Effects of temperature on UV-B-induced DNA damage and photorepair in Arabidopsis
thaliana
Shaohua (given name) Wang (family name)1,2, Ping Wang2,*
1. Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085,
China. E-mail: jesc@263.net (first author’s email address)
2. School of Life Sciences and Technology, Jinan University, Guangzhou 510632, China
Received 06 November 2006; revised 12 December 2006; accepted 24 January 2007
Abstract: DNA damage in the form of cyclobutane pyrimidine dimers (CPDs)
(Abbreviations should be explained when it is shown first time in following three parts:
Abstract, main text part, and figure and table. BTW, if it only shown once in the
Abstract, it is not necessary using abbreviation in the Abstract) and photoproducts (6--4
PPs) induced by UV-B radiation in Arabidopsis thaliana at different temperatures was
investigated using the technologies with specific monoclonal antibodies. CPDs and 6--4 PPs
increased during 3 hr UV-B exposure, but further exposure led to decreases. Contrary to the
commonly accepted view that DNA damage induced by UV-B radiation is temperatureindependent because of its photochemical nature, we found UV-B-induction of CPDs and 6-4 PPs in Arabidopsis to be slower at a low than at a high temperature. Photorepair of CPDs at
24℃ was much faster than that at 0 and 12℃, with 50% CPDs removal during 1 hr exposure
to white light. Photorepair of 6--4 PPs at 12℃ was very slow as compared with that at 24℃,
and almost no removal of 6--4 PPs was detected after 4 hr exposure to white light at 0℃.
There was evidence to suggest that temperature-dependent DNA damage and photorepair
could have important ecological implications.
Key words: cyclobutane pyrimidine dimers; Arabidopsis thaliana; DNA repair
---------------------------------*
Corresponding author. E-mail: jesc@263.net
Introduction
There is growing awareness of the potential biological effects of stratospheric ozone
depletion as part of global climatic change (Leun et al., 1995 (three or more authors). The
most important consequence of the ozone depletion is an increase in the amount of UV-B
1
(280--315 nm) radiation reaching the surface of earth. Even modest increase in UV-B
radiation is likely to cause significant biological damage (Björn et al., 1999a)). Investigations
during the past two decades have demonstrated that UV-B radiation has many direct and
indirect effects on plants, including damage to DNA. The most common DNA lesion caused
by exposure to UV-B is the formation of dimers between adjacent pyrimidines in the same
strand, i.e., the cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6--4) pyrimidinone
adducts (6--4 PPs) (Britt, 1996 (one author)). Unrepaired dimers are leathal to cells because
they deform the DNA helix, interfering with both replication and transcription. Both types of
DNA damage can be reversed by subsequent exposure to radiation ranged from 360--420 nm
(UV-A to blue light). This phenomenon is termed photoreactivation or photorepair and is due
to the actions of one or more proteins tremed “photolysis”. These enzymes specifically
recognize and bind to pyrimidine dimers (Britt and Mori, 1999 (two authors)).
DNA damage and repair has been investigated in several plant species (Mitchell et al.,
1993; Pang et al., 1991; Takeuchi, 1996; Taylor, 1996; Björn et al., 1996, 1999), but
information on the effects of environmental factors such as temperature is limited. Interaction
of increased solar UV-B radiation with other climatic change factors such as global warming
is scarce.
In the present study, we examined the temperature effects on the formation and photo
repair of DNA damage induced by UV-B radiation in Arabidopsis thaliana.
1 Materials and methods
1.1 Soil samples
Soil samples were collected from surface layer (0-20 cm in depth) of a paddy field
located in Sumen Village, Binjiang Country, Guixi City, Jiangxi Province, central subtropical
China (N 28°20.307′ and E 117°14.133′). This about 260 hm2 paddy fields have been
contaminated with Cu and Cd by sewerage from an adjacent smelting factory for more than
20 years (Hu et al., 2004). The paddy soil was developed from red sandstone with 13.0% clay,
40.5% silt and 46.5% sand, and the main properties of soil are shown in Table 1. The total
concentrations of Cu and Cd significantly exceed the environmental quality standard for
agricultural soils (Cu 50 mg/kg and Cd 0.3 mg/kg in GB 15618-1995) issued by State
Environmental Protection Administration of China. Unfortunately, rice is still planted on the
contaminated soils by local farmers due to drive of compensation mechanism and poverty.
Table 1 Properties of the soil used in the greenhouse experiment
2
Property
Value
pH
7.9
Organic matter content (g/kg)
14.4
Total N (g/kg)
1.1
Olsen-P (mg/kg)
19.0
NH4OAc-extractable K (mg/kg)
85.2
Total Cd (mg/kg)
0.08
Total As (mg/kg)
5.5
1.2 Plant material and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used in all experiments. Seeds
were surface-sterilized with 75% ethanol, rinsed with water, and incubated for 2 days at 4℃,
then distributed in commercial mixture medium and covered with glass for 48 hr to ensure
high humidity for an even germination. After growing for 10 days, young plants were
transplanted to 6 cm × 6 cm plastic pots (5 plants in each pot) and grown in a greenhouse
under 800 µmol/(m2·sec) photosynthetically active radiation (PAR, 400--700 nm), supplied
by 400 W dysprosium lamps (Osram Powerstar, Germany). Spctrum of this type of
dysprosium was shown in our former paper (Li, 2002).
Fully expanded leaves were used as plant materials. Detached leaves with abaxial
surface up floating on distilled water in Petri dish, were exposed to UV-B radiation at a
distance of 20 cm from the lamps, and without any other illumination.
1.3 UV-B and white light irradiation
UV-B radiation (also containing UV-A) was obtained from 6 UVB-313 lamps (QPANEL, USA) and filtered through 0.13 mm cellulose diacetate. All radiation below 280 nm
was filtered out. Measurement of spectral irradiance was same as our privious report (Li,
2002b). Irradiance of the UV-B region (280--315 nm) was 2.95 W/m2. White light, 150 W/m2
in the interval 400--700 nm, used for photorepair experiments, was supplied by a 400 W lamp
(Osram Powerstar, Germany) and filtered through a 10 cm depth of water in a transparent
polystyrene container to remove excess infrared radiation. Radiation measurements were
carried out with a model 754-6S spectroradiometer (Optronic Laboratories, USA). Spectral
irradiances of UV-B and white light for photorepair experiments were shown in our privious
report (Li, 2002).
In the isotherm experiments, 4 g of samples were mixed with 100 mL Pb 2+ solution (80,
400, 800, 2000 and 4000 mg/L) at 200 r/min and 25℃ for the equilibrium time. To gain a
3
comprehensive knowledge of the Pb2+ adsorption process, the adsorbents included S, P, SP1
(0, 2, 4, 6, 9 and 10 days), SP2 (10 days) and SP3 (10 days). The Pb2+ solution after
adsorption was collected and measured using the method mentioned above, with each
treatment being in triplicates. The total number of treatments in the experiment was 150
(5×10×3). The Pb2+ adsorbed amount (Q) was calculated by Eq. (1): (write equations
reactions, formulas using MathType)
Q  (C0  C ) V / m
(1)
where, C0 (mg/L) and C (mg/L) are the initial and final Pb2+ concentrations, respectively; V (L)
is the solution volume in the flask, m (g) is the dry mass of the absorbent, including S, P, and
SP.
The foregoing steps were repeated four times to obtain the irreversible parameter of
adsorption-desorption (H), which was calculated by Eq. (2) (Yang and Zhang, 2007):
H
bdes
bads
(2)
where, H is the irreversible parameter of adsorption-desorption, and bdes and bads are
adsorption constant and desorption constant, respectively.
2 Results and discussion (results and discussion can be written separately)
2.1 UV-B induced DNA damage in Arabidopsis thaliana
Arabidopsis thaliana plants grown in greenhouse were exposed to UV-B radiation in
dark room at 24℃. Both types of dimeric pyrimidine photoproducts were induced in plant
leaves. The CPDs content of leaves increased during 3 hr UV-B exposure and a smaller
increase of 6--4 PPs was observed (Fig. 1). Further exposure of UV-B radiation led to
decrease of both types of DNA damage. The decrease is probably due to photorepair activity
driven by the UV-A radiation supplied together with the UV-B. It was deduced from our
result that A. thaliana, as quatitified by dimer formation in DNA, was very sensitive to UV-B
radiation.
----------------------------------
---------------------------------Fig. 1 UV-B induced DNA damage in leaves of Arabidopsis thaliana. Plants were irradiated with UV-B.
2.2 Effect of temperature on DNA damage
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Figure 2 shows that UV-B-induced DNA damage in A. thaliana depends on
temperature. When detached leaves were exposed to UV-B radiation for 2 hr at 12 and 24℃,
more CPDs and 6--4 PPs accumulated than at 0℃ (t-test, P < 0.01) (Fig. 2a), but the
difference of 6--4 PPs formation between at 12 and 24℃ was not significant. Both CPDs and
6--4 PPs were induced by UV-B radiation even at 0℃ (Fig. 2b).
----------------------------------
---------------------------------Fig. 2 Effects of pH value of medium on pollen germination of litchi. (a) concentration of 50 mg/L; (b)
concentration of 100 mg/L.
3 Conclusions
Temperature is one of the major environmental factors controling survial, growth,
reproduction, and thus geographic distribution of plants. The study of combined temperature
and UV-B radiation could be of importance with respect to possible effects of climatic change,
especially global warming and increasing levels of UV-B radiation caused by the depeletion
of stratospheric ozone layer. The present investigation provided molecular evidence for
temperature-dependence of UV-B-induced DNA damage and photorepair.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No.
30200030) and the Natural Science Foundation of Educational Department of Guangdong
government (No. 20070506). The authors would like to thank Prof. Dennis Tabor for his
analytical support, Shirley Wasson for XPS analysis, and Marc Calvi for his work related to
the CEM measurements. The efforts of Matt Clayton from Arcadis G&M and Bob Frazier
from US EPA during the experimental endeavor are gratefully acknowledged.
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List of Figure Captions
Fig. 1 Relationship of degradation and mineralization.
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Fig. 2 Effect of lambda-cyhalothrin on RAW 264.7 cell viability. (a) RAW 264.7 cells were incubated with
different concentrations of lambda-cyhalothrin for 3 days, followed by the MTT assay; (b) RAW 264.7
cells were incubated in lambda-cyhalothrin at a concentration of 10−7 mol/L for 0, 1, 2 and 3 days, followed
by the MTT assay. Asterisk above adjacent bars indicate a significant difference (p < 0.05, n = 5) between
the treatment and control. Different letters above asterisk indicate a significant difference (p < 0.05, n = 5)
among each concentration.
Fig. 1 BPA degradation as function of ultrasonic power densities. (Experimental conditions: initial BPA
concentration of 1mg/L, ultrasonic frequency of 800 kHz, and power intensity of 3 W/cm2.)
1.0
-1
0.5W/mL, k=0.0307 min
-1
1.0W/mL, k=0.0687 min
-1
2.0W/mL, k=0.191 min
0.8
c/c0
0.6
0.4
0.2
0.0
0
20
40
60
80
100
120
Time (min)
Fig. 2 BPA degradation as function of ultrasonic power densities. (Experimental conditions: initial BPA
concentration of 1mg/L, ultrasonic frequency of 800 kHz, and power intensity of 3 W/cm2.)
Additional information
Unit format
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Time
Mass
High
Area
Volume
Pressure (suggest only use Pa)
Concentration (using mol/L
instead of M and N)
Year
Month
Week
Day
Hour
Minutes
Second
Kilogram
Gram
Milligram
Microgram
Meter
Centimeter
Millimeter
Micrometer
Square meter
Hectare
Cubic meter
Liter
Milliliter
Micro liter
Pascal
Milligram per liter
Mole per liter
JES format
year
month(s)
week(s)
day(s)
hr
min
sec
kg
g
mg
μg
m
cm
mm
μm
m2
ha
m3
L
mL
μL
Pa
mg/L
mol/L
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