Contributions of Visible and Ultraviolet Parts of Sunlight
to Photoinhibition
Marja Hakala-Yatkin, Mika Mäntysaari, Heta Mattila and Esa Tyystjärvi*
Photoinhibition is light-induced inactivation of PSII, and
action spectrum measurements have shown that UV light
causes photoinhibition much more efficiently than visible
light. In the present study, we quantified the contribution of
the UV part of sunlight in photoinhibition of PSII in leaves.
Greenhouse-grown pumpkin leaves were pretreated with
lincomycin to block the repair of photoinhibited PSII, and
exposed to sunlight behind a UV-permeable or UV-blocking
filter. Oxygen evolution and Chl fluorescence measurements
showed that photoinhibition proceeds 35% more slowly
under the UV-blocking than under the UV-permeable filter.
Experiments with a filter that blocks UV-B but transmits
UV-A and visible light revealed that UV-A light is almost fully
responsible for the UV effect. The difference between leaves
illuminated through a UV-blocking and UV-transparent
filter disappeared when leaves of field-grown pumpkin
plants were used. Thylakoids isolated from field-grown and
greenhouse-grown plants were equally sensitive to UV light,
and measurements of UV-induced fluorescence from leaves
indicated that the protection of the field-grown plants was
caused by substances that block the passage of UV light to
the chloroplasts. Thus, the UV part of sunlight, especially the
UV-A part, is potentially highly important in photoinhibition
of PSII but the UV-screening compounds of plant leaves
may offer almost complete protection against UV-induced
photoinhibition.
Keywords: Action spectrum • Cucurbita maxima •
Photoinhibition • Photosystem II • Solar radiation • Ultraviolet
radiation.
Abbreviations: PPFD, photosynthetic photon flux density.
Introduction
UV radiation (200–400 nm) is harmful to all organisms primarily
due to its ability to cause DNA damage and production of
reactive oxygen species (for reviews see Ichihashi et al. 2006,
Rünger and Kappes 2008, Solovchenko and Merzlyak 2008).
UV-B light (280–315 nm) causes several adverse effects on plant
growth and photosynthesis, and attenuation of UV-B from
ambient level may lead to increase in biomass production by
one-third in various land plants (Mazza et al. 2000, Xiong and
Day 2001, Day et al. 2001, Zhao et al. 2003, for review, see Kakani
et al. 2003). On the other hand, UV-A light (315–400 nm) is
considered less harmful than UV-B radiation.
Photosynthesis of land plants, measured on leaf area basis,
has often been found to remain unaffected by an increase in
UV-B radiation if the applied dose has been realistic considering
the possible UV-B levels reaching the Earth's surface (Allen et al.
1998, Searles et al. 2001, Xiong and Day 2001). Photoinhibition
is a reaction in which the photochemical activity of PSII is lost
so that recovery occurs only via synthesis of the D1 protein
(for recent reviews, see Tyystjärvi 2008, Vass and Aro 2008).
Both visible and UV light cause photoinhibition, and the action
spectrum of photoinhibition, measured from isolated thylakoid
membranes, shows that the photoinhibitory efficiency has
a low peak in red light (650–700 nm), remains fairly constant
when going from red to blue–green light (from 650 to 450 nm)
and increases substantially with decreasing wavelength from
450 nm to UV-C (220–280 nm) (Jones and Kok 1966, Hakala
et al. 2005, Ohnishi et al. 2005). UV-A light of 360 nm is ∼10
times as efficient as visible light (Hakala et al. 2005). Measurements of photoinhibition in intact Arabidopsis leaves also
showed an extensive increase in photoinhibitory efficiency
with decreasing wavelength throughout the blue region
towards UV (Sarvikas et al. 2006) and results from intact cells of
the cyanobacterium Synechocystis sp. PCC 6803 point in the
same direction (Tyystjärvi et al. 2002).
Measurements of the action spectrum of photoinhibition
both in vitro (Jones and Kok 1966; Jung and Kim 1990; Hakala
et al. 2005; Ohnishi et al. 2005) and in vivo (Tyystjärvi et al. 2002;
Sarvikas et al. 2006) suggest that UV light may make a significant contribution to photoinhibition under sunlight. Because
sunlight contains much more UV-A than UV-B, while photoinhibitory efficiency increases with decreasing wavelength
Regular Paper
Molecular Plant Biology, Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland
*Corresponding author: E-mail, esatyy@utu.fi; Tel, +358-2-333-5771
(Received July 7, 2010; Accepted August 19, 2010)
Plant Cell Physiol. 51(10): 1745–1753 (2010) doi:10.1093/pcp/pcq133, available online at www.pcp.oxfordjournals.org
© The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 51(10): 1745–1753 (2010) doi:10.1093/pcp/pcq133 © The Author 2010.
1745
M. Hakala-Yatkin et al.
throughout the UV range, the action spectra suggest that the
decisive part of the UV spectrum might be the UV-A rather
than the UV-B part. Calculations, based on comparison of the
actual spectrum of sunlight with an in vivo action spectrum
of photoinhibition, suggested that UV wavelengths would
contribute to 84% of photoinhibition under direct sunlight in
young Arabidopsis leaves (Sarvikas et al. 2006).
Information about the contribution of UV light to photoinhibition in nature is important for predicting the potential
effects of depletion of stratospheric ozone. Furthermore, large
decrease in the rate of photoinhibition, obtained if the most
photoinhibitory but at least productive UV light is filtered off
from solar radiation, might improve plant yield in greenhouses.
Earlier photoinhibition experiments in full sunlight and UVfiltered sunlight (Herrmann et al. 1996, Häder et al. 1996,
1997a,b, 1998) have shown that in red, brown and green algae,
exclusion of UV-B radiation significantly protects against
decrease in photosynthesis in sunlight. However, in those
experiments, concurrent recovery of photoinhibited PSII centers was allowed to function during illumination, and therefore
it is not known whether blocking UV light slows down the
damaging reaction of photoinhibition or whether UV light has
an adverse effect on the repair of photoinhibited PSII centers.
Moreover, UV-B was found to interfere with the repair of
photoinhibited PSII in Antarctic phytoplankton (Bouchard
et al. 2005).
In the present study, we exposed intact pumpkin leaves
to natural sunlight, using either a UV-permeable plexiglas or
the same plexiglas covered with a filter foil that blocked all
radiation <400 nm but allowed 95% of visible light to pass.
B
Transmission, %
A
The importance of UV-B radiation was tested by using a Mylar
sheet, which is transparent in the visible and UV-A ranges of
light but opaque in UV-B. The leaves were treated with lincomycin, an antibiotic that inhibits chloroplast protein synthesis,
thus blocking the concurrent repair of photoinhibited PSII.
Chlorophyll fluorescence was measured from control and
illuminated leaf disks, and PSII electron transfer activity was
instantly measured from thylakoids isolated from control
and treated leaves. The results showed that UV light very
significantly contributes to photoinhibition when leaves of
greenhouse-grown plants are exposed to sunlight. On the
other hand, protective substances of field-grown pumpkin
plants were found to eliminate the photoinhibitory effect of
UV light.
Results and Discussion
UV radiation of sunlight participates in
photoinhibition of pumpkin leaves
Experiments of this study aimed at direct measurement of
the contribution of solar UV light in the damaging reaction of
photoinhibition of PSII. Lincomycin-treated pumpkin leaves
were set to the horizontal position and exposed to sunlight
either under a UV-permeable bare plexiglas plate or under
a UV-blocking film attached to the plexiglas (see Fig. 1 for
the experimental setup, the transmission spectra and the estimated spectrum of illumination in the greenhouse used to
grow the plants). Samples for oxygen evolution and fluorescence measurements were taken at the beginning and after
100
80
60
Acrylic
40
Acrylic + Mylar
Acrylic + UV shield
20
0
200
300
400
500
600
700
800
380
400
Transmission, %
C
100
D
10
80
Relative irradiance
Wavelength, nm
1
60
40
20
0
280
1746
300
320 340 360
Wavelength, nm
380
400
0.1
)
/
0.01
0.001
280
(
I
300
320
340
360
Wavelength, nm
Plant Cell Physiol. 51(10): 1745–1753 (2010) doi:10.1093/pcp/pcq133 © The Author 2010.
Fig. 1 Experimental details. Experimental
setup for measuring photoinhibition
under natural sunlight (A). Transmission
spectra of the VS UVT acrylic with and
without the UV shield film and the Mylar
film, as indicated, in the 200–800 nm
range (B) and in the 280–400 nm range
(C). The spectra of the acrylic and UV
shield were measured before the
experiments (black lines in B) and after
all experiments (cyan lines in B). The
black and cyan lines coincide for the plain
acrylic plate. (D) Estimated spectrum of
illumination in the greenhouse, calculated
by assuming that two-thirds of intensity
between 400 and 700 nm is from sunlight
(blue line) or that one-third is from
sunlight (red line), in comparison with
the spectrum of unfiltered terrestrial
sunlight delivering the same power
between 400 and 700 nm as the two
calculated spectra (dashed line).
Photoinhibition in sunlight
3 and 6 h of illumination. The treatments were carried out in
June, July and August in Turku, Finland, starting between 7 and
11 a.m. Solar elevation varied from 20° to 52° during the treatments. Experiments were done in both sunny and cloudy
weather, avoiding only rainy days. The treatments led to a gradual decrease in oxygen evolution, measured from thylakoids
isolated from the treated leaves (Fig. 2). As expected, the
decrease in oxygen evolution was faster on sunny days. The
time-course of the loss of oxygen evolution was roughly similar
to the first-order reaction observed under constant light
in laboratory conditions (see e.g. Sarvikas et al. 2010). Due to
differences in illumination during different days, parallel
treatments under UV-permeable and UV-blocking cover were
always compared.
Blocking UV radiation significantly slowed down the loss of
oxygen evolution, on both cloudy and sunny days (Fig. 2). To
evaluate the efficiency of the protection, we fitted the decrease
in oxygen evolution to the first-order reaction equation; this
leads to an estimate of the initial rate of photoinhibition in
each case. For comparison of results from different days, we
calculated a relative quantum yield for photoinhibition by
dividing the rate constant, obtained from the fit, by the average
A
100
600
60
400
200
20
0
0
0
1
2
3
4
5
Illumination time, h
6
The decisive spectral range is UV-A, not UV-B
To measure the importance of UV-B light in photoinhibition
of PSII, we illuminated the leaves using the bare plexiglas plate
and a plate covered with a Mylar sheet (Krause et al. 1999,
Mazza et al. 2000). Mylar transmits 83% of visible and UV-A
light (Fig. 1B) but blocks most of the UV-B range (280–315 nm)
(Fig. 1C). Pairwise comparison of rate constants, obtained
B
100
800
80
600
60
400
40
200
20
0
Irradiance, W m-2
Oxygen evolution, % of control
40
Irradiance, W m-2
800
80
solar power during the experiment. In this calculation, we also
took into account that the UV-blocking filter transmitted 5%
less visible light than the bare plexiglas plate. According to this
analysis, the quantum yield of photoinhibition was 54% higher
under the UV-permeable bare plexiglas than under the UVblocking film (Table 1), indicating that ∼35% of photoinhibition of intact greenhouse-grown leaves under sunlight was
caused by the UV part of sunlight. The relative photoinhibitory
contribution of UV light was similar on sunny and cloudy days
(Fig. 2). According to the standard solar spectrum of the
American Society for Testing and Materials (http://rredc.nrel
.gov/solar/spectra/am1.5/), the 280–700 nm region of the
terrestrial solar spectrum contains 7.3% UV-A and only 0.1%
UV-B radiation. In comparison with the small proportion of
UV radiation in sunlight, the 35% contribution of UV-induced
photoinhibition is large, even though much smaller than
our earlier estimate obtained with young Arabidopsis leaves
(Sarvikas et al. 2006). Multiplication of the spectrum of terrestrial sunlight with the in vitro action spectrum of photoinhibition measured by Jones and Kok (1966) predicts that the
contribution of UV to photoinhibition is 31%, which is very
near to the result of the present study. However, differences in
UV sensitivity between species are well known (Kakani et al.
2003). Large differences in UV sensitivity have been observed
in aquatic bryophytes according to the collection site and the
collection date of the samples (Martinez-Abaigar et al. 2009)
and even between Arabidopsis accessions originating from
nearby geographical regions (Jansen et al. 2010).
Table 1 Relative quantum yield of photoinhibition in lincomycintreated pumpkin leaves under natural sunlight passing through
a UV-permeable or a UV-blocking filter
Growth site
Relative quantum yield of Contribution of
photoinhibition
UV light to
photoinhibition,
UVUV%
blocking
permeable
filter
filter
t-test
Greenhouse
0.479 ± 0.07
0.740 ± 0.14
35
7.9 × 10−4
Field
0.494 ± 0.11
0.526 ± 0.15
6
0
0
1
2
3
4
5
6
Illumination time, h
Fig. 2 Photoinhibition of greenhouse-grown pumpkin leaves.
Lincomycin-treated leaves were illuminated with sunlight passing
through a UV-permeable filter (open symbols) or through a UVblocking filter (solid symbols) in the course of a sunny day (A) and
a cloudy day (B), and oxygen evolution was measured from thylakoids
isolated from the illuminated leaves. The error bars show SD from two
leaves treated simultaneously. The dotted lines show solar irradiance.
Typical results are shown.
0.34
The rate constant of photoinhibition was estimated by fitting the decrease in the
light-saturated rate of oxygen evolution to the first order equation, and the
relative quantum yield was calculated by dividing the rate constant by the average
solar power during the experiment, also correcting for the 5% attenuation of
visible light due to the UV-blocking filter. Each quantum yield estimate represents
the mean and SD of six independent experiments, each done with two pumpkin
leaves. The t-test shows the probability of obtaining a different quantum yield
with the UV-blocking filter by chance.
Plant Cell Physiol. 51(10): 1745–1753 (2010) doi:10.1093/pcp/pcq133 © The Author 2010.
1747
M. Hakala-Yatkin et al.
leaves of pumpkin plants grown in an open garden. In this case,
we found no statistically significant difference between leaves
illuminated through the UV-blocking and UV-permeable cover,
although the results point to ∼6% slower photoinhibition
under the UV-blocking cover (Fig. 4, Table 1). Thus, solar UV
radiation virtually fails to induce photoinhibition in field-grown
pumpkin leaves, indicating that growth in the field induces
an efficient protective mechanism. The inducible UV protection lowers the rate constant of photoinhibition of pumpkin
leaves in sunlight by ∼30%. The efficiency of this mechanism is
comparable to the efficiency of non-photochemical quenching
of excitation energy, which may lower the rate constant of
photoinhibition caused by visible light by ∼25% (Tyystjärvi
et al. 2005, Sarvikas et al. 2006). The finding that PSII of fieldgrown pumpkin plants is protected against solar UV radiation
is in agreement with the conclusion that the adverse effects of
UV radiation on vascular plants of the Antarctic Peninsula
are mainly targeted outside of PSII (Xiong and Day 2001).
The relative quantum yield of photoinhibition under the
UV-blocking cover was similar in field-grown and greenhousegrown leaves (Table 1), indicating that growth in the
field does not induce mechanisms that would efficiently
A
100
80
Oxygen evolution and Fv /FM,
% of control
100
80
400
40
Oxygen evolution, % of control
The pumpkin plants used for sunlight photoinhibition experiments of Fig. 1, as well as Arabidopsis plants used in our earlier
measurements of the in vivo action spectrum of photoinhibition (Sarvikas et al. 2006), were grown indoors. In order to
determine the contribution of UV radiation in photoinhibition
of field-grown plants, we carried out similar treatments to
200
20
0
0
0
1
2
3
4
Illumination time, h
5
6
B
100
800
80
600
60
400
40
60
200
20
40
0
20
0
1
2
3
4
5
6
0
Illumination time, h
0
0
1
2
3
4
5
6
Illumination time, h
Fig. 3 Photoinhibition of greenhouse-grown pumpkin leaves shown as
a decrease in oxygen evolution (circles) and FV/FM (triangles).
Lincomycin-treated leaves were illuminated with sunlight passing
through a UV-permeable filter (open symbols) or through a UV-B
blocking Mylar filter (solid symbols) in the course of a sunny day.
1748
600
60
Irradiance, W m-2
Field-grown pumpkin leaves are protected
against UV radiation
800
Irradiance, W m-2
after correction for the lower transmission of Mylar in the visible and UV-A ranges, showed that the mean contribution of
UV-B radiation in photoinhibition under sunlight was 2 ± 6%,
but this difference between Mylar and bare plexiglas is not
statistically significant. The decrease in oxygen evolution and
ratio of variable to maximum Chl fluorescence (FV/FM) during
one experimental day under a UV-transparent filter and the
Mylar filter are shown in Fig. 3. Direct calculation on the basis
of the in vitro action spectrum of Jones and Kok (1966) and
the standard solar spectrum predicts that UV-B contributes
by 1.2% to photoinhibition in sunlight. In the earlier published
data, plants or algae have been treated with different wavelengths of light while the repair cycle of PSII was allowed to run
normally during the treatments. These studies have produced
widely varying results, depending on the species. In Antarctic
phytoplankton assemblages, UV-A caused somewhat less inhibition than UV-B (Holmhansen et al. 1993) and in shade leaves
of tropical plants, UV-B was found to be decisive (Krause et al.
1999). On the other hand, UV-A caused two thirds of the UV
damage to photosynthesis in phytoplankton of Lake Titicaca
(Helbling et al. 2001). Our data show that the UV-B contribution to photoinhibition of PSII in intact pumpkin leaves is the
same order of magnitude as predicted by the in vitro action
spectrum. However, the inhibitory efficiency of UV-B radiation
is not limited to the damaging reaction of photoinhibition of
PSII (Allen et al. 1998).
Fig. 4 Photoinhibition in field-grown pumpkin leaves. Lincomycintreated leaves were illuminated with sunlight passing through
a UV-permeable filter (open symbols) or through a UV-blocking filter
(solid symbols) in the course of a sunny day (A) and a cloudy day (B),
and oxygen evolution was measured from thylakoids isolated from
the illuminated leaves. The error bars show SD from two leaves treated
simultaneously. The dotted lines show solar irradiance. Typical results
are shown.
Plant Cell Physiol. 51(10): 1745–1753 (2010) doi:10.1093/pcp/pcq133 © The Author 2010.
Photoinhibition in sunlight
slow down the damaging reaction of photoinhibition induced
by visible light.
To get insight into the UV protection mechanism functioning in field-grown pumpkin leaves, we used strong UV-A light
(365 nm) as photoinhibitory light for greenhouse-grown and
field-grown pumpkin leaves and for thylakoids isolated from
the same leaves. UV-A light was chosen for this experiment
because the experiments done in sunlight indicate that UV-A is
mainly responsible for the UV contribution in photoinhibition.
The results showed three times faster photoinhibition under
UV-A light in greenhouse-grown than in field-grown leaves
(Fig. 5), confirming the higher sensitivity of greenhouse-grown
leaves. The result was similar whether oxygen evolution or
FV/FM was used to measure photoinhibition. When isolated
thylakoids were exposed to the same UV-A intensity as the
leaves, the rate constant of photoinhibition was ∼10 times
higher than in leaves, indicating that constitutive protection
offered by the leaf structure drastically slows down photoinhibition under UV-A light. Furthermore, thylakoids isolated
from greenhouse-grown and field-grown pumpkin leaves were
equally sensitive to UV-A light (Fig. 5), indicating that the
inducible protection of pumpkin leaves functions exclusively
outside the thylakoid membrane. Plant species may differ
also in this respect. Short daily exposure to direct sunlight
induced large increases in thylakoid-bound carotenoids in a
shade-grown tropical plant, Anacardium excelsum (Krause et al.
1999) but in two rainforest trees, Tetragastris panamensis
80
60
40
1.0
20
0
0
5
10
15
20
25
30
Illumination, min
Fig. 5 Photoinhibition in UV-A light. Leaves (circles and triangles) and
isolated thylakoids (diamonds) of greenhouse-grown (open symbols)
and field-grown (solid symbols) pumpkin plants were illuminated
with 365 nm light and photoinhibition was measured with oxygen
evolution (triangles, diamonds) or with FV/FM (circles). Each data point
represents an average of three and eight independent experiments
in thylakoid and leaf experiments, respectively, and the error bars,
drawn if larger than the symbol, show SD. The lines represent the best
fit of the data to a first-order equation, with rate constants of
0.10 ± 0.012 min−1 and 0.12 ± 0.006 min−1 for thylakoids isolated from
greenhouse-grown and field-grown plants, respectively, and
0.01 ± 0.003 min−1 and 0.003 ± 0.002 min−1 for leaves of greenhousegrown and field-grown plants, respectively, calculated from the oxygen
evolution data.
Fluorescence intensity, reI. units
Oxygen evolution, % of control
100
and Calophyllum longifolium, growth under near-ambient
UV-B did not markedly affect carotenoid levels but increased
the UV absorbance of ethanolic extracts, when compared
with plants grown under reduced UV-B (Krause et al. 2007).
The finding that the protective mechanism functions outside the thylakoid membrane suggested that the passage of
UV light to chloroplasts is blocked in leaves of field-grown
pumpkin plants. We measured the relative transmittance of
UV-A light in greenhouse-grown and field-grown pumpkin
leaves by illuminating the adaxial surface of the leaf with UV-A
light of very low intensity and measuring Chl fluorescence at
685 nm. The same principle was used earlier by Mazza et al.
(2000). To overcome complications like amount and distribution of Chl in the leaf structure, we used 400–450 nm light as an
internal standard of the fluorescence yield by dividing the fluorescence intensity obtained with 365 nm excitation with the
fluorescence intensity obtained with very dim 400–450 nm
excitation. Fig. 6 shows that the intensity of UV-A-excited Chl
a fluorescence of greenhouse-grown leaves was seven times
higher than fluorescence of field-grown leaves. Thus, chloroplasts of greenhouse-grown leaves are exposed to much higher
UV-A intensity than chloroplasts of field-grown leaves at the
same intensity of sunlight, which explains why the UV part of
sunlight fails to cause photoinhibition in field-grown plants.
The inducible screening compounds are phenolics, flavonoids
and anthocyanins found in cuticle waxes and in vacuoles of
epidermal cells and the upper cell layers of the mesophyll
(Solovchenko and Merzlyak 2008).
Acclimation to UV radiation can be induced by UV radiation
itself (Jenkins 2009). Our greenhouse has ordinary glass windows to let sunlight in and is additionally illuminated with so
called daylight lamps (Philips HPI-T Plus). We estimated the
spectrum of illumination in the greenhouse using the transmission spectrum of standard window glass obtained from the
0.8
0.6
0.4
0.2
0.0
Greenhouse
Field
Growth site
Fig. 6 Demonstration of the presence of UV-absorbing compounds
in field-grown plants. Intensity of 685 nm fluorescence induced by
365 nm illumination in leaves of greenhouse-grown and field-grown
pumpkin plants. The values were normalized by dividing by the
intensity of 685 nm fluorescence obtained with 450 nm excitation.
Plant Cell Physiol. 51(10): 1745–1753 (2010) doi:10.1093/pcp/pcq133 © The Author 2010.
1749
M. Hakala-Yatkin et al.
National Research Council of Canada, http://www.nrc-cnrc.gc.ca,
the standard spectrum of terrestrial sunlight from the
American Society for Testing and Materials and the emission
spectrum of the lamps, obtained from the manufacturer.
Measurements in the greenhouse showed that the contribution of sunlight, passing through the windows, varies between
one-third and two-thirds, and therefore we simulated the
actual illumination spectrum by assuming these two extremes.
In both cases, the simulation shows that the UV-A region is
fairly similar in the greenhouse and in the field, whereas UV-B
radiation is virtually fully absent only in the greenhouse
(Fig. 1D). Thus, it may be that although UV-A is mainly responsible for photoinhibition of PSII, it is UV-B radiation, not UV-A,
that triggers the synthesis of UV-protective substances in
pumpkin. However, the greenhouse illumination is also much
less intense than sunlight, and therefore it is possible that
high light intensity per se contributes to triggering of the
synthesis of UV-protective substances in field-grown plants.
A
100
80
O2 evolution and Fv/FM, % of control
60
1750
20
0
0
1
2
3
4
5
6
5
6
Illumination time, h
B
100
80
60
Photoinhibition in diffuse light mainly affects
the uppermost cell layers
40
20
0
0
1
2
3
4
Illumination time, h
Oxygen evolution, % of control
In addition to measuring photoinhibition as a decrease in
oxygen evolution, we also measured the ratio of variable to
maximum Chl fluorescence (FV/FM) from the same leaves.
With regard to the importance of solar UV radiation, the results
of fluorescence measurements agreed with those of oxygen
evolution, showing that solar UV radiation significantly contributes to photoinhibition in greenhouse-grown plants but
not in field-grown plants (Fig. 7A, B).
During illumination of pumpkin leaves in sunlight, the photoinhibitory decrease in FV/FM occurred much more quickly
than the decrease in oxygen evolution during illumination
(Fig. 7A, B). A large difference was obtained in both
greenhouse-grown and field-grown leaves, under both UVblocking and UV-permeable filter (Fig. 7C). This difference
contrasts with the majority of earlier laboratory data showing
that loss of FV/FM closely approximates loss of oxygen
evolution during photoinhibition of leaves of higher plants
(Krause et al. 1992, Schnettger et al. 1994, Tyystjärvi et al. 1999,
Sarvikas et al. 2010). We hypothesized that diffuse light like
sunlight that mostly hits the leaf at a small angle penetrates
less efficiently to a leaf than collimated light straight from the
top of the leaf. Due to inefficient penetration to lower cell
layers, diffuse light inhibits the inner layers slowly, compared
with the top layer, while collimated light causes more even
photoinhibition throughout the leaf. Thus, Chl fluorescence,
emitted by the topmost chloroplasts, shows faster inhibition in
diffuse light than oxygen evolution measured from thylakoids
isolated from the leaf. To test this hypothesis, we compared
photoinhibition induced by illuminating lincomycin-treated
pumpkin leaves with white light directly from the top to photoinhibition induced by placing a leaf inside an integrating sphere
where illumination is diffuse. Illumination at a 45° angle was
also tested. The results confirmed that in diffuse light, FV/FM
decreases much more quickly than oxygen evolution while
40
80
C
60
40
20
0
0
20
40
60
80
Fv/FM, % of control
Fig. 7 Relationship between loss of oxygen evolution and decrease
in FV/FM during photoinhibition in sunlight. Decrease in oxygen
evolution activity (circles) and FV/FM (stars) during illumination of
lincomycin-treated pumpkin leaves in sunlight under a UV-permeable
cover (open symbols) or under a UV-blocking cover (solid symbols)
(A and B). Leaves were detached from greenhouse-grown (A) and
field-grown pumpkins (B), and oxygen evolution was measured from
thylakoids isolated from treated leaves. The error bars show SD from
two leaves treated simultaneously. Typical results are shown. (C)
Oxygen evolution (% of control) as a function of FV/FM (% of control)
after 3 h of illumination of lincomycin-treated pumpkin leaves
in sunlight under a UV-permeable cover (open symbols) or under
a UV-blocking cover (solid symbols). Squares are results from
greenhouse-grown plants and triangles from field-grown plants.
Oxygen evolution was measured from thylakoids isolated from treated
leaves and FV/FM was measured from the leaf surface.
Plant Cell Physiol. 51(10): 1745–1753 (2010) doi:10.1093/pcp/pcq133 © The Author 2010.
Photoinhibition in sunlight
Table 2 Influence of the direction of incident light on photoinhibition
measured with oxygen evolution or with fluorescence
kPI from
oxygen
evolution
kPI from
FV/FM
kPI obtained from
FV/FM divided by
kPI obtained from
oxygen evolution
90°
0.33 ± 0.03
0.41 ± 0.06
1.26
45°
0.28 ± 0.06
0.25 ± 0.10
0.89
Diffuse light
0.09 ± 0.01
0.17 ± 0.01
1.86
Lincomycin-treated pumpkin leaves were illuminated with collimated light at
a 90° or 45° angle (PPFD 1350 µmol m 2s−1) or with diffuse light (750 µmol m−2 s−1)
obtained by placing the leaf in an integrating sphere. Photoinhibition was
measured as loss of oxygen evolution, measured from thylakoids isolated from
treated leaves, or as decrease in FV/FM, measured from the adaxial leaf surface.
The rate constant of photoinhibition (kPI) was obtained by fitting the decrease
in oxygen evolution or FV/FM to the first-order equation. The values represent the
mean and SD of at least three independent experiments.
illumination with collimated light, either straight from the
top or at a 45° angle, causes approximately equal lowering of
oxygen evolution and FV/FM, when both parameters are compared with the respective control values (Table 2).
In conclusion, our results show that UV radiation is a highly
efficient inducer of photoinhibition. However, comparison of
isolated thylakoids, greenhouse-grown plants and field-grown
plants shows that evolution has equipped land plants with constitutive and inducible UV screens that may fully eliminate the
photoinhibitory power of the UV part of solar radiation.
Materials and Methods
Plant material
Pumpkin plants (Cucurbita pepo L.) were grown in a research
greenhouse at the mean photosynthetic photon flux density
(PPFD) of 150 µmol photons m−2 s−1 in a 16 h light period. The
greenhouse is illuminated by sunlight through ordinary glass
windows on one wall, and additionally with 400 W Philips HPI-T
Plus daylight lamps from the ceiling. Leaves of field-grown
pumpkin plants were obtained from the Botanical Garden of
the University of Turku; the leaves used for the experiments
were collected from an open setting. Thylakoids were isolated
according to Hakala et al. (2005).
Illumination of leaves
Illumination with sunlight. Before illumination, leaves were
incubated in dim light overnight with the petiole in 2.4 mM
lincomycin to inhibit the repair of photoinhibited PSII. For each
photoinhibition experiment in sunlight, four leaves were taped
on styrox tables in horizontal position, with the petioles in lincomycin solution. Two leaves were kept under a 3 mm plate of
UV-permeable acrylic (VS UVT, Altuglas, Le Garenne, France)
and two leaves under a plate from the same acrylic covered
with a UV-protection film (Long Life for Art, Eichstetten,
Germany) (Fig. 1). To measure the effect of UV-B light alone,
the UV-permeable acrylic was covered with a layer of clear
0.0015 mm thick Mylar sheet (DuPont Teijin Films, Chester,
VA, USA). The experiments were done in Turku, Finland
(60°27′ N, 22°17′ E). The leaves were sprayed with water every
30 min to prevent desiccation and to lower the leaf temperature. After 0, 3 and 6 h of illumination, two 3.5 cm2 disks
were cut from every leaf, one for thylakoid isolation and
one for fluorescence measurement. Weather data were
obtained from the weather station of Process Design & Systems
Engineering Laboratory, Åbo Akademi University, Turku
(http://at8.abo.fi/cgi-bin/en/get_weather).
Illumination with UV light. To measure photoinhibition
induced with UV-A light, lincomycin-treated leaves and isolated thylakoid membranes were illuminated with ENF-280C
lamp (Spectronics, Westbury, NY, USA) that emits a wide
spectral peak centered at 365 nm. The lamp was placed at 1 cm
distance from the leaf surface, and the photon flux density of
the UV-A light (measured earlier by Hakala et al. 2005) was
170 µmol m−2s−1. The same lamp at the same distance was used
for illumination of isolated thylakoids (1 ml, 50 µg Chl ml−1).
Illumination with white light. White light was used for comparison of diffuse and collimated light. For illumination with collimated light (PPFD 1350 µmol m−2 s−1), a lincomycin-treated
pumpkin leaf was placed under a high-pressure Xenon lamp
(model 6258; Oriel, Stanford, CT, USA) and illuminated through
a UV-blocking filter. For oblique illumination, the leaf was
placed at a 45° angle to the lamp in otherwise identical conditions. Diffuse light was obtained by placing a leaf inside a 60 cm
diameter integrating sphere illuminated by white light from
a slide projector. A diffusor cone was placed in the input
light path. PPFD, measured from an empty sphere, was
750 µmol m−2 s−1.
Measurements of oxygen evolution
and Chl fluorescence
The light-saturated rate of oxygen evolution was measured
with an oxygen electrode (Hansatech, King's Lynn, UK) as
described by Hakala et al. (2005). Chl concentration was
10 µg ml−1, and 125 µM 2,6-dichlorobenzoquinone was used as
electron acceptor. The ratio of variable to maximum fluorescence (FV/FM) was measured from leaf disks with FluorPen
(PS Instruments, Brno, Czech Republic) after 40 min of dark
incubation.
Quantum yield of photoinhibition
An estimate of the rate constant of photoinhibition was
obtained by fitting the photoinhibitory loss of PSII oxygen
evolution, measured from thylakoids isolated from treated
leaves, to the first-order reaction equation. Each experiment,
with data from two leaves measured after 0, 3 and 6 h of illumination, was fitted separately. The best fit is obtained by taking
into account all three time points (e.g. data in Fig. 2A yield
two rate constant values). The average solar power (kW m−2)
during the experiment was calculated as an average of the
Plant Cell Physiol. 51(10): 1745–1753 (2010) doi:10.1093/pcp/pcq133 © The Author 2010.
1751
M. Hakala-Yatkin et al.
data obtained for every half hour. The relative quantum yield of
photoinhibition was calculated by dividing the rate constant of
photoinhibition by the average solar power during the illumination treatment. Comparisons between the treatments were
done by averaging the quantum yield values from several independent experiments.
Measurement of UV-induced fluorescence
For the measurement of relative UV-A transmission, a leaf
disk was excited with a 365 nm illuminator (LM-26E, Cell
Biociences, Inc., Santa Clara, CA, USA) and the emission spectrum of the leaf in the 600–800 nm range was measured with
an S2000 spectrophotometer (Ocean Optics, Dunedin, FL,
USA). To ensure that the UV-induced fluorescence remained
constantly at the F0 level, the intensity of the UV light was
attenuated until fluorescence yield at 685 nm did not change
when the UV-A light was switched on after placing a darkadapted leaf disk in the measuring position. Eventual differences in leaf Chl concentration and in optical properties of
the leaf samples were compensated for by dividing the amplitude of 685 nm fluorescence by fluorescence excited by a weak
beam of 450 nm light. The 450 nm light was obtained by placing
a LS450 filter (Corion) in front of a slide projector and using
a light guide to illuminate the leaf disk.
Funding
This work was supported by the Academy of Finland and by
the Finnish Cultural Foundation.
Acknowledgements
Taina Tyystjärvi is thanked for important comments on the
manuscript. Kurt Lundqvist (Åbo Akademi University) is
thanked for access to the weather data and Sinikka Vento
(Botanical Garden) for the permission to use their pumpkin
leaves.
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