Plant CellPhysiol. 37(2): 181-187 (1996)
JSPP © 1996
Induction and Repair of Damage to DNA in Cucumber Cotyledons
Irradiated with UV-B
Yuichi Takeuchi1 2, Mina Murakami2, Nobuyoshi Nakajima 3 , Noriaki Kondo 3 and Osamu Nikaido 4
1
2
3
4
Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, Sapporo, 005 Japan
Course of Environmental and Biological Sciences, Graduate School of Science and Engineering, Hokkaido Tokai University,
Sapporo, 005 Japan
Biotechnology Research Team, National Institute for Environmental Studies, Tsukuba, 305 Japan
Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, 920 Japan
Photoinduced lesions in DNA, namely, cyclobutane
pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts [(6-4)photoproducts], in cucumber
cotyledons that had been irradiated with naturally occurring levels of UV-B (290-320 nm) were quantitated by enzyme-linked immunosorbent assays with monoclonal antibodies specific to each type of photolesion. Induction of
these photolesions was dependent on temperature and their
extent was reduced by simultaneous irradiation with white
light. The dark repair of both types of photolesion was undetectable. Light-dependent removal of (6-4)photoproducts was very slow, with 50% removal in 4 h. By contrast,
50% of initial CPDs were removed within 15 min. Both
photorepair processes were dependent on the intensity of
white light and were sensitive to temperature. These results
indicate that high photolyase activity is present in cucumber cotyledons and that repair activities in cucumber cotyledons are different from those reported in Arabidopsis, in
which (6-4)photoproducts are photorepaired more rapidly
than CPDs.
Key words: Cucumber — Cyclobutane pyrimidine dimer —
DNA damage — DNA repair — Monoclonal antibody —
Pyrimidine-(6-4)-pyrimidone photoproduct.
Plants use sunlight for photosynthesis and, as a consequence, they are exposed to the UV radiation that is
present in sunlight. The UV-B region of sunlight has received much attention in recent years because it has been
predicted that a decrease in the ozone layer, as a result of
contamination of the atmosphere by chlorofluorocarbons
(Molina and Rowland 1974), will lead to an increase in the
UV-B radiation (290-320 nm) that reaches the earth's surface (Blumthaler and Amback 1990, Gleason et al. 1993).
UV radiation directly alters the structure of DNA, in
addition to damaging both proteins and membranes. Two
Abbreviations: CPD, cyclobutane pyrimidine dimer; ELISA,
enzyme-linked immunosorbent assay; PBS, phosphate-buffered
saline; PPFD, photosynthetic photon flux density; (6-4)photoproduct, pyrimidine-(6-4)-pyrimidone photoproduct.
major products of the direct damage to DNA are dimers
of adjacent pyrimidines, namely, cyclobutane pyrimidine
dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts t(6-4)photoproducts] (Michell 1988). Studies of
the biological effects of CPDs have been facilitated by the
relative abundance of this type of photolesion, as compared to other types of UV-induced photodamage, and by
the specific recognition in vitro of CPDs by certain prokaryotic endonucleases (Sancar and Sancar 1988). Recent
evidence suggests that another type of UV-induced photolesion in DNA, the (6-4)photoproduct, might also be an important determinant of the lethal and mutagenic effects of
UV on various organisms (for review, see Mitchell and
Nairn 1989).
UV-induced lesions block both the transcription and
the replication of DNA. Therefore, living organisms have
developed specialized mechanisms for the repair of these
photolesions. Studies of Escherichia coli, yeast and mammalian cells have revealed four distinct mechanisms by
which cells cope with UV-induced damage to DNA (for
review, see Sancar and Sancar 1988). Damage due to CPDs
and (6-4)photoproducts is repaired via the nucleotide excision repair pathway, in which a nuclease makes an incision on each side of a lesion and then releases the lesion
from the double helix as a small oligomer. This repair pathway is often referred to as "dark repair" to distinguish it
from light-dependent repair processes, and such a process
remains to be characterized in plants. A mutant of Arabidopsis that is defective in dark repair has, however, recently been isolated (Britt et al. 1993, Chen et al. 1994).
Photorepair directly reverses the dimerization of pyrimidines. The enzyme involved, photolyase, uses energy
from light at wavelengths from 300 to 500 nm. Photolyases
have been purified from several microbial systems and characterized (for review, see Sancar and Sancar 1988), and
a photolyase in E. coli has been shown to be unable to
reverse the formation of (6-4)photoproducts (Brash et al.
1985). A photolyase specific to (6-4)photoproducts has,
however, been identified in cell extracts of Drosophila
(Todo et al. 1993). Photorepair has been reported in many
plant species (see Pang and Hays 1991), but few biochemical and molecular biological investigations of this process
have been reported. The activity of a CPD photolyase in
181
182
Y. Takeuchi et al.
Arabidopsis has been demonstrated and characterized in
vivo and in vitro (Pang and Hays 1991), and the sequence
of a cDNA for a photolyase from white mustard (Sinapis
alba) has also been reported (Batschauer 1993). In addition, photorepair of (6-4)photoproducts has been reported
in an excision repair-deficient mutant of Arabidopsis (Chen
et al. 1994).
As described above, detailed investigations of the
repair of DNA photolesions, in particular of (6-4)photoproducts, in plant cells have been limited to Arabidopsis
(Britt et al. 1993, Chen et al. 1994). Moreover, factors that
affect the repair processes have not been fully defined. In
the present study, using enzyme-linked immunosorbent
assays (ELISAs) with monoclonal antibodies specific to
CPDs and (6-4)photoproducts, we examined the effects of
temperature and white light on the induction and repair of
two types of photolesion after the exposure of cucumber
cotyledons to UV-B irradiation.
Materials and Methods
Plant material—Seeds of cucumber (Cucumis sativus L. cv.
Hokushin) were germinated and seedlings were allowed to develop
on wet paper towels for 5 days at 25°C in darkness. Cotyledons
were excised from the seedlings and five of them were placed on
filter paper on the bottom of a 5-cm-wide, stainless-steel Petri
dish. Two ml of 20 mM potassium phosphate buffer (pH 6.0) containing 20 mM KC1 and 100//M zeatin were added to each dish as
the growth medium (Takeuchi and Amino 1984, Takeuchi et al.
1985, 1993). Each dish was covered with a UV-transmitting filter
(UV 28, 5 x 5 cm2; Hoya Co. Ltd., Tokyo, Japan) to filter out
almost completely all radiation at wavelengths below 290 nm and
then the dish was sealed with Parafilm™ (American National
Can, CT., U.S.A.).
Irradiation with UV-B—Cotyledons were irradiated with UVB at 25°C. UV light was supplied by a fluorescent sunlamp (FL
20SE; Toshiba, Tokyo, Japan), which was suspended 35 cm
above the dishes. The spectral UV flux density was measured with
a double monochromator spectroradiometer (PGD-25C; Japan
Spectroscopic Co. Ltd., Tokyo, Japan). The spectral distribution
of UV radiation is shown in Figure 1A. In the case of simultaneous irradiation with white light and UV-B together, two coolwhite fluorescent lamps (FL 20SS-BRN/18; Toshiba) were located
on each side of the sunlamp. The photosynthetic photon flux density (PPFD) was 125 j/mol m~2 s" 1 .
Samples to be repaired were exposed to UV-B for 15min
without white light, as described above, and then they were incubated in darkness or under white light (PPFD, 110/wnol m~2 s~')
at 25°C. After reactivation for various periods of time, cotyledons
were frozen in liquid nitrogen and then lyophilized.
Extraction of DNA— DNA was extracted from the cotyledons by the procedure of Honda and Hirai (1990) with slight modifications. Five freeze-dried cotyledons were homogenized in 0.6
ml of extraction buffer (100 mM Tris-HCl buffer, pH 8.0, containing 50 mM EDTA, 500 mM NaCl and 10 mM 2-mercaptoethanol)
in a glass homogenizer. The homogenate was transferred to a 1.5ml microtube, 80^1 of 10% (w/v) SDS were added, and the tube
was incubated at 65 °C for 10 min. Then 200 /A of 5 M potassium
acetate were added and the mixture was incubated on ice for 20
min. After centrifugation at 15,000 x g for 20 min at 0°C, the
350
400
10
1
I
!
4-
/\
2 -
\
.1.. /
300
400
500
J
600
\
700
Wavelength (nm)
Fig. 1 Spectral distributions of the UV light and white light employed for the induction and photorepair of photolesions.
(A), Cucumber cotyledons were irradiated with UV light with
(solid line) or without (dashed line) white light. (B), In the experiments to examine photorepair, cotyledons that had been irradiated with UV light were incubated under white light.
supernatant was passed through one layer of Kimwipe™ (Jujo
Kimberly K. K., Tokyo, Japan) and collected in a new microtube,
mixed with 400 /A of isopropanol and incubated on ice for 15 min.
After centrifugation at 15,000 x g for 15 min at 0°C, the pellet was
dissolved in 400 /A of 10TE (50 mM Tris-HCl buffer, pH 8.0, containing 10 mM EDTA) and centrifuged at 15,000 x g for 20 min at
0°C. The supernatant was transferred to a new microtube, supplemented with 10/A of a solution of RNase A (DNase-free; Nippon
Gene Co., Ltd., Toyama, Japan; 1.0 mg ml" 1 ) and incubated at
room temperature for 30 min. Then the sample solution was extracted with 400/il of a mixture of phenol and chloroform ( 1 : 1 ,
w/v). The aqueous phase was transferred to another microtube
and 240 |il of a 20% (w/v) solution of polyethylene glycol 6,000
(Wako Pure Chemical Ind., Ltd., Osaka, Japan) containing 2.5 M
NaCl were added. After incubation for 15 min at room temperature, the mixture was centrifuged at 15,000xg for 10 min. The
pelleted DNA fraction was rinsed twice with 500 ftl of 70% (v/v)
ethanol by centrifugation at 15,000xg for 10 min and then dried
in a vacuum desiccator.
Determination of DNA content—Since the yield of DNA in
the procedure described above was not consistent, the DNA content of cucumber cotyledons was determined by the method of
Naito et al. (1978). Ten cotyledons were homogenized in 10 ml of
10% (w/v) trichloroacetic acid (TCA) in a glass homogenizer.
After centrifugation of the homogenate at 2,000Xg for 10 min,
the pellet was washed with 7% TCA and then with 95% (v/v) ethanol. The residue was suspended in a mixture of ethanol and
UV-B-induced DNA damage and repair mechanisms
diethyl ether ( 2 : 1 , v/v) and incubated at 50°C for 20 min. This
treatment was repeated once more to decolorize the residue completely. The residue was then suspended in 5 ml of 0.3 M KOH
and incubated at 30°C overnight. After neutralization with 3 M
HC1, 60% (w/v) perchloric acid (PCA) was added to the mixture
to a final concentration of 5% (w/v). The mixture was centrifuged
to remove RNA, and the residue was digested with 3 ml of 5%
PCA at 90°C for 10 min. The DNA in the supernatant obtained
by centrifugation was quantitated by the method of Giles and
Myers (1965), with calf thymus DNA as the standard.
Irradiation of standard DNA with monochromatic UV light
—For preparation of standard DNA for ELISA of CPDs and
(6-4)photoproducts, DNA from bacteriophage lambda (Takara
Shuzo Co., Ltd., Kyoto, Japan) was irradiated with monochromatic UV light at 260 nm (0-40 Jm~ 2 ) at the Okazaki
Large Spectrograph (OLS) of the National Institute for Basic Biology (Okazaki, Japan).
Two ml of a solution of ADNA (50 n% ml" 1 in PBS) were placed in a 5-cm-wide, stainless-steel Petri dish and irradiated vertically via the front surface of an aluminum mirror oriented at an
angle of 45 °. The fiuence rate was measured with a photon density
meter (HK-1, custom-made at the Institute for Physical and Chemical Research, Wako, Japan; Hashimoto et al. 1982). The design
and performance of the OLS were described in detail by Watanabe et al. (1982).
ELISA—CPDs and (6-4)photoproducts were detected with
the TDM-2 and 64M-2 monoclonal antibodies, respectively. The
source and specificity of each monoclonal antibody have been described in detail elsewhere (Matsunaga et al. 1993, Mizuno et al.
1991, Mori et al. 1988, 1991). The TDM-2 antibody binds 5-TT-3'
and 5'-CT-3' cyclobutane dimers, while the (6-4)photoproducts
formed in 5-TT-3'and 5-TC-3'sequences can be detected by 64M2. A direct ELISA, adapted to include a streptavidin-biotin system, was performed by the method of Matsunaga et al. (1991).
DNA was dissolved in 1.0 ml of PBS, and its concentration
was determined by measuring the absorbance at 260 nm. Samples
of DNA were denatured by heating for 10 min in boiling water
with subsequent rapid cooling in an ice bath. Fifty /A of the solution of DNA (0.05 jig m P 1 for TDM-2; 2.0 fig ml" 1 for 64M-2)
were placed in the wells of polyvinylchloride flat-bottom microtiter plates (Dynatech, Alexander, VA), that had been precoated
with 1% (w/v) protamine sulfate (50/il well"1), and plates were incubated at 37°C overnight. After drying, the plates were washed
four times with PBS that contained 0.05% (v/v) Tween 20 (PBST), and then with 1% (v/v) newborn calf serum in PBS (150/il
well"1) at 37CC for 60 min to prevent non-specific binding of the
antibody, and then they were washed again. One hundred fi\ of a
solution of monoclonal antibody, diluted 1 : 1,000 in PBS-T, were
added to wells, and plates were incubated at 37°C for 60 min.
Then plates were treated with the biotinylated F(ab')2 fragment of
antibodies against mouse IgG (1 : 1,000 in PBS-T; Zymed, San
Francisco, CA), and then with streptavidin-peroxidase conjugate
(1 : 10,000 in PBS-T; Zymed). Finally, after three washes with
PBS-T and two subsequent washes with citrate-phosphate buffer
(pH 5.0), 100^1 of substrate solution, consisting of 0.04% (w/v)
o-phenylene diamine and 0.007% (v/v) H2O2 in citrate-phosphate
buffer, were added to each well. After a 15-min incubation at
37°C, 50 jd of 2 M H2SO4 were added to stop the reaction and the
absorbance at 490 nm was measured. The background value was
subtracted from the average absorbance in each of four identically
prepared wells.
183
Results
Induction of CPDs and of (6-4)photoproducts by UVB—Rates of induction of CPDs and (6-4)photoproducts in
cucumber cotyledons that had been irradiated with UV-B
at wavelengths longer than 290 nm, with or without white
light, are shown in Figure 2. The amounts of CPDs and (64)photoproducts increased at an almost constant rate for at
least the first 15 min of UV-B irradiation. Simultaneous irradiation with white light considerably reduced the levels
of photolesions, but the extent of such a reduction differed
between CPDs and (6-4)photoproducts. The extent of reduction of CPDs was about 50% while that of the (6-4)photoproducts was less than 20%.
The effects of temperature on the rates of induction of
the photolesions are shown in Figure 3. At 15°C, the rates
of induction of photolesions were similar to those observed
when cotyledons were cooled on ice. However, at higher
temperatures up to 35°C, the rates of induction increased
slightly as the temperature was raised. The difference between the amount of CPDs formed upon simultaneous irradiation by white light and upon irradiation without white
light, which is presumed to reflect the activity of the photorepair process, was largest at 25 °C.
Dark repair of CPDs and the (6-4)photoproducts—In
darkness, the levels of both types of photolesion were reduced to approximately 50% of the initial levels after a 24h incubation (Fig. 4). The DNA content of etiolated cucum-
-10
10
20
30 0
10
20
30
Duration of irradiation (min)
Fig. 2 Effects of simultaneous irradiation with white light (WL)
on the induction of photolesions by UV-B.
Cucumber cotyledons were irradiated with UV-B for various periods of time at
C
25 C with (o) or without (•) white light. DNA was extracted from
the cotyledons and the relative amounts of CPDs and (6-4)photoproducts were determined by ELISA with monoclonal antibodies
TDM-2 and 64M-2, respectively. Amounts are expressed as equivalents to damage to ADNA caused by the indicated fiuence of UV
light at 260 nm.
Y. Takeuchi et al.
184
100
-50
on Ice 15
25
35 on Ice 15
35
Temperature (%)
30
Fig. 3 Effects of temperature on the induction of photolesions.
The dishes in which cotyledons had been placed were set on ice or
in a growth cabinet temperatures from 15 to 35°C, and the cotyledons were irradiated with UV-B for 15 min with (O) or without (•)
white light.
ber cotyledons was 22.8 ± 5.4//g per cotyledon (average
from four separate samples±SD), and it increased 1.8-fold
after a 24-h incubation in darkness. From these results, we
concluded that the decrease in relative levels of photolesions in darkness was not due to the actual removal of
photolesions but to the synthesis of DNA de novo. It was
60
90
120
PPFD(ymolm" 2 s -1 )
Fig. 5 Effects of the intensity of white light on photorepair.
Cotyledons were irradiated with UV-B for 15 min without simultaneous irradiation with white light and then they were incubated
under white light at various intensities at 25°C for 30 min and for
6 h for quantitation of CPDs (o) and (6-4)photoproducts (D), respectively.
also apparent that the rate of dark repair of both CPDs
and (6-4)photoproducts, presumably via excision repair,
was low or undetectable in cucumber cotyledons.
100
CPDS
(6-4)photoproducts
O
100
o"o
-^ 80 ti
o
dark
3
2 60
o
O
t
1
1
o
' light
°\
I
o
20 -
o
o,
H
""••9
—/t
15
1—
24 0
24
20
25
30
35
Tempera ture(°C)
Time (h)
Fig. 4 Time course of repair of photolesions.
Cotyledons
were irradiated with UV-B for 15 min at 25 °C without simultaneous irradiation with white light and then they were incubated in
darkness (•) or in the light (PPFD, 110/imol m~ 2 s~', O). DNA
was extracted at various times during the repair period.
Fig. 6 Effects of temperature on photorepair.
Cotyledons
were irradiated with UV-B for 15 min without simultaneous irradiation with white light and then they were incubated under white
light (PPFD, 110/imol m~ 2 s~') at various temperatures for 30
min and for 6 h for quantitation of CPDs (o) and (6-4)photoproducts (•), respectively.
UV-B-induced DNA damage and repair mechanisms
Photorepair of CPDs and (6-4)photoproducts—The
rates of repair of both types of photolesion were significantly enhanced by exposure to white light (Fig. 4), but the rate
of this enhancement differed between CPDs and the (6-4)photoproducts. CPDs were eliminated much faster than
the (6-4)photoproducts, with approximately 50% of the initial CPDs being repaired within the first 15 min after irradiation. The light-dependent removal of the (6-4)photoproducts was much slower, with 50% removal in 4 h. The rates
of photorepair of both types of lesion were dependent on
the intensity of white light (Fig. 5). Although the duration
of the necessary incubation differed between the photorepair of CPDs and that of (6-4)photoproducts, the former
being 30 min and the latter being 6 h, the curves showing
the dependence of repair activity on the intensity of white
light resembled one another for the two types of photolesion. The effects of temperature on the photorepair of
CPDs and of the (6-4)photoproducts are shown in Figure
6. For both types of photolesion, high repair activity
was observed at 25-30°C. At lower (15-20°C) and higher
(35°C) temperatures, both photorepair activities were
much lower.
Discussion
Although DNA damage induced by UV-C (wavelengths below 280 nm) is not physiologically relevant to
plants that grow in sunlight, irradiation with UV-C from
germicidal lamps has often been used to study UV-induced
damage to DNA in animals and bacteria, as well as in
plants. However, since numerous results have been reported indicating that the .responses to UV-C irradiation
are different, both quantitatively and qualitatively, from
those to UV-B, responses to UV-C cannot be considered to
provide a model of the physiological responses to UV-B
(Stapleton 1992). Furthermore, the lower limit of wavelengths of solar UV radiation that reaches the earth's surface is generally considered to be approximately 290 nm
(Caldwell 1971). Therefore, in the present study, we examined DNA damage induced by UV-B irradiation at
wavelengths longer than 290 nm. In the present study,
plants were irradiated at 300 nm at a rate of 12.7 mW m~2
s~' nm" 1 (Fig. 1A), which was almost equal to the highest
recorded value for solar UV radiation at Tsukuba (36°N
latitude) in 1992 (12.0mWm~ 2 s~' nm" 1 , at noon on
August 8; Annual Report of Ozone Layer Monitoring,
1994, Japan Meteorological Agency). Thus, substantial
damage to DNA was induced by a naturally occurring level
of UV-B.
Among the various available methods for quantitation
of photolesions, the immunological method is the only one
that allows detection of both CPDs and (6-4)photoproducts that have been formed in DNA as a consequence
of UV irradiation at physiological doses (Mitchell et al.
185
1985a, b) because of the low yield, in particular, of the latter lesions. In the present study, the levels of photolesions
were expressed in terms of equivalence to lesions induced
by a given fluence of UV irradiation at 260 nm in ADNA.
Since the immunological assays were indirect assays, we
could not determine the actual yields of photolesions in
DNA prepared from cucumber cotyledons that had been irradiated with UV-B. However, assuming that the rate of induction of CPDs corresponds to 0.003% of the total number of thymine residues per J m~2 (unpublished data) and
that the rate of induction of the (6-4)photoproducts is one
third of that of CPDs (Mitchell 1988), we can calculate that
the amounts of CPDs and (6-4)photoproducts equivalent
to those produced at 1 J m~2 of UV light at 260 nm (Fig. 2,
3) were 11 x 10"18 and 3.8 x 10"18 mol (ng DNA)" 1 , respectively (Mori et al. 1991).
Dark repair of (6-4)photoproducts has been reported
to occur much more rapidly than that of CPDs in Arabidopsis, with approximately 50% of the initial lesions being
eliminated within 2 h (Britt et al. 1993). Therefore, Arabidopsis resembles other biological systems in that the rate of
dark repair of CPDs is low or undetectable, whereas the
(6-4)photoproducts are rapidly removed (Mitchell et al.
1985a, Mitchell and Nairn 1989). In cucumber cotyledons,
damage due to CPDs and (6-4)photoproducts was repaired
at a low or undetectable rate in darkness (Fig. 4).
In Arabidopsis, (6-4)photoproducts were repaired
more rapidly than CPDs in the light (Chen et al. 1994).
Moreover, high activity of CPD photorepair has been reported in alfalfa seedlings grown outdoors (Takayanagi
et al. 1994). In cucumber cotyledons, photorepair of CPDs
was very rapid, with 50% removal in 15 min (Fig. 4), and
the rate of photorepair was dependent on both the intensity
of white light (Fig. 5) and the temperature (Fig. 3, 6), suggesting that high activity of a CPD photolyase is present in
etiolated cucumber cotyledons. These results indicate that
the activities for dark repair and photorepair of CPDs and
(6-4)photoproducts are different in Arabidopsis and cucumber, and they suggest the importance of further examinations of DNA repair in many other plant species.
A new enzyme that specifically catalyzes the photorepair of the (6-4)photoproducts was recently identified in
Drosophila (Todo et al. 1993). However, the mechanism of
the photorepair of (6-4)photoproducts has not yet been
characterized in any plant. In cucumber cotyledons, the dependence on light intensity and the sensitivity to temperature of the photorepair of the (6-4)photoproducts were
similar to those of the photorepair of CPDs (Fig. 5, 6),
suggesting that the (6-4)photoproducts might be photorepaired by a process similar to that involved in photorepair of CPDs, with photolyase playing an important role.
The wavelength at which UV light most efficiently
induces formation of CPDs and (6-4)photoproducts is
around 260 nm, and (6-4)photoproducts are converted to
186
Y. Takeuchi et al.
the Dewar isomers by UV light at longer wavelengths.
Moreover, photoisomerization is efficiently induced by UV
light at 310-340 nm, with a peak at about 320 nm (Matsunaga et al. 1991). Therefore, the Dewar isomers of the (6-4)photoproducts might play an important role in cytotoxicity
and in the generation of mutations in cells exposed to the
UV-A and UV-B in sunlight. In the present experiments,
the light source employed for the induction of photolesions
emitted UV light that is effective for photoisomerization
(Fig. 1A). However, significant photoisomerization is only
observed after extended irradiation at high fluences of UVB (Matsunaga et al. 1993). Thus, significant conversion of
the (6-4)photoproducts to their Dewar isomers during UVB irradiation probably did not occur. Nonetheless, there
remains the possibility that some (6-4)photoproducts might
have been converted to Dewar isomers during photoreactivation under white light. In fact, the decrease in the
amounts of (6-4)photoproducts in the light was consistent
with that of CPDs between 15-25CC but was larger at 3035°C (Fig. 6), suggesting that there might exist a non-enzymatic process, separate from the photorepair, that is catalyzed by an enzyme similar to photolyase.
In addition to the predicted higher levels of UV-B
radiation, increases in temperature due to the greenhouse
effect can also be anticipated in our future environment. Although DNA-repair activity must be dependent on various
environmental conditions, such as temperature and plant
growth conditions, there have been only a few reports on
this subject. The dependence on temperature of photorepair of CPDs has been reported in Arabidopsis (Pang
and Hays 1991). The present study showed that photorepair of CPDs, as well as that of (6-4)photoproducts, in cucumber cotyledons was sensitive to temperature. In alfalfa
seedlings, the repair mechanisms have been reported to be
determined by initial levels of CPDs (Quaite et al. 1994),
and rates of induction and photorepair of CPDs differ
between seedlings grown outdoors and those grown in a
UV-free environmental chamber (Takayanagi et al. 1994).
Moreover, it has also been reported that photolyase activity changes during the growth of plants (Pang and Hays
1991, Saito and Werbin 1969). In addition to temperature,
numerous environmental factors are known to weaken or
enhance the responses of plants to UV radiation (Tenunura
1983). Therefore, it is important to investigate the factors
that affect DNA-repair activity if we are to characterize the
likely effects on plants of the increased UV-B radiation that
will be a consequence of depletion of the ozone layer.
The irradiation with monochromatic UV light was carried out
under the Cooperative Research Program of the National Institute
for Basic Biology for the Okazaki Large Spectrograph (95-521).
The authors thank Dr. M. Watanabe and Mr. M. Kubota, National Institute for Basic Biology, for help with the UV irradiation.
References
Batschauer, A. (1993) A plant gene for photolyase: an enzyme catalyzing
the repair of UV-light induced DNA damage. Plant J. 4: 705-709.
Blumthaler, M. and Amback, W. (1990) Indication of increasing solar UVB radiation flux in alpine regions. Science 248: 206-208.
Brash, D.E., Flanklin, W.A., Sancar, G.B., Sancar, A. and Haseltine,
W.A. (1985) E.coli DNA photolyase reverses cyclobutane pyrimidine
dimers but not pyrimidine-pyrimidone (6-4) photoproducts. J. Biol.
Chem. 260: 11438-11441.
Britt, A.B., Chen, J.-J., Wykoff, D. and Mitchell, D. (1993) A UV-sensitive
mutant of Arabidopsis defective in the repair of pyrimidine-pyrimidinone(6-4) dimers. Science 261: 1571-1574.
Caldwell, M.M. (1971) Solar UV irradiation and the growth and development of higher plants. In Photophysiology. Edited by Giese, A.C. pp.
131-177. Academic Press, New York.
Chen, J.-J., Mitchell, D.L. and Britt, A.B. (1994) A light-dependent pathway for the elimination of UV-induced pyrimidine (6-4) pyrimidinone
photoproducts in Arabidopsis. Plant Cell 6: 1311-1317.
Giles, K.W. and Myers, A. (1965) An improved diphenylamine method for
estimation of deoxyribonucleic acid. Nature 206: 93.
Gleason, J.F., Bhartia, P.K., Herman, J.R., McPeters, R., Newman,
P., Stolarski, R.S., Flynn, L., Labow, G., Larko, D., Seftor, C ,
Wellenmeyer, C , Komhyr, D., Miller, A.J. and Planet, W. (1993)
Record low global ozone in 1992. Science 260: 523-526.
Hashimoto, T., Yatsuhashi, H. and Kato, H. (1982) A high-sensitivity photon density meter for monochromatic lights. Abstracts Annu. Meeting,
Jap. Soc. Plant Physiol. p. 38.
Honda, H. and Hirai, A. (1990) A simple and efficient method for identification of hybrids using nonradioactive rDNA as probe. Jap. J. Breed.
40: 339-348.
Matsunaga, T., Hatakeyama, Y., Ohta, M., Mori, T. and Nikaido, O.
(1993) Establishment and characterization of a monoclonal antibody
recognizing the Dewar isomers of (6-4)photoproducts. Photochem. Photobiol. 57: 934-940.
Matsunaga, T., Hieda, K. and Nikaido, O. (1991) Wavelength dependent
formation of thymine dimers and (6-4)photoproducts in DNA by monochromatic ultraviolet light ranging from 150 to 365 nm. Photochem.
Photobiol. 54: 403-410.
Mitchell, D.L. (1988) The relative cytotoxicity of (6-4)photoproducts and
cyclobutane dimers in mammalian cells. Photochem. Photobiol. 48: 5157.
Mitchell, D.L., Haipek, C.A. and Clarkson, J.M. (1985a) (6-4)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more
efficiently than cyclobutane pyrimidine dimers. Mutat. Res. 143: 109112.
Mitchell, D.L., Haipek, C.A. and Clarkson, J.M. (1985b) Further characterization of a polyclonal antiserum for DNA photoproducts: the use of
different labelled antigens to control its specificity. Mutat. Res. 146: 129—
133.
Mitchell, D.L. and Nairn, R.S. (1989) The biology of the (6-4) photoproduct. Photochem. Photobiol. 49: 805-819.
Mizuno, T., Matsunaga, T., Ihara, M. and Nikaido, O. (1991) Establishment of a monoclonal antibody recognizing cyclobutane-type thymine
dimers in DNA: a comparative study with 64M-1 antibody specific for
(6-4)photoproducts. Mutat. Res. 254: 175-184.
Molina, M.J. and Rowland, F.S. (1974) Stratospheric sink for chlorofluoromethanes: chlorine atom-catalysed destruction of ozone. Nature 249:
810-812.
Mori, T., Matsunaga, T., Hirose, T. and Nikaido, O. (1988) Establishment
of a monoclonal antibody recognizing ultraviolet light-induced (6-4)photoproducts. Mutat. Res. 194: 263-270.
Mori, T., Nakane, M., Hattori, T., Matsunaga, T., Ihara, M. and
Nikaido, O. (1991) Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6-4)photoproduct from the same mouse immunized with ultraviolet-irradiated
DNA. Photochem. Photobiol. 54: 225-232.
Naito, K., Tsuji, H. and Hatakeyama, I. (1978) Effects of benzyladenine
on DNA, RNA, protein, and chlorophyll contents in intact bean leaves:
differential responses to benzyladenine according to leaf age. Physiol.
UV-B-induced DNA damage and repair mechanisms
Plant. 43: 367-371.
Pang, Q. and Hays, J.B. (1991) UV-B-inducible and temperature-sensitive
photoreactivation of cyclobutane pyrimidine dimers in Arabidopsis
thaliana. Plant Physiol. 95: 536-543.
Quaite, F.E., Takayanagi, S., Ruffini, J., Sutherland, J.C. and
Sutherland, B.M. (1994) DNA damage levels determine cyclobutyl
pyrimidine dimer repair mechanisms in alfalfa seedlings. Plant Cell 6:
1635-1641.
Saito, N. and Werbin, H. (1969) Radiation spectrum for a DNA-photoreactivating enzyme isolated from higher plants. Radiat. Bot. 9: 421-424.
Sancar, A. and Sancar, G.B. (1988) DNA repair enzymes. Annu. Rev. Biochem. 57: 29-67.
Stapleton, A.E. (1992) Ultraviolet radiation and plants: burning questions.
Plant Cell 4: 1353-1358.
Takayanagi, S., Trunk, J.G., Sutherland, J.C. and Sutherland, B.M.
(1994) Alfalfa seedlings grown outdoors are more resistant to UVinduced DNA damage than plants grown in a UV-free environmental
chamber. Photochem. Photobiol. 60: 363-367.
Takeuchi, Y. and Amino, S. (1984) Analysis of UDP-sugar content in cu-
187
cumber cotyledons in relation to growth rate. Plant Cell Physiol. 25:
1589-1593.
Takeuchi, Y., Ikeda, S. and Kasahara, H. (1993) Dependence on
wavelength and temperature of growth inhibition induced by UV-B irradiation. Plant Cell Physiol. 34: 913-917.
Takeuchi, Y., Saito, M., Kondo, N. and Sugahara, K. (1985) Inhibition of
zeatin-induced growth of cucumber cotyledons by sulfite ions. Plant Cell
Physiol. 26: 123-130.
Teramura, A.H. (1983) Effects of ultraviolet-B radiation on the growth
and yield of crop plants. Physiol. Plant. 58: 415-427.
Todo, T., Takemori, H., Ryo, H., Ihara, M., Matsunaga, T., Nikaido,
O., Sato, K. and Nomura, T. (1993) A new photoreactivating enzyme
that specifically repairs ultraviolet light-induced (6-4)photoproducts.
Nature 361: 371-374.
Watanabe, M., Furuya, M., Miyoshi, Y., Inoue, Y., Iwahashi, I. and
Matsumoto, K. (1982) Design and performance of the Okazaki Large
Spectrograph for photobiological research. Photochem. Photobiol. 36:
491-498.
(Received October 13, 1995; Accepted December 28, 1995)