日出間 純

Ozone depletion increases terrestrial solar ultraviolet B (UV-B; 280-315 nm) radiation, intensifying the risks plants face from DNA damage, especially covalent cyclobutane pyrimidine dimers (CPD). Without efficient repair, UV-B destroys genetic integrity, but plant breeding creates rice cultivars with more robust photolyase (PHR) DNA repair activity as an environmental adaptation. So improved strains of Oryza sativa (rice), the staple food for Asia, have expanded rice cultivation worldwide. Efficient light-driven PHR enzymes restore normal pyrimidines to UV-damaged DNA by using blue light via flavin adenine dinucleotide to break pyrimidine dimers. Eukaryotes duplicated the photolyase gene, producing PHRs that gained functions and adopted activities that are distinct from those of prokaryotic PHRs yet are incompletely understood. Many multicellular organisms have two types of PHR: (6-4) PHR, which structurally resembles bacterial CPD PHRs but recognizes different substrates, and Class II CPD PHR, which is remarkably dissimilar in sequence from bacterial PHRs despite their common substrate. To understand the enigmatic DNA repair mechanisms of PHRs in eukaryotic cells, we determined the first crystal structure of a eukaryotic Class II CPD PHR from the rice cultivar Sasanishiki. Our 1.7 angstrom resolution PHR structure reveals structure-activity relationships in Class II PHRs and tuning for enhanced UV tolerance in plants. Structural comparisons with prokaryotic Class I CPD PHRs identified differences in the binding site for UV-damaged DNA substrate. Convergent evolution of both flavin hydrogen bonding and a Trp electron transfer pathway establish these as critical functional features for PHRs. These results provide a paradigm for light-dependent DNA repair in higher organisms.

Rice cultivars vary widely in their sensitivity to ultraviolet B (UVB, 280-320 nm). Specifically, many indica rice cultivars from tropical regions, where UVB radiation is higher, are hypersensitive to UVB. Photoreactivation mediated by the photolyase enzyme is the major pathway for repairing UVB-induced cyclobutane pyrimidine dimers (CPDs) in plants. Still, these UVB-sensitive cultivars are less able to repair CPDs through photoreactivation than UVB-resistant cultivars. Here, we produced CPD photolyase-overexpressing transgenic rice plants with higher CPD photolyase activity using UVB-sensitive rice Norin 1 (japonica) and UVB-hypersensitive rice Surjamkhi (indica) as parental line (PL) plants. The results show that these transgenic rice plants were much more resistant to UVB-induced growth inhibition than were PL cultivars. The present findings strongly indicate that UVB-resistance, caused by an increase in CPD photolyase activity, can be achieved in various rice cultivars. However, there was a difference in the level of reduction of UVB-induced growth inhibition among rice cultivars; the level of reduction of growth inhibition in transgenic rice plants generated from the indica strain was lower than that of transgenic rice plants generated from japonica strains. These results indicate that the growth of the UVB-hypersensitive indica strain was strongly inhibited by other factors in addition to CPD levels.

Plants use sunlight as energy for photosynthesis; however, plant DNA is exposed to the harmful effects of ultraviolet-B (UV-B) radiation (280-320 nm) in the process. UV-B radiation damages nuclear, chloroplast and mitochondrial DNA by the formation of cyclobutane pyrimidine dimers (CPDs), which are the primary UV-B-induced DNA lesions, and are a principal cause of UV-B-induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival while exposed to UV-B-containing sunlight. Nuclear repair of the UV-B-induced CPDs involves the photoreversal of CPDs, photoreactivation, which is mediated by CPD photolyase that monomerizes the CPDs in DNA by using the energy of near-UV and visible light (300-500 nm). To date, the CPD repair processes in plant chloroplasts and mitochondria remain poorly understood. Here, we report the photoreactivation of CPDs in chloroplast and mitochondrial DNA in rice. Biochemical and subcellular localization analyses using rice strains with different levels of CPD photolyase activity and transgenic rice strains showed that full-length CPD photolyase is encoded by a single gene, not a splice variant, and is expressed and targeted not only to nuclei but also to chloroplasts and mitochondria. The results indicate that rice may have evolved a CPD photolyase that functions in chloroplasts, mitochondria and nuclei, and that contains DNA to protect cells from the harmful effects of UV-B radiation.

Plants use sunlight as energy for photosynthesis; however, plant DNA is exposed to the harmful effects of ultraviolet-B (UV-B) radiation (280-320 nm) in the process. UV-B radiation damages nuclear, chloroplast and mitochondrial DNA by the formation of cyclobutane pyrimidine dimers (CPDs), which are the primary UV-B-induced DNA lesions, and are a principal cause of UV-B-induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival while exposed to UV-B-containing sunlight. Nuclear repair of the UV-B-induced CPDs involves the photoreversal of CPDs, photoreactivation, which is mediated by CPD photolyase that monomerizes the CPDs in DNA by using the energy of near-UV and visible light (300-500 nm). To date, the CPD repair processes in plant chloroplasts and mitochondria remain poorly understood. Here, we report the photoreactivation of CPDs in chloroplast and mitochondrial DNA in rice. Biochemical and subcellular localization analyses using rice strains with different levels of CPD photolyase activity and transgenic rice strains showed that full-length CPD photolyase is encoded by a single gene, not a splice variant, and is expressed and targeted not only to nuclei but also to chloroplasts and mitochondria. The results indicate that rice may have evolved a CPD photolyase that functions in chloroplasts, mitochondria and nuclei, and that contains DNA to protect cells from the harmful effects of UV-B radiation.

UV-B responses of three rice (Oryza sativa L.) cultivars (Sasanishiki, Norin 1 and Surjamkhi) with different photolyase activity were investigated. Carbon dioxide assimilation data support that Sasanishiki was less sensitive to UV-B than Norin 1 and Surjamkhi. UV-B radiation sharply decreased the content of Rubisco protein in Surjamkhi and has no effect in Sasanishiki. The photochemical activities of photosystem (PS) 1 and PS 2 was slightly affected by UV-B treatment. The content of H2O2 and the activities of antioxidant enzymes, catalase (CAT), peroxides (POX) and superoxide dismutase (SOD) were enhanced after UV-B treatment. The activities of CAT and POX isoenzymes in Sasanishiki were more enhanced by UV-B radiation than those in Norin 1 and Surjamkhi.

UV-B responses of three rice (Oryza sativa L.) cultivars (Sasanishiki, Norin 1 and Surjamkhi) with different photolyase activity were investigated. Carbon dioxide assimilation data support that Sasanishiki was less sensitive to UV-B than Norin 1 and Surjamkhi. UV-B radiation sharply decreased the content of Rubisco protein in Surjamkhi and has no effect in Sasanishiki. The photochemical activities of photosystem (PS) 1 and PS 2 was slightly affected by UV-B treatment. The content of H2O2 and the activities of antioxidant enzymes, catalase (CAT), peroxides (POX) and superoxide dismutase (SOD) were enhanced after UV-B treatment. The activities of CAT and POX isoenzymes in Sasanishiki were more enhanced by UV-B radiation than those in Norin 1 and Surjamkhi.

Light-induced activation of class II cyclobutane pyrimidine dimer (CPD) photolyases of Arabidopsis thaliana and Oryza sativa has been examined by UV/Vis and pulsed Davies-type electron-nuclear double resonance (ENDOR) spectroscopy, and the results compared with structure-known class I enzymes, CPD photolyase and (6-4) photolyase. By ENDOR spectroscopy, the local environment of the Flavin adenine dinucleotide (FAD) cofactor is probed by virtue of proton hyperfine couplings that report on the electron-spin density at the positions of magnetic nuclei. Despite the amino-acid sequence dissimilarity as compared to class I enzymes, the results indicate similar binding motifs for FAD in the class II photolyases. Furthermore, the photoreduction kinetics starting from the FAD cofactor in the fully oxidized redox state, FAD(ox), have been probed by UV/Vis spectroscopy. In Escherichia coli (class I) CPD photolyase, light-induced generation of FADH(center dot) from FAD(ox), and subsequently FADH(-) from FADH(center dot), proceeds in a step-wise fashion via a chain of tryptophan residues. These tryptophans are well conserved among the sequences and within all known structures of class I photolyases, but completely lacking from the equivalent positions of class II photolyase sequences. Nevertheless, class II photolyases show photoreduction kinetics similar to those of the class I enzymes. We propose that a different, but also effective, electron-transfer cascade is conserved among the class II photolyases. The existence of such electron transfer pathways is supported by the observation that the catalytically active fully reduced flavin state obtained by photoreduction is maintained even under oxidative conditions in all three classes of enzymes studied in this contribution. (C) 2010 Elsevier B.V. All rights reserved.

Light-induced activation of class II cyclobutane pyrimidine dimer (CPD) photolyases of Arabidopsis thaliana and Oryza sativa has been examined by UV/Vis and pulsed Davies-type electron-nuclear double resonance (ENDOR) spectroscopy, and the results compared with structure-known class I enzymes, CPD photolyase and (6-4) photolyase. By ENDOR spectroscopy, the local environment of the Flavin adenine dinucleotide (FAD) cofactor is probed by virtue of proton hyperfine couplings that report on the electron-spin density at the positions of magnetic nuclei. Despite the amino-acid sequence dissimilarity as compared to class I enzymes, the results indicate similar binding motifs for FAD in the class II photolyases. Furthermore, the photoreduction kinetics starting from the FAD cofactor in the fully oxidized redox state, FAD(ox), have been probed by UV/Vis spectroscopy. In Escherichia coli (class I) CPD photolyase, light-induced generation of FADH(center dot) from FAD(ox), and subsequently FADH(-) from FADH(center dot), proceeds in a step-wise fashion via a chain of tryptophan residues. These tryptophans are well conserved among the sequences and within all known structures of class I photolyases, but completely lacking from the equivalent positions of class II photolyase sequences. Nevertheless, class II photolyases show photoreduction kinetics similar to those of the class I enzymes. We propose that a different, but also effective, electron-transfer cascade is conserved among the class II photolyases. The existence of such electron transfer pathways is supported by the observation that the catalytically active fully reduced flavin state obtained by photoreduction is maintained even under oxidative conditions in all three classes of enzymes studied in this contribution. (C) 2010 Elsevier B.V. All rights reserved.

We visualized flavonol distribution in the abaxial epidermis of onion scales without chemical processes via detection of blue-light-induced green autofluorescence. Our visualizing results indicated an unequal intercellular distribution of flavonols among epidermal cells causing a patch distribution in the epidermis, and indicated that flavonol accumulation in ultraviolet irradiated-onion scales was in uniformity with epidermal cells, probably to compensate for their stress-hypersensitiveness.

The cyclobutane pyrimidine dimer (CPD) is a major type of DNA damage induced by ultraviolet B (UVB) radiation. CPD photolyase, which absorbs blue/UVA light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (Oryza sativa) to UVB radiation. Here, we purified native class II CPD photolyase from rice leaves. As the final purification step, CPD photolyase was bound to CPD-containing DNA conjugated to magnetic beads and then released by blue-light irradiation. The final purified fraction contained 54-and 56-kD proteins, whereas rice CPD photolyase expressed from Escherichia coli was a single 55-kD protein. Western-blot analysis using anti-rice CPD photolyase antiserum suggested that both the 54-and 56-kD proteins were the CPD photolyase. Treatment with protein phosphatase revealed that the 56-kD native rice CPD photolyase was phosphorylated, whereas the E. coli-expressed rice CPD photolyase was not. The purified native rice CPD photolyase also had significantly higher CPD photorepair activity than the E. coli-expressed CPD photolyase. According to the absorption, emission, and excitation spectra, the purified native rice CPD photolyase possesses both a pterin-like chromophore and an FAD chromophore. The binding activity of the native rice CPD photolyase to thymine dimers was higher than that of the E. coli-expressed CPD photolyase. These results suggest that the structure of the native rice CPD photolyase differs significantly from that of the E. coli-expressed rice CPD photolyase, and the structural modification of the native CPD photolyase leads to higher activity in rice.

UV radiation induces the formation of two classes of photoproducts in DNA, the cyclobutane pyrimidine dimer (CPD) and the pyrimidine 6-4 pyrimidone photoproduct. CPDs in plants are repaired by class II CPD photolyase via a UV-A/blue light-dependent mechanism. The genes for the class II CPD photolyase have been cloned from higher plants such as Arabidopsis, Cucumis sativus (cucumber), Oryza sativa (rice) and Spinacia oleracea (spinach). Flavin adenine dinucleotide (FAD) has been identified as a cofactor. Here we report the isolation and characterization of the CPD photolyase cDNA from soybean (Glycin max). The sequence of amino acids predicted from the cDNA sequence was highly homologous to sequences of higher plant class II CPD photolyases. When the cDNA was expressed in a photolyase-deficient Escherichia coli, photoreactivation activity was partially restored by illumination with a fluorescent light. The purified enzyme showed CPD binding and light-dependent photoreactivation activities in vitro. When soybean CPD photolyase was heat-treated in vitro from 25 degrees C to 45 degrees C for 3 min, thymine dimer-binding activity and photoreactivation activity were decreased, and FAD was released from the enzyme. On the other hand, when the enzyme-CPD complex was heat-treated, photoreactivation activity was stable. We argue that FAD in the soybean CPD photolyase is labile for temperature, but once the enzyme-CPD complex has formed, FAD becomes tightly bound to the enzyme or complex.

We investigated the UVB-sensitivity in 12 rice strains belonging to two cultivated species (O.sativa and O.glaberrima) and three wild species (O.barthii, O.meridionalis and O.rufipogon) of rice possessing the AA genome, while focusing on the CPD photolyase activity and the genotypes of CPD photolyase. Although the UVB sensitivity, CPD photolyase activity, and CPD photolyase genotype varied widely among these rice species, the sensitivity to UVB radiation depended on the activity of the CPD photolyase, regardless of grass shape, habitat, or species. The rice strains examined here clearly divided into three groups based on the CPD photolyase activity, and the activity of the strains greatly depended on amino acid residues at positions 126 and 296, with the exception of the W1299 strain ( O.meridionalis). The amino acid residues 126 and 296 of CPD photolyase in Sasanishiki strain ( O.sativa), which showed higher enzymatic activity and more resistance to UVB, were glutamine (Gln) and Gln, respectively. An amino acid change at position 126 from Gln to arginine ("Nori"- type) in the photolyase led to a reduction of enzymatic activity. Additionally, an amino acid change at position 296 from Gln to histidine led to a further reduction in activity. The activity of the W1299 strain, which possesses a "Nori"- type CPD photolyase, was the highest among the strains examined here, and was similar to that of the Sasanishiki. The CPD photolyase of the W1299 contains ten amino acid substitutions, compared to Sasanishiki. The alterations in amino acid residues in the W1299 CPD photolyase compensated for the reduction in activity caused by the amino acid substitutions at positions 126. Knowledge of the activity of different CPD photolyase genotypes will be useful in developing improved rice cultivars.

Rice cultivars vary widely in their sensitivity to ultraviolet B (UVB) and this has been correlated with cyclobutane pyrimidine dimer (CPD) photolyase mutations that alter the structure/function of this photorepair enzyme. Here, we tested whether CPD photolyase function determines the UVB sensitivity of rice (Oryza sativa) by generating transgenic rice plants bearing the CPD photolyase gene of the UV-resistant rice cultivar Sasanishiki in the sense orientation (S-B and S-C lines) or the antisense orientation (AS-D line). The S-B and S-C plants had 5.1- and 45.7-fold higher CPD photolyase activities than the wild-type, respectively, were significantly more resistant to UVB-induced growth damage, and maintained significantly lower CPD levels in their leaves during growth under elevated UVB radiation. Conversely, the AS-D plant had little photolyase activity, was severely damaged by elevated UVB radiation, and maintained higher CPD levels in its leaves during growth under UVB radiation. Notably, the S-C plant was not more resistant to UVB-induced growth inhibition than the S-B plant, even though it had much higher CPD photolyase activity. These results strongly indicate that UVB-induced CPDs are one of principal causes of UVB-induced growth inhibition in rice plants grown under supplementary UVB radiation, and that increasing CPD photolyase activity can significantly alleviate UVB-caused growth inhibition in rice. However, further protection from UVB-induced damage may require the genetic enhancement of other systems as well.

Rice cultivars vary widely in their sensitivity to ultraviolet B (UVB) and this has been correlated with cyclobutane pyrimidine dimer (CPD) photolyase mutations that alter the structure/function of this photorepair enzyme. Here, we tested whether CPD photolyase function determines the UVB sensitivity of rice (Oryza sativa) by generating transgenic rice plants bearing the CPD photolyase gene of the UV-resistant rice cultivar Sasanishiki in the sense orientation (S-B and S-C lines) or the antisense orientation (AS-D line). The S-B and S-C plants had 5.1- and 45.7-fold higher CPD photolyase activities than the wild-type, respectively, were significantly more resistant to UVB-induced growth damage, and maintained significantly lower CPD levels in their leaves during growth under elevated UVB radiation. Conversely, the AS-D plant had little photolyase activity, was severely damaged by elevated UVB radiation, and maintained higher CPD levels in its leaves during growth under UVB radiation. Notably, the S-C plant was not more resistant to UVB-induced growth inhibition than the S-B plant, even though it had much higher CPD photolyase activity. These results strongly indicate that UVB-induced CPDs are one of principal causes of UVB-induced growth inhibition in rice plants grown under supplementary UVB radiation, and that increasing CPD photolyase activity can significantly alleviate UVB-caused growth inhibition in rice. However, further protection from UVB-induced damage may require the genetic enhancement of other systems as well.

Class I and class II CPD photolyases are enzymes which repair pyrimidine dimers using visible light. A detailed characterization of class I CPD photolyases has been carried out, but little is known about the class II enzymes. Photolyases from rice are suitable for functional analyses because systematic breeding for long periods in Asian countries has led to the selection of naturally occurring mutations in the CPD photolyase gene. We report the biochemical characterization of rice mutant CPD photolyases purified as GST-form from Escherichia coli. We identified three amino acid changes, Gln126Arg, Gly255Ser, and Gln296His, among which Gln but not His at 296 is important for complementing phr-defective E. coli, binding UV-damage in E. coli, and binding thymine dimers in vitro. The photolyase with Gln at 296 has an apoenzyme:FAD ratio of 1 : 0.5 and that with His at 296 has an apoenzyme:FAD ratio of 1 : 0.12-0.25, showing a role for Gln at 296 in the binding of FAD not in the binding of thymine dimer. Concerning Gln or Arg at 126, the biochemical activity of the photolyases purified from E. coli and complementing activity for phr-defective E. coli are similarly proficient. However, the sensitivity to UV of cultivars differs depending on whether Gln or Arg is at 126. The role of Gln and Arg at 126 for photoreactivation in rice is discussed.

Background Depletion of the stratospheric ozone layer leads to an increase in ultraviolet-B (UVB: 280-320 nm) radiation reaching the earth's surface, and the enhanced solar UVB radiation predicted by atmospheric models will result in reduction of growth and yield of crops in the future. Over the last two decades, extensive studies of the physiological, biochemical and morphological effects of UVB in plants, as well as the mechanisms of UVB resistance, have been earned out. Scope In this review, we describe recent research into the mechanisms of UVB resistance in higher plants, with an emphasis on rice (Oryza sativa), one of the world's most important staple food crops. Recent studies have brought to light the following remarkable findings. UV-absorbing compounds accumulating in the epidermal cell layers have traditionally been considered to function as UV filters, and to play an important role in countering the damaging effects of UVB radiation. Although these compounds are effective in reducing cyclobutane pyrimidine dimer (CPD) induction in plants exposed to a challenge exposure to UVB, certain levels of CPD are maintained constitutively in light conditions containing UVB, regardless of the quantity or presence of visible light. These findings imply that the systems for repairing DNA damage and scavenging reactive oxygen species (ROS) are essential for plants to grow in light conditions containing UVB. Conclusion CPD photolyase activity is a crucial factor determining the differences in UVB sensitivity between rice cultivara. The substitution of one or two bases in the CPD photolyase gene can alter the activity of the enzyme, and die associated resistance of die plant to UVB radiation. These findings open up die possibility, in the near future, of increasing die resistance of rice to UVB radiation, by selective breeding or bioengineering of the genes encoding CPD photolyase.

Rice qUVR-10, a quantitative trait locus (QTL) for ultraviolet-B (UVB) resistance on chromosome 10, was cloned by map-based strategy. It was detected in backcross inbred lines (BILs) derived from a cross between the japonica variety Nipponbare (UV resistant) and the indica variety Kasalath (UV sensitive). Plants homozygous for the Nipponbare allele at the qUVR-10 locus were more resistant to UVB compared with the Kasalath allele. High-resolution mapping using 1850 F-2 plants enabled us to delimit qUVR-10 to a < 27-kb genomic region. We identified a gene encoding the cyclobutane pyrimidine dimer (CPD) photolyase in this region. Activity of CPD photorepair in Nipponbare was higher than that of Kasalath and nearly isogenic with qUVR-10 [NIL(qUVR-10)], suggesting that the CPD photolyase of Kasalath was defective. We introduced a genomic fragment containing the CPD photolyase gene of Nipponbare to NIL(qUVR10). Transgenic plants showed the same level of resistance as Nipponbare did, indicating that the qUVR-10 encoded the CPD photolyase. Comparison of the qUVR-10 sequence in the Nipponbare and Kasalath alleles revealed one probable candidate for the functional nucleotide polymorphism. It was indicated that single-base substitution in the CPD photolyase gene caused the alteration of activity of CPD photorepair and UVB resistance. Furthermore, we were able to develop a UV hyperresistant plant by overexpression of the photolyase gene.

Sensitivity to ultraviolet-B (UVB) radiation (280-320 nm) varies widely among rice cultivars. We previously indicated that UV-resistant rice cultivars are better able to repair cyclobutane pyrimidine dimers (CPDs) through photorepair than are UV-sensitive cultivars. In this paper, we report that UVB sensitivity in rice, in part, is the result of defective CPD photolyase alleles. Surjamkhi (indica) exhibited greater sensitivity to UVB radiation and was more deficient in CPD photorepair ability compared with UV-resistant Sasanishiki (japonica). The deficiency in CPD photorepair in Surjamkhi resulted from changes in two nucleotides at positions 377 and 888 in the photolyase gene, causing alterations of two deduced amino acids at positions 126 and 296 in the photolyase enzyme. A linkage analysis in populations derived from Surjamkhi and Sasanishiki showed that UVB sensitivity is a quantitative inherited trait and that the CPD photolyase locus is tightly linked with a quantitative trait locus that explains a major portion of the genetic variation for this trait. These results suggest that spontaneously occurring mutations in the CPD photolyase gene cause different degrees of sensitivity to UVB in rice, and that the resistance of rice to UVB radiation could be increased by increasing the photolyase function through conventional breeding or bioengineering.

Variation in growth, grain size and grain storage protein content of rice (Oryza sativa L.) in response to elevated UV-B radiation under sunlight was examined in a cool rice-growing region of Miyagi Prefecture, Japan, in 1999, 2001 and 2002. Tiller number, dry mass, panicle number, grain yield and grain size significantly decreased under elevated UV-B radiation in 2001 and 2002. The effects of elevated UV-B radiation on the reduction of each growth parameter were greatly enhanced by daily lower temperature during the ripening stage in those two years. On the contrary, total grain nitrogen content and grain storage protein content significantly increased under elevated UV-B radiation in 2001 and 2002. Among grain storage proteins, glutelin content significantly increased but albumin-globulin and prolamin contents did not. It was thus evident that not only grain size but also grain storage protein of rice was markedly influenced due to elevated UV-B radiation.

There is a cultivar difference in the response to ultraviolet-B (UVB: 280-320 nm) in rice (Oryza sativa L.). Among Japanese lowland rice cultivars, Sasanishiki, a leading Japanese rice cultivar, is resistant to the damaging effects of UVB while Norin 1, a close relative, is less resistant. We found previously that Norin 1 was deficient in cyclobutane pyrimidine dimer (CPD) photorepair ability and suggested that the UVB sensitivity in rice depends largely on CPD photorepair ability. In order to verify that suggestion, we examined the correlation between UVB sensitivity and CPD photolyase activity in 17 rice cultivars of progenitors and relatives in breeding of UV-resistant Sasanishiki and UV-sensitive Norin 1. The amino acid at position 126 of the deduced amino acid sequence of CPD photolyase in cultivars including such as Norin 1 was found to be arginine, the CPD photolyase activities of which were lower. The amino acid at that position in cultivars including such as Sasanishiki was glutamine. Furthermore, cultivars more resistant to UVB were found to exhibit higher photolyase activities than less resistant cultivars. These results emphasize that single amino acid alteration from glutamine to arginine leads to a deficit of CPD photolyase activity and that CPD photolyase activity is one of the main factors determining UVB sensitivity in rice.

Growth of a near-isogenic line (NIL) for the purple leaf gene Pl of rice with a genetic background of Taichung 65 (T-65) rice was significantly retarded by supplementary ultraviolet-B radiation (UV-B), despite the fact that the amounts of UV-absorbing compounds and anthocyanins in NIL were significantly higher than those in T-65. In order to understand the role of flavonoids in UV-B induced damage protection in T-65 and the NIL, both the (1) relationships between changes in the steady state of cyclobutane pyrimidine dimer (CPD) levels and changes in accumulation of anthocyanins and UV-absorbing compounds in leaves with leaf age, and (2) the susceptibility to CPD induction by UV-B radiation and the ability to photorepair CPD were examined. Although supplementary UV-B elevated the steady state of CPD levels in leaves in both strains, the level in the leaf of the NIL was higher than that in T-65 at any time. The susceptibility to CPD induction by short-term (challenge) UV-B exposure was lower in the NIL than in T-65. On the other hand, the CPD photorepair was also lower in the leaves of the NIL than in those of T-65. The decrease in CPD-photorepair in the NIL was due to a lowering of the leaf-penetrating blue/UV-A radiation, which is effective for photoreactivation by photolyase, by anthocyanins. Thus, accumulation of anthocyanins and UV-absorbing compounds did not effectively function as screening against damage caused by elevated UV-B radiation in the NIL, and the retardation of growth in the NIL resulted from its lower ability to photorepair CPD by higher amounts of anthocyanins.

Ultraviolet radiation induces the formation of two classes of photoproducts in DNA-the cyclobutane pyrimidine dimer (CPD) and the pyrimidine [6-4] pyrimidone photoproduct (6-4 product). Many organisms produce enzymes, termed photolyases, which specifically bind to these lesions and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. Two classes of photolyases (class I and class 11) repair CPDs. A gene that encodes a protein with class 11 CPD photolyase activity in vitro has been cloned from several plants including Arabidopsis thaliana, Cucumis sativus and Chlamydomonas reinhardtii. We report here the isolation of a homolog of this gene from rice (Oryza sativa), which was cloned on the basis of sequence similarity and PCR-based dilution-amplification. The cDNA comprises a very GC-rich (75%) 5' region, while the 3' portion has a GC content of 50%. This gene encodes a protein with CPD photolyase activity when expressed in E. coli. The CPD photolyase gene encodes at least two types of mRNA, formed by alternative splicing of exon 5. One of the mRNAs encodes an ORF for 506 amino acid residues, while the other is predicted to code for 364 amino acid residues. The two RNAs occur in about equal amounts in O. sativa cells.

The effects of supplementary ultraviolet-B (UV-B) radiation on the changes in synthesis and degradation of ribulose -1,5-bisphosphate carboxylase/oxygenase (Rubisco) and light-harvesting chlorophyll a /b binding protein of PSII (LHCII) were examined, as well as mRNA levels for small and large subunits of Rubisco (rbcS and rbcL , respectively) and LHCII (cab ) with leaf age in UV-sensitive rice (Norin 1) and UV-resistant rice (Sasanishiki). Both Rubisco and LHCII were actively synthesized until the leaf had fully expanded, and then decreased with increasing leaf age. Synthesis of Rubisco, but not LHCII, was significantly suppressed by UV-B in Norin 1. The degradation of Rubisco was enhanced by UV-B around the time of leaf maturation in the two cultivars. The levels of rbcS and rbcL were reduced by UV-B at the early stages after leaf emergence in both cultivars. Cab transcripts were first present at high levels in the two cultivars, but drastically decreased due to UV-B treatment immediately after leaf emergence in Norin 1. It was shown that synthesis and degradation of Rubisco and LHCII greatly changed with leaf age: Rubisco synthesis was significantly suppressed by supplementary UV-B radiation at the transcription step during the early leaf stages. It was also suggested that the difference in UV-B sensitivity in Rubisco synthesis between the two rice cultivars might be due to specific suppression both transcriptionally and post-transcriptionally.

Among Indica rice cultivars (Oryza sativa L. cvs.) that belong to the aus ecotype from the tropical Bengal region, where the amount of ultraviolet-B (UV-B) radiation in the solar radiation is relatively great, Marich-bati cultivar has exhibited resistance to UV-B radiation, while Surjamkhi cultivar appeared to be less resistant. We have examined the susceptibility to cyclobutane pyrimidine dimer (CPD) induction by UV-B radiation and the ability to photorepair CPDs using these two cultivars. UV-B radiation produced similar dimer levels in the leaves of the two cultivars. In contrast, the ability to photorepair CPDs in the UV-sensitive Surjamkhi cultivar was lower than that in the UV-resistant Marich-bati cultivar. These results were similar to our previous data, namely, that a UV-sensitive Japanese rice cultivar (Oryza sativa L. cv. Norin 1) cultivated in the moderate climate of Japan is deficient in its ability to photorepair CPDs. Thus, these results suggest that a strong correlation exists between the sensitivity to UV-B and the photorepair deficiency, and that a low ability in CPD photorepair may be a principal factor in determining the UV-B sensitivity in rice plants.

The aim of this study was to investigate the regulatory effect of ultraviolet-B (UV-B) radiation on a number of key stress response genes found in the epidermis and mesophyll of Pisum sativum L., Argenteum mutant. This mutant was chosen for the ease with which the entire epidermis can be removed from the mesophyll tissue. An additional goal was to explore the potential modifying effect of pre-acclimation of plants to UV-B radiation prior to exposure by UV-B during treatment. Results showed that mRNA accumulation was similar during acute short-term UV-B exposure for chalcone synthase (Chs) and short-chain alcohol dehydrogenase (SadA) in both epidermis and mesophyll. In contrast, the mRNA levels differed considerably between tissues for phenylalanine ammonia lyase, chalcone isomerase and lipid transfer protein. After 24 h incubation in visible light after cessation of UV-B exposure, the regulation of mRNA levels also differed between Chs and SadA, the former showing no expression in the epidermis and the latter none in the mesophyll. Acclimation to low UV-B levels before acute exposures resulted in delayed induction of Chs and SadA. Measurements of UV-B-induced cyclobutane pyrimidine dimers (CPDs) showed a greater formation in epidermis than in mesophyll. In addition, acclimation at low UV-B levels resulted in significantly higher basal levels of CPDs than in non-acclimated plants in both mesophyll and epidermis and also in increased damage in concomitant acute exposures. The lack of correlation between the number of CPDs and levels of transcripts for defence genes, indicates that DNA damage does not control transcription of these genes.

Measuring DNA damage in higher plants is important in assessing the impacts of environmental conditions, e,g,, increased UV resulting from ozone depletion, and in testing the relationship of productivity to DNA damage and repair. Sunlight exposure of plants produces UV-induced DNA damages measurable by treating DNA with damage-specific enzymes and dispersion of DNA molecules in denaturing media. Such DNA must be enzyme-digestible, with few single strand breaks. DNA isolation must preclude repair, providing a "snapshot" of DNA damage. We developed a method for isolating DNA from several crop plants, both monocots and dicots - alfalfa (Medicago sativa L,), pea (Pisum sativum L,), rice (Oryza sativa L,), soybean [Glycine mar (L,) Merr,], sorghum [Sorghum bicolor (L.) Moench], and spinach (Spinacia oleraceae L,), This method is simple, readily deals with multiple samples, and avoids organic solvents. We show that pyrimidine dimers can readily be quantified in DNA prepared by this method. This method should also be useful for other experiments requiring high molecular length, enzymatically digestible plant DNA.

An investigation was made of the variations in growth and grain yield in response to increased exposure to W-B radiation of Japanese lowland rice (Oryza saliva L.) in a cool rice-growing region. Two cultivars, UV-resistant cv. 'Sasanishiki' and UV-sensitive cv. 'Norin 1', were examined in a lowland field at Kashimadai (37 degrees 28'E, 141 degrees 06'E) in Miyagi Prefecture, Japan, for four cropping seasons from 1994 to 1997. The two cultivars were grown in a lowland field with or without supplemental UV-B radiation, which was provided by UV-B-emitting fluorescent lamps, with a 0.1-mm-thick cellulose diacetate film as a filter. In both cultivars, significant decreases in tiller number as the result of supplemental W-B radiation were observed during the tillering stage in 1994, 1995 and 1997. Furthermore, decreases in grain size from supplemental UV-B radiation were recorded in all seasons. The trend towards small grain size was pronounced in 1996. In that year, the mean daily middle temperatures were lower throughout most of the cropping season and the mean daily hours of sunshine during the tillering stage and between the end of the panicle differentiation stage and the beginning of the ripening stage were shorter. In 1993 when the temperature and the amount of sunshine were both lower, the tiller number, the dry mass of aboveground parts and the panicle number were significantly reduced by supplemental unfiltered UV-B radiation. There was a cultivar difference in the inhibitory effects of supplemental UV-B radiation on growth between the sensitive cultivar Norin 1 and the resistant cultivar Sasanishiki. These results indicate that supplemental UV-B radiation has a positive effect on the growth and grain development of rice, which may be enhanced by unusual climatic conditions such as lower temperature and less sunshine, in cool rice-growing regions. (C) 2001 Elsevier Science B.V. All rights reserved.

An investigation was made of the variations in growth and grain yield in response to increased exposure to UV-B radiation of Japanese lowland rice (Oryza sativa L.) in a cool rice-growing region. Two cultivars, UV-resistant cv. 'Sasanishiki' and UV-sensitive cv. 'Norin 1', were examined in a lowland field at Kashimadai (37° 28′E, 141°06′E) in Miyagi Prefecture, Japan, for four cropping seasons from 1994 to 1997. The two cultivars were grown in a lowland field with or without supplemental UV-B radiation, which was provided by UV-B-emitting fluorescent lamps, with a 0.1-mm-thick cellulose diacetate film as a filter. In both cultivars, significant decreases in tiller number as the result of supplemental UV-B radiation were observed during the tillering stage in 1994, 1995 and 1997. Furthermore, decreases in grain size from supplemental UV-B radiation were recorded in all seasons. The trend towards small grain size was pronounced in 1996. In that year, the mean daily middle temperatures were lower throughout most of the cropping season and the mean daily hours of sunshine during the tillering stage and between the end of the panicle differentiation stage and the beginning of the ripening stage were shorter. In 1993 when the temperature and the amount of sunshine were both lower, the tiller number, the dry mass of aboveground parts and the panicle number were significantly reduced by supplemental unfiltered UV-B radiation. There was a cultivar difference in the inhibitory effects of supplemental UV-B radiation on growth between the sensitive cultivar Norin 1 and the resistant cultivar Sasanishiki. These results indicate that supplemental UV-B radiation has a positive effect on the growth and grain development of rice, which may be enhanced by unusual climatic conditions such as lower temperature and less sunshine, in cool rice-growing regions. © 2001 Elsevier Science B.V.

Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase-dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase.