Effects of Temperature on UV-B-Induced DNA Damage and Photorepair in Arabidopsis Thaliana

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: [email protected] (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 temperature- independent 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: [email protected]

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

1 most important consequence of the ozone depletion is an increase in the amount of UV-B (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.

2 Table 1 Properties of the soil used in the greenhouse experiment 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 (Q- PANEL, 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 Pb2+ 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)

2+ where, C0 (mg/L) and C (mg/L) are the initial and final Pb 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):

bdes H = (2) bads 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

4 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.

6 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 0.5W/mL, k=0.0307 min-1 1.0W/mL, k=0.0687 min-1 -1 0.8 2.0W/mL, k=0.191 min

c / c 0.4

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

7 Unit format JES format Time Year year Month month(s) Week week(s) Day day(s) Hour hr Minutes min Second sec Mass Kilogram kg Gram g Milligram mg Microgram μg High Meter m Centimeter cm Millimeter mm Micrometer μm Area Square meter m2 Hectare ha Volume Cubic meter m3 Liter L Milliliter mL Micro liter μL Pressure (suggest only use Pa) Pascal Pa Concentration (using mol/L Milligram per liter mg/L instead of M and N) Mole per liter mol/L