6b), the similarity of the increase in SOSG
fluorescence at the two temperatures (Fig. 6c, d) suggests
that the actual rate of 1O2 production in NT, CT and HL
leaves was higher at 4 °C than at 22 °C, especially at PPFD
1000 µmol m−2 s−1.
Modulation of PSII charge recombination reactions by
cold-acclimation (Sane et al. 2003) has been suggested
to diminish 1O2 production (Ivanov 2008). To assess the
recombination reactions, thermoluminescence was measured from the same thylakoid membranes used for the
1
O2 assay. In all samples, the peak of the B-band, measured in the absence of DCMU, was between 25 °C and
28 °C whereas the Q-band (measured in the presence of
DCMU) peaked around 7–9 °C with no systematic differences between accessions or between NT and CT plants
(Fig. 7). However, the thermoluminescence yield of the
B-band was 4–46% and the yield of the Q-band 26–46%
lower for the CT than NT plants (relative peak intensities
are listed in the legend of Fig. 7).
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Planta (2020) 252:19
Fig. 4 Chlorophyll (a) and flavonol (b) contents measured from intact
leaves of A. thaliana accessions (Rschew, Tenela, Columbia-0 and
Coimbra) after 7 or 14 days, as indicated, of cold-treatment at 4 °C
(CT) or from control plants of similar ages grown at 21 °C (NT). The
error bars show SD values from at least four biological replicates.
Statistically significant differences are marked with **P < 0.05 or
***P < 0.01 on top of the horizontal lines that show between which
samples the difference is significant; the horizontal curly bracket indicates a whole group of four accessions. The significance of the differences between different accessions are shown only within the same
treatment group, and significances between NT and CT plants are
shown only between the corresponding age groups
Fig. 3 Photoinhibition at 22 °C (a) or at 4 °C (b, c) in the presence
of lincomycin, quantified by the light-saturated oxygen evolution of
PSII in the presence of an artificial electron acceptor. PSII activity
was measured from thylakoid membranes isolated from leaves of four
non-cold-treated (NT; a, b) or cold-treated (CT; c) A. thaliana accessions, at different time points during the 45-min illumination (PPFD
2000 µmol m−2 s−1). The error bars show SD from at least four biological replicates. The lines show a fit to the first order reaction equation. The control values in µmol O2 (mg chlorophyll)−1 h−1 (± SD)
were 160 (37.5), 162 (26.4), 180 (20.2) and 132 (31.2) for Rschew,
Tenela, Columbia-0 and Coimbra, respectively, in a, 169 (67.6), 148
(67.1), 162 (37.4) and 135 (24.7) in b and 210 (21.7), 186 (22.9), 164
(28.7) and 162 (37) in c
Excitation pressure at 4 °C and 22 °C
To understand the role of excitation pressure in photoinhibition, the reduction state of QA during 45-min high-light
13
treatment was measured from Columbia-0 (Fig. 8). In NT
plants illuminated at PPFD 2000 µmol m−2 s−1, the yield
of PSII electron transfer and coefficients of photochemical quenching, qP and qL, were slightly lower at 4 °C than
at 22 °C, and the difference was statistically significant
(P < 0.05) at the 30-min time point (Fig. 8a, b). The data
indicate that QA was almost completely reduced at 4 °C in
both CT and NT Columbia-0 (Fig. 8b, c). To obtain plants
with a larger difference in the excitation pressure between
the two temperatures, we repeated the experiments under
PPFD 1000 µmol m−2 s−1 with HL Columbia-0 (grown at
PPFD 1000 µmol m−2 s−1) in the presence of lincomycin. In
this case, a much higher proportion of QA remained reduced
at 4 °C than at 22 °C (Fig. 8d, e). To explore the relationship
Planta (2020) 252:19
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Fig. 5 Net CO2 assimilation rates at 22 °C during 42-min illumination in high light (PPFD 2000 µmol m−2 s−1), measured from intact
light-acclimated leaves of non-cold-treated (a) or cold-treated (b) A.
thaliana accessions (Rschew, Tenela, Columbia-0 and Coimbra). The
plants were illuminated with a light intensity close to that of growth
light (PPFD 100 µmol m−2 s−1) for 1 min after which the high light
was switched on (at the time point 0). The lines show averages and
the colored areas show SD values from at least three biological replicates
between excitation pressure and photoinhibition of PSII,
light-induced damage to PSII was quantified from the HL
plants after 45-min illumination at 1000 µmol m−2 s−1 in the
presence of lincomycin, using both fluorescence and oxygen evolution methods. The oxygen evolution capacity of
PSII declined slightly faster at 22 °C than at 4 °C, but the
difference was not statistically significant (Fig. 8f). Fv/Fm
values, in turn, indicated slightly faster photoinhibition at
4 °C (P = 0.046; Fig. 8f).
corroborates earlier in vitro (Tyystjärvi et al. 1994) and
in vivo (Tyystjärvi 2013) results. Values of PSII electron
transfer yield and photochemical quenching, measured in
Columbia-0, indicate that QA was more reduced at 4 °C than
at 22 °C during the 45-min high light treatment (Fig. 8a, b).
Thus, the finding that the damaging reaction of photoinhibition is faster at the higher, not at the lower temperature,
indicates that the excitation pressure hypothesis, according
to which the damage to PSII is caused by reduction of the
electron transfer chain (see Kornyeyev et al. 2003), does not
explain the temperature dependence of photoinhibition of
PSII in our data.
The use of a high PPFD for photoinhibition treatment,
as done in our study, is justified by the finding that kPI is
directly proportional to PPFD (for a review, see Tyystjärvi
2013). Therefore, when comparing different samples, the
choice of treatment PPFD is somewhat irrelevant. However,
due to high intensity of light used in the treatments, the difference in the reduction state of QA between 4 and 22 °C was
small in NT plants, possible because they were grown under
much lower light intensity. Therefore, we quantified both
excitation pressure and photoinhibition also from high-lightgrown (HL) Columbia-0 accession. The 45-min high light
treatments were given at the growth light of the HL plants,
both at the growth temperature (22 °C) and at 4 °C. In the
case of HL plants, QA was clearly more reduced in high light
at 4 °C than at 22 °C (Fig. 8d, e). Despite the big difference
in excitation pressure, photoinhibition rate, measured as a
decline in the oxygen evolution capacity of PSII, did not differ much between the two temperatures (Fig. 8f). However,
Fv/Fm measurements showed faster photoinhibition at lower
temperature, even though the difference was small (Fig. 8f).
Discussion
Rate of photodamage in vivo is slower or equal
at 4 °C than at 22 °C, in plants grown at 21 °C
Temperature affects the rate of the damaging reaction of
photoinhibition. We used two parallel methods for measuring photoinhibition of PSII: the chlorophyll a fluorescence
parameter, Fv/Fm, and PSII-dependent oxygen evolution
measured from thylakoids isolated from illuminated leaves.
Comparison of the present data obtained from non-treated
plants grown at 21 °C (NT) of all accessions showed a trend
of slower damage to PSII in the presence of lincomycin at
4 °C than at 22 °C (Table 1). Fluorescence data showed a
larger kPI at 22 °C than 4 °C for Columbia-0 and Coimbra and oxygen evolution data for Rschew and Columbia-0
(Table 1; Fig. 2). However, the experimental noise in both
Fv/Fm and oxygen data does not allow more than identification of a trend. In no case was the kPI value significantly
larger at 4 °C than 22 °C (Table 1). A slight increase in
the rate of the damaging reaction at higher temperatures
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Planta (2020) 252:19
Fig. 6 Production of 1O2 by isolated thylakoid membranes in vitro
(a), by a methylene blue solution (b) and by leaf disks in vivo (c,
d). The measurements were done at 20–22 °C or 4 °C, as indicated,
from A. thaliana accessions (Rschew, Tenela, Columbia-0 and Coimbra) grown at 21 °C (NT), cold-treated for 2 weeks (CT), grown and
developed at 4 °C (CD) or grown in high light at 20 °C (HL). Production of 1O2 in vitro (a) was measured with a histidine-based method
in high light (PPFD 4000 µmol m−2 s−1). 1O2 production by methylene blue and by A. thaliana leaves in vivo (b–d) was measured as an
increase in SOSG fluorescence after 0–45 min illumination with red
light of PPFD 2000 µmol m−2 s−1 (b, c) or 1000 µmol m−2 s−1 (d).
After each illumination period, SOSG fluorescence was excited with
500 nm light and recorded at 535–555 nm. The error bars show SD
values from at least three biological replicates. Statistically significant
differences in a are marked with **P < 0.05. The significances of the
differences between different accessions are shown only within the
same treatment group, and significances between NT and CT plants
are shown only between the corresponding temperatures
Tsonev and Hikosaka (2003) and Kornyeyev et al. (2003)
observed significantly faster decline in Fv/Fm at low temperature than at optimal temperature in vivo in Chenopodium
album and Gossypium hirsutum, respectively, at several
PPFD values. It is possible that the discrepancy between our
results and this negative temperature dependence originates
from the methods used for assessing photoinhibition. Fv/Fm
may reflect low-temperature-induced fluorescence quenching that does not relax during a typical dark-adaptation time
(20‒60 min). Such slowly relaxing decline of Fv/Fm might
be related to the sustained NPQ that develops in some evergreen species at low temperatures (for a review, see Verhoeven 2014) and might be important also in A. thaliana (Malnoë et al. 2018). Even in our data, Fv/Fm results from HL
plants alone (Fig. 8f) might allow concluding that excitation
pressure enhances photoinhibition. Due to these complications, the use of methods based on oxygen evolution of PSII
as a measure of photoinhibition is advisable, especially in
studies involving cold-treatments.
Upon a (sudden) decrease in temperature, light absorption
continues but carbon fixation and other enzymatic reactions
slow down. Therefore, exposure to high light at low temperature can induce severe stress for plants (see e.g. Alboresi
et al. 2011), even though the rate of light-induced damage to
PSII would not be accelerated compared to optimal conditions. In non-acclimated plants, low temperature commonly
slows down the enzymatic reactions of concurrent recovery of photoinhibitory damage (Greer et al. 1986). In addition, ROS produced at low temperatures may specifically
inhibit translation in the chloroplast (Kojima et al. 2009).
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Planta (2020) 252:19
Page 11 of 17
19
Fig. 7 Normalized thermoluminescence bands recorded in the
absence (a, c) or in the presence of DCMU (b, d) from thylakoid
membranes of non-cold-treated (NT; a, b) or cold-treated (CT; c, d)
A. thaliana accessions (Rschew, Tenela, Columbia-0 and Coimbra).
The colored areas show SD values from at least three biological repli-
cates. Maximum luminescence intensities (arbitrary units; ± SD) were
2.1 (0.60), 1.8 (0.45), 1.7 (0.15) and 2.8 (0.21) for Rschew, Tenela,
Columbia-0 and Coimbra, respectively, in a, 2.5 (0.77), 2.2 (0.23),
2.2 (0.17) and 2.6 (0.39) in b, 2.0 (0.26), 1.5 (0.20), 1.4 (0.25) and
1.5 (0.2) in c, and 1.8 (0.65), 1.2 (0.41), 1.6 (0.16) and 1.9 (0.08) in d
Therefore, net loss of PSII activity may occur (Öquist et al.
1993; Allakhverdiev and Murata 2004). The results of the
present study confirm faster loss of PSII activity at 4 °C than
at 22 °C in NT plants when no protein synthesis inhibitor
was used (Fig. 1a, b). As the rate of the damage to PSII was
faster at 22 °C than at 4 °C (Table 1), these results simply
confirm that the repair cycle of PSII functioned more slowly
at 4 °C than at 22 °C.
be considered as intermediate cold-tolerant and cold-susceptible, respectively. Knaupp et al. (2011) observed that
14 days of similar cold-treatment enhanced stability of PSII
during consequent freezing and thawing, and accordingly,
control activities of thylakoids isolated from CT plants were
higher than those of NT plants (see the legend of Fig. 3).
This, however, would not affect the measured in vivo photoinhibition rate because photoinhibition is a first-order
reaction (Tyystjärvi and Aro 1996). To conclude, 2-week
cold-treatment enhanced photoinhibition-tolerance in some
A. thaliana accessions, even though the effect was rather
small in the present study.
Duration of the cold-treatment has to be taken into
account when comparing the present results with literature (Gray et al. 1996; Krause et al. 1999; Savitch et al.
2000; Venema et al. 2000; Sane et al. 2003; Yang et al.
2010). Freezing tolerance is enhanced rapidly upon a coldtreatment (Ristic and Ashworth 1993) but, for example,
low-temperature grown A. thaliana plants are reported to
be able to keep QA more oxidized than plants that have
been shifted to the low temperature (Savitch et al. 2001).
Two‑week cold‑treatment may increase
photoinhibition tolerance in A. thaliana accessions
Two-week cold-treatment at 4 °C slowed down photoinhibition of PSII by slowing down the damaging reaction of
photoinhibition of PSII in all accessions when measured
with Fv/Fm, and the difference was statistically significant
in the two most cold-tolerant accessions Rschew and Tenela
(Fig. 2; Table 1), similarly as reported previously for cyanobacteria (Vonshak and Novoplansky 2008). Oxygen evolution data, in turn, showed protection by the cold-treatment
only in the accessions Columbia-0 and Coimbra, which can
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Planta (2020) 252:19
Fig. 8 Chlorophyll a fluorescence parameters (a–f) and oxygen
evolution (f) during 45-min illumination at 22 °C (a, d, f) or at
4 °C (b, c, e, f) measured from detached leaves of non-cold-treated
(NT; a, b), cold-treated for 2 weeks (CT; c) or grown in high light
at 20 °C (HL; d, e) A. thaliana accession Columbia-0. PPFD was
2000 µmol m−2 s−1 in a–c and 1000 µmol m−2 s−1 in d–f. F0 and
Fm were measured after 30 min dark-acclimation [time point 0 min
(30 min D)] and all other parameters were measured in the light,
15, 30 or 45 min after switching on the light, as indicated (a–e). F
is the fluorescence yield under illumination. Fv/Fm is the maximum
quantum yield of PSII electron transfer, calculated as (Fm − F0)/Fm.
ɸII is the quantum yield of PSII electron transfer in the light, calculated as (Fm’ − F)/Fm’. F0′ is the value of F0 in the light, calculated
as 1/(1/F0 − 1/Fm + 1/Fm’). qP and qL are estimates of photochemical quenching; qP is calculated as (Fm’ − F)/(Fm’ − F0′) and qL as
qP × F0′/F. F0, Fm and Fm’ values may differ between treatment
groups due to different settings of the fluorometer. Occasional belowzero qP or qL values in (a–c) were interpreted as zeros. HL Columbia-0 leaves were incubated overnight with the petioles in lincomycin
(d–f), and photoinhibition after the 45-min illumination was quantified by the chlorophyll a fluorescence parameter Fv/Fm from leaves
after 30 min in dark and by the light-saturated oxygen evolution of
PSII in the presence of an artificial electron acceptor measured from
thylakoids isolated from the illuminated leaves (f). The error bars
show SD from at least three biological replicates. In d, e F0′, qP and
qL have been calculated from averages
Accordingly, we did not observe alleviation in QA reduction during the 45-min high light treatment at 4 °C in CT
Columbia-0 compared to NT Columbia-0 (Fig. 8c). Also,
leaves fully developed at a low temperature were found to
be more tolerant against photoinhibition than leaves coldtreated for 20 days (e.g. Gray et al. 2003).
Direct comparison between NT and CT plants in the present study is complicated by the fact that the plants might
be at slightly different developmental stages. For example,
chlorophyll content (per leaf area) increased in NT plants
at 21 °C during the 2-week measurement period while in
CT plants there was very little or no increase in chlorophyll
13
Planta (2020) 252:19
during the 2 weeks at 4 °C (Fig. 4). It has been shown that
younger leaves are more susceptible to photoinhibition but
in fully developed leaves, such as those used in the present
study, the differences may be small (Bielczynski et al. 2017).
Therefore, the differences in photoinhibition tolerance
between accessions in the present study can be assumed to
be due to the cold-treatment and not because of the developmental stage of a leaf.
Decrease in the amount of functional PSII units during exposure to the 45-min illumination with strong light
at 22 °C (Figs. 1‒3) did not lead to a decrease in net CO2
assimilation (Fig. 5). The lack of change in the rate of carbon
fixation during the illumination, both in NT and CT plants,
suggests that A. thaliana leaves contain more PSII than
needed to saturate the needs of the carbon fixation reactions
in high light (see also Chow 2001). Net CO2 assimilation at
22 °C was slower in CT leaves than in their NT counterparts
(Fig. 5), similarly as observed previously after a 6-day coldtreatment (Velitchkova et al. 2020). No recovery occurs after
5 days in optimal conditions (Velitchkova et al. 2020), suggesting that short cold-treatments cause cold stress or induce
persistent down-regulation of carbon fixation in A. thaliana.
Decrease in thermoluminescence yield did not lower
1
O2 production after 2‑week cold‑treatment
What causes the improvement of photoinhibition-tolerance
by exposure to low temperatures? In plants, 1O2 is produced
mainly in the recombination reactions of PSII, and 1O2 has
been suggested to cause photodamage to PSII (e.g. Vass and
Cser 2009). Increased production of 1O2 in the npq1lut2
mutant of A. thaliana leads to oxidative damage to thylakoid
proteins in high light at 10 °C (Alboresi et al. 2011). In different plant species including A. thaliana (Janda et al. 2000;
Ivanov et al. 2001; Sane et al. 2003), cold-acclimation causes
a decrease in the redox gap between the QA and QB electron acceptors of PSII, which favors a direct, non-radiative
recombination route (Rappaport and Lavergne 2009) for the
elimination of the charge in QA− (Ivanov 2008). The direct
route does not produce 1O2 and, therefore, the observed
decrease in photoinhibition caused by cold-acclimation
might be due to a decrease in 1O2 production (for a more
comprehensive discussion about the relationship between
recombination reactions and 1O2, see Vass and Cser 2009).
Accordingly, Ramel et al. (2012) observed a decrease in both
production of 1O2 and oxidation of beta-carotene after 99 h
at 7 °C. Unfortunately, 1O2 was measured with SOSG in
white light, a condition that has been shown to induce 1O2
production by the sensor itself (e.g. Ragás et al. 2009).
We did not observe shifts in the peak temperatures of the
thermoluminescence bands (Fig. 7), contrary to the previous
observations (Sane et al. 2003). The shifts were observed
already after 7 days of cold-treatment (Sane et al. 2003). It
Page 13 of 17
19
might be possible that temperature induced modifications of
PSII are observed only when thermoluminescence is measured from leaves, as done by Sane et al. (2003), but not
from isolated thylakoids as used in the present study. We
did, however, observe a decrease in the thermoluminescence
yield for both B and Q-bands after the 2-week treatment at
4 °C, resembling that reported by Ivanov et al. (2001) and
Sane et al. (2003). The intensity of the B-band decreased
only little, ~ 4%, in Rschew but the decrease was more pronounced in Tenela (18%), Columbia-0 (20%) and Coimbra
(46%). We also observed strong alleviation of photoinhibition in CT Coimbra, compared to NT Coimbra. However,
in other accessions the decrease in the thermoluminescence
peak intensity did not correlate with the rate of photoinhibition (Fig. 7, Table 1). Furthermore, cold-treatments did
not cause a decrease in the intrinsic capacity of the thylakoids (presumably PSII) to produce 1O2 during illumination
(Fig. 6). Neither was 1O2 production in vivo in Columbia-0
affected by 2-week cold-treatment or by growth under high
light (PPFD 1000 µmol m−2 s−1) at 20 °C. To conclude, the
present cold-treatment at rather low light, or growth at high
light but at optimal temperature, did not lead to diminished
1
O2 production. Possibly a combination of high light and low
temperature is needed to alter the redox properties of PSII
in A. thaliana (see also Velitchkova et al. 2020). We cannot,
however, exclude the possibility that the in vivo results are
affected e.g. by different diffusion of SOSG into NT, CT or
HL leaves, due to the different optical and structural properties of the leaves.
If the rate of repair is insignificant at low temperatures,
then results from illumination treatments without an inhibitor of (chloroplast) protein synthesis could be taken to represent the amount of damage to PSII. However, in the present
data, addition of lincomycin clearly increased photoinhibition, indicating that the repair cycle is active at 4 °C even in
NT plants (Figs. 1, 2). Also the finding that genes coding for
a protease involved in PSII repair cycle (FtsH) are up-regulated at a low temperature (Soitamo et al. 2008) supports
this view. Consequently, it remains unclear whether diminished 1O2 production by modified recombination reactions
(Ivanov et al. 2001; Sane et al. 2003) decreases PSII damage
or protects the repair reactions (see e.g. Kojima et al. 2009).
Sensitivity to ROS may not equal sensitivity
to photoinhibition of PSII
The roles of ROS in the low-temperature-induced protection
against photoinhibition are interesting also for the mechanism of photoinhibition. The kPI values of the four accessions, measured by both Fv/Fm and oxygen evolution, were
mostly in the order Rschew < Tenela < Columbia-0 < Coimbra (Table 1). Furthermore, Coimbra was more susceptible
to the damage caused by high light than the other accessions
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Page 14 of 17
when the PSII repair cycle was allowed to run (Fig. 1). Thus,
the two cold-tolerant accessions (Rschew and Tenela) were
somewhat more tolerant to photoinhibition than the other
two accessions (Columbia-0 and Coimbra). Previously, it
has been shown that Tenela is sensitive to oxidative stress
(Brosché et al. 2010), which is probably linked to increased
production of hydrogen peroxide at low temperatures (Distelbarth et al. 2013). However, non-acclimated Tenela was
not particularly sensitive to photoinhibition of PSII (Figs. 1,
2, 3), which supports the suggestion that oxidative stress
and photoinhibition are not (always) related (Hakkila et al.
2014). In fact, the rate of light-induced damage to PSII may
not be defined by ROS but rather by direct light absorption
of the oxygen-evolving complex of PSII (Hakala et al. 2005).
In vivo, 1O2 production may be affected e.g. by the
reduction state of QA. We measured in vivo 1O2 production with SOSG, from leaves of Columbia-0. At PPFD
1000 µmol m−2 s−1, the rate of 1O2 production in high light
seemed faster at 4 °C than at 22 °C (considering the strong
temperature dependence of the reaction between SOSG and
1
O2; Fig. 6b), coinciding with a big difference in the reduction state of QA between the temperatures (Figs. 6 and 8). At
PPFD 2000 µmol m−2 s−1, when the difference in QA reduction was small, also the difference in 1O2 production between
the temperatures was smaller. The results support the idea
that the reduction of QA enhances 1O2 production. However,
photoinhibition proceeded rather similarly or faster at 22 °C
compared to 4 °C (Figs. 2, 3, 8f).
The sensitivity to oxidative stress may also be governed
by a genetic program (Brosché et al. 2010). Hydrogen peroxide, which has a relatively long lifetime in cells, is an
important signaling molecule (for a review, see e.g. Černý
et al. 2018). Further complications in the roles of ROS in
cold-acclimation are exemplified by discrepant findings on
the relationship between ROS metabolism and freezing tolerance (Distelbarth et al. 2013; Hashempour et al. 2014).
Do flavonols quench 1O2 and/or protect plants
from photoinhibition?
Synthesis of many flavonol species is induced in coldness
(e.g. Schulz et al. 2015), and their amount was shown to
correlate with freezing tolerance (Korn et al. 2008). Flavonols are also able to physically and chemically quench 1O2
in vitro (Tournaire et al. 1993), and it has been suggested
that they function as 1O2 quenchers also in vivo (Majer et al.
2014). Accordingly, Havaux and Kloppstech (2001) reported
that mutants unable to synthetize anthocyanins and flavonols
had lower Fv/Fm values and more lipid peroxidation at low
temperatures than wild-type plants.
In Columbia-0 and Coimbra one can see a negative correlation between flavonol amounts and the rate constant
13
Planta (2020) 252:19
Fig. 9 Rate constants of photoinhibition (quantified as a loss of oxygen evolution capacity of PSII) plotted against the amount of epidermal flavonols of four A. thaliana accessions (Rschew, Tenela,
Columbia-0 and Coimbra). Values are measured after 2 weeks of
cold-treatment at 4 °C (CT) or from control plants of similar ages
grown at 21 °C (NT). The data are from Fig. 4 and from Table 1
of photoinhibition, suggesting that flavonols can in some
accessions protect against photoinhibition (Fig. 9). However, no general correlation was found. In Columbia-0,
cold-acclimation did not decrease 1O2 production in vivo
(Fig. 6). Of course, the optical method used in the present study reflects mostly epidermal flavonols and, therefore, the possible effects of chloroplast-located flavonols
(probably in small amounts compared to epidermal flavonols) able to quench 1O2 (Agati et al. 2007) may not be
observed. In addition, it has been shown that some flavonol
species are more efficient in quenching ROS than others
(Majer et al. 2014). Therefore, a method measuring the
total amount of flavonols, as used here, may not give a full
answer. To resolve the roles of flavonols in cold-tolerance,
more research is required.
Author contribution statement ET, KBM and HM conceived
and designed research. HM, KBM, IK, AM, KN and DŠ
conducted experiments. HM, ET and KBM analyzed data.
HM wrote the manuscript with contributions from ET and
KBM. All authors read and approved the manuscript.
Acknowledgements Open access funding provided by University of
Turku (UTU) including Turku University Central Hospital. We thank
Prof. Arnd G. Heyer, University of Stuttgart, Germany, for providing seeds of natural accessions of Arabidopsis thaliana. This work
was supported by Academy of Finland (grant 307335; to ET), Turku
University Foundation (12353; to HM), University of Turku Graduate
School (to HM), Vilho, Yrjö and Kalle Väisälä Foundation (to HM),
European Union COST Action (TD1102; to ET, HM and KBM), the
Ministry of Education, Youth and Sports of CR within the National
Sustainability Program I (NPU I, LO1415; to KBM, AM, KN and
DŠ) and the project “SustES—Adaptation strategies for sustainable
ecosystem services and food security under adverse environmental
conditions” (CZ.02.1.01/0.0/0.0/16_019/0000797; to KBM).
Planta (2020) 252:19
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
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