Photochemistry and Photobiology, zyxwvuts 1999, 69(6): 681-685 zyxwvu Photoregulation of DNA Photolyases in Broom zyxw Sorghum Seedlings Megumi Hada*’, Gunther Buchholz2, Tohru Hashimoto3, Osamu Nikaido4and Eckard Wellmannz ’Department of Biology, Faculty of Science, Kobe University, Kobe, Japan; ‘Institute of Biology 11, Freiburg University, Freiburg, Germany; 3Department of Life Science, Kobe Women’s University, Kobe, Japan and 4Divisionof Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan Received 20 October 1998; accepted 5 March 1999 ABSTRACT zyxwvutsr Using dark-grown 3 day-old zyxwvutsrq Sorghum bicolor seedlings, photorepair of cyclobutane pyrimidine dimers (CPD) by CPD-photolyase and pyrimidine-(6-4)pyrimidinone pho- toproducts ([6-4]PP) by [6-4]PP-photolyase was studied in vivo and in vitro. The two types of DNA photoproducts were measured with specific monoclonal antibodies. Sor- ghum seedlings have different photorepair enzymes for CPD and [6-4]PP. The CPD-photolyase was found to be increased in response to light with maximal efficiency in the UVAhlue spectral range, whereas [6-4]PP-photolyase was not influenced by light. INTRODUCTION zyxwvutsr Sunlight is essential for plant growth and, therefore, plants are exposed to the potentially damaging UV spectral part of solar radiation throughout the life cycle. Destruction of the stratospheric ozone layer mainly by chlorofluorocarbons ( 1) results in an increase in UVB radiation reaching the surface of the Earth (2,3). Ultraviolet-B induces damage in DNA as well as in proteins and cell membranes. Typical damage in DNA by UVB irradiation includes cyclobutane pyrimidine dimers (CPD)? and pyrimidine-(6-4)pyrimidinone photo- products ([6-4]PP). These types of damage inhibit DNA rep- lication and transcription (4). Therefore, living organisms have developed efficient mechanisms for the repair of these photolesions. Nucleotide excision repair involves endonu- cleolytic steps, release of the damaged oligonucleotide and DNA resynthesis (5). Photorepair directly reverses the di- merization of pyrimidines. The enzymes involved in photo- repair, photolyases, use energy from the blue to the UVA waveband range (6). Although the existence of photorepair has been reported in many plants (4), information on the *To whom correspondence should be addressed at: Department of Biology, Faculty of Science, Kobe University, Tsurukabuto, Nada-ku, Kobe 657-8501, Japan. Fax: 8 1-78-803-0444; e-mail: mhada@biol.kobe-u.ac.jp zyxwvutsrqpon tAhbreviations: CPD, cyclobutane pyrimidine dimer; CTAB, hex- adecyltrimethylammonium bromide; ELISA, enzyme-linked irn- munosorbent assay: [6-4]PP. pyrimidine-(6-4)pyrimidinone pho- toproduct. zyxwvutsrqp 0 1999 American Society for Photobiology 003 1-8655/99 zyxwvutsrq %5.00+0.00 effects of environmental factors such as light on the expres- sion of this UV protective function is limited. In Arabidopsis thaliana leaves, UVB-containing white light induces an increase in CPD photorepair as compared to UV-free-grown controls (7). Alfalfa seedlings grown out- doors showed faster CPD photorepair than seedlings grown in a UV-free environmental chamber (8). In light-grown pea seedlings, CPD photorepair was higher than that in etiolated seedlings (9). In bean hypocotyls and mustard cotyledons CPD-photolyase activity was found to be phytochrome-reg- ulated (10,ll). However, in the only report on a monocot plant, no photoinduction of CPD-photolyase could be found in wheat leaves (9). A light-dependent enzyme activity specific to the removal of [6-4]PP has been found in Drosophila melanogaster (12,13). Plants also appear to possess this second photolyase activity. Chen et al. (14) observed fast [6-4]PP elimination under white light in Arabidopsis. Cucumber and wheat also showed photorepair of [6-4]PP (1516). In contrast to CPD- photolyase, the amount of [6-4]PP-photolyase activity was not affected by light in Arabidopsis (14), pea and wheat (9). In this report we examined the effects of prior light treat- ments on photorepair of CPD and [6-4]PP in dark-grown Sorghum bicolor seedlings using in vivo and in vitro assays. In contrast to a report on wheat (9), another monocot, we observed the inducibility by light of CPD-photolyase but not of [6-4]PP-photolyase in Sorghum seedlings. MATERIALS AND METHODS Plant material and irradiation. Seeds of S. hicolor Moench. cv. Acme Broomcom, 1991 crop from Kobe University Farm at Kasai, were soaked for 24 h with circulating tap water in a 24°C water bath and sown in vermiculite in pots, 8 X 8 X 7 (height) cm3. Seedlings were grown for 3 days at 24°C until they reached a height of -7 cm at the time of irradiation. In the experiment involving prior irradiation for induction of pho- tolyase, seedlings were irradiated from above covered with trans- mission cutoff filters. Thirty millimeter segments from the first internode of irradiated or nonirradiated seedlings were excised under a green safelight, leav- ing the 20 mrn top part of the internode. Cell division does not occur in this part of the internode. For UV irradiation, 20 segments were placed on filter paper moistened with deionized water in a plastic vat, covered with a 3 mm quartz plate and irradiated with UVB for 10 min. For photorepair experiments, UVB-irradiated segments were immediately exposed to UVA light. Light sources. Ultraviolet-B radiation was obtained from a Philips TL40W/12 fluorescent tube (A,,, = 310 nm, half-bandwidth = 40 68 1