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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.

*Summary 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.

The detection of quantitative trait loci (QTLs) associated with UV-B resistance in rice should allow their practical application in breeding for such a complex trait, and may lead to the identification of gene characteristics and functions. Considerable variation in UV-B resistance exists within cultivated rice (Oryza sativa L.), but its detailed genetic control mechanism has not been well elucidated. We detected putative QTLs associated with the resistance to enhanced UV-B radiation in rice, using 98 BC(1)F(5) (backcross inbred lines; BILs) derived from a cross between Nipponbare (a resistant japonica rice variety) and Kasalath (a sensitive indica rice variety). We used 245 RFLP markers to construct a framework linkage map. BILs and both parents were grown under visible light with or without supplemental UV-B radiation in a growth chamber. In order to evaluate UV-B resistance, we used the relative fresh weight of aerial parts (RFW) and the relative chlorophyll content of leaf blades (RCC). The BIL population exhibited a wide range of variation in RFW and RCC. Using composite interval mapping with a LOD threshold of 2.9, three putative QTLs associated with both RFW and RCC were detected on chromosomes 1, 3 and 10. Nipponbare alleles at the QTLs on chromosome 1 and 10 increased the RFW and RCC, while the Kasalath allele at the QTL on chromosome 3 increased both traits. Furthermore, the existence of both QTLs on chromosomes 1 and 10 for UV-B resistance was confirmed using chromosome segment substitution lines. Plants with Kasalath alleles at the QTL on chromosome 10 were more sensitive to UV-B radiation than plants with them on chromosome 1. These results also provide the information not only for the improvement of UV-B resistance in rice though marker-associated selection, but also for the identification of UV-B resistance mechanisms by using near-isogenic lines.

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 II) repair CPDs. A gene that encodes a protein with class II 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.

A cDNA clone derived from a novel gene (uvi-1) that is inducible by near-UV light was isolated by a differential screening procedure from a cDNA library of the fungus Bipolaris oryzae and characterized further. Sequence analysis of the clone revealed that uvi-1 encodes a protein with a putative molecular mass of 17 kDa; the UVI-1 protein shows significant similarity to a putative protein encoded by a cDNA which is expressed during appressorium formation in the rice blast fungus, Magnaporthe grisea. The corresponding genomic clone was also isolated, and Southern analysis of genomic DNA indicated the presence of a single copy of the uvi-1 gene in B. oryzae. Northern analysis showed that the uvi-1 transcripts are induced by exposure to near-UV light, but not by blue or red light. Furthermore, accumulation of uvi-1 transcripts is observed during differentiation of the appressorium.

We produced near isogenic lines (NILs) for three purple leaf genes, Pl, Plw and Pli of rice with a genetic back-ground of T-65 to characterize each allele. Three NILs each had characteristic tissue-specific anthocyanin pigmentation. T-65 Plw was newly found to make the root purple when the root was exposed to light. To explore the inducible characteristics of the three genes, we examined the responses of the three NILs to UV-B irradiation. Although the accumulation of anthocyanin increased in all NILs with the increase in the intensity of UV-B irradiation, we observed differential responses for the accumulation of anthocyanin in the NILs: T-65 Pli accumulated the largest amounts of anthocyanin under weak and intermediate intensity of UV-B, though this NIL reduced anthocyanin accumulation under a high intensity of UV-B. T-65 Pl or Plw increased anthocyanin accumulation with increasing intensity of UV-B. Anthocyanin accumulation of the Pl NIL was higher than that of the Plw NIL under each intensity of UV-B. In addition, the total biomass of all the irradiated NILs decreased with the increasing dose of UV-B irradiation and the decrease was negatively correlated with the increase in anthocyanin accumulation in the dose range of UV-B irradiation.

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.

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair.

Two types of fungi based on the light required for induction of conidiophore formation are known as ‘photo-induced’ and ‘non-photo-induced’ sporulators. Conidial development in a ‘photo-induced’ strain of Bipolaris oryzae (formerly, Helminthosporium oryzae) is controlled by antagonistic action of blue and near-UV radiation mediated through the ‘mycochrome’ system at two developmental stages: conidiophore induction and conidiophore maturation. Otherwise, conidial development in a ‘non-photo-induced’ strain of B. oryzae, unlike that in the ‘photo-induced’ strain, is controlled by antagonistic action of blue and near-UV radiation at the conidiophore maturation stage alone. In this study, we collected fungi, B. oryzae, from brown lesion spots on rice leaves cultivated in paddy fields, and investigated the possible existence of a new ecotype different from known ‘photo-induced’ and ‘non-photo-induced’ sporulators. We also investigated the sensitivity of conidial development of each isolate to blue and near-UV radiation. The isolates were identified as B. oryzae by morphology of hyphae and conidia and the symptomatic brown lesion spots on rice leaves caused by each isolate. There was no distinction between ‘photo-induced’ and ‘non-photo-induced’ strains on rDNA base. Moreover, the existence of a new type of ‘non-photo-induced’ sporulator different from the one presently known was confirmed. In this newly identified sporulator, conidial development was not affected by light irradiation. Among 153 isolates, one isolate belonged to a previously known ‘non-photo-induced’ sporulator, five isolates belonged to a new ‘non-photo-induced’ sporulator ecotype, and over 95% of the isolates were “photo-induced” sporulators. Among ‘photo-induced’ strains, there were several subtypes having different sensitivities to near-UV radiation required for inducing conidiophore formation.

Genetic analysis of resistance to the inhibitory effects of UV radiation on growth of rice (Oryza sativa L.) cultivars was carried out. Some experimental plants were grown in visible radiation supplemented with UV radiation containing a large amount of UV-B and a small amount of UV-C in a phytotron, while others were grown without UV radiation. The degree of resistance to UV radiation was estimated in terms of the degree of reduction caused by supplemental UV radiation in the fresh weight of the aboveground plant parts and the chlorophyll content per unit fresh weight. Fresh weight and chlorophyll content in F2 plants generated by reciprocally crossing cv. Sasanishiki, a cultivar more resistant to UV radiation, and Norin 1, a cultivar less resistant to such radiation exhibited a normal frequency distribution. The heritabilities of these two properties in F2 plants were low under conditions of non-supplemental UV radiation. Under elevated UV radiation, the F2 population shifted to the lower range of fresh weight and chlorophyll content, and the means were close to those of Norin 1. The heritabilities of these two properties were the same in the reciprocal crosses, indicating that maternal inheritance was not involved. Inheritance of chlorophyll content per unit fresh weight was further determined in F3 lines generated by self-fertilizing F2 plants of Sasanishiki and Norin 1. The results showed that the F3 population was segregated into three genotypes, namely, resistant homozygotes, segregated heterozygotes and sensitive homozygotes, with a ratio of 1:65:16. It was thus evident that the resistance to the inhibitory effect of elevated UV radiation in these rice plants was controlled by recessive polygenes.

Flowering response and plant form of photomorphogenic mutants (hy1, hy2, hy3, hy4 and hy5) of Arabidopsis thaliana (L.), a long-day plant, were examined in long and short days. There were only slight differences among genotypes including Landsberg wild type with respect to the flowering time under long days. The effect of 1 h light-(night)-breaks of far-red, red, blue and white light given in the middle of the dark period of plants grown under short days, was studied. Effects of far-red light applied at the end or the beginning of the main photoperiod on flowering and plant form were also examined. The light-breaks with all the above mentioned light qualities promoted floral initiation of all the genotypes including the wild type in terms of both the flowering time and the number of rosette leaves. In general, far-red light was most effective. It is possible to classify the hy-mutants into 3 groups by their responses to light-breaks under short day conditions: (a) Mutants hy2 and hy3, which have a reduced number of rosette leaves, and flower early. Red light is as effective as far-red light. The wavelength of light-breaks is relatively unimportant for flowering response. (b) Mutants hy4, hy5 and Landsberg wild type, which have a greater number of rosette leaves, and flower relatively late. The effectiveness of light-breaks is in the following order, far-red, blue, and red light, which is in reverse order to the transformation of phytochrome to the Pfr form. (c) Mutant hy1, which behaves anomalously with respect to relations between flowering time and number of rosette leaves; late flowering with reduced number of rosette leaves. Red, blue and far-red light are effective, but white light is ineffective for reducing the number of rosette leaves. When far-red light was given in the middle of the night or at the end of the main photoperiod, it markedly reduced the number of rosette leaves compared to those grown under short days for all the genotypes, while when applied at the beginning of the main photoperiod far-red light did not affect the number of rosette leaves. Different effects on the plant form dependent on the time of treatment with far-red light-breaks are also discussed.

Application of an inhibitor of gibberellin biosynthesis alleviated the UV-B injury in rice seedlings when tested for chlorophyll content per unit fresh and dry weight. A genetic study was conducted by crossing Tan-ginbozu, a gibberellin-deficient mutant, against two floating rice cultivars that presumably contain large amounts of endogenous gibberellins. The heritabilities of plant height, and chlorophyll content per unit fresh and dry weight were high in the F2 plants in non UV-B irradiated control populations. When those plants were treated with UV-B irradiation, the F2 population tended to shift to the lower range in the chlorophyll content per unit weight, and the value of heritability fluctuated depending on the cross and characters measured. Selection of plants that are highly resistant to UV-B irradiation can be made possible by finding the range of UV-B energy that increases the difference between sensitive and resistant, while also increasing the value of the heritability.

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 F2 plants enabled us to delimit qUVR-10 to a

Verticillium agaricinum (Link) Corda, grown in a yeast extract-sucrose medium, conidiated abundantly in darkness after irradiation with near ultraviolet (290–400 nm) for 15 min or blue light (400–550 nm) for 60 min. Few conidia were formed in total darkness. Exposure to 30 min of near ultraviolet light suppressed conidiation. Conidiation was also suppressed by phosphate in excess of 10−4M irrespective of light condition. After irradiation with near ultraviolet light for more than 30 min, there was a cessation of growth and a change in colony color from yellow to reddish. The color does not appear to be due to a carotenoid because the colonies turned from red to yellow when covered with acid. At pH lower than 6.0 the pigment has an absorption maximum around 390 nm, whereas at higher pH it is around 540 nm. Thus, it appears that irradiation of V. agaricinum with near ultraviolet may cause an increase in pH, which in turn produces the change of colony color from yellow to reddish.

Coniditation the fungus, Alternaria cichorii Nattras (IMI 225641), was repeatedly induced and inhibited by alternating doses of near ultraviolet and blue radiation. The final response largely depended on the last kind of radiation administered. When the final radiation was near ultraviolet, conidiophore formation was induced, and conidia were then produced in the subsequent dark period; when it was blue, the induction of conidiation was suppressed. The inductive effect of near ultraviolet was promoted by anaerobiosis, but the reversing effect by blue radiation was not influenced by molecular oxygen. Thus, the “mycochrome” system apprears to control induction of conidiation in Alternaria cichorii, and oxidation-reduction processes may be involved in its function.

Conidiation in A. cichorii Nattras is reversibly stimulated by near UV radiation (NUV .apprx. 313 nm) and inhibited by blue light (.apprx. 450 nm) and apparently is a mycochrome-mediated process. After induction with plane-polarized NUV, blue light polarized perpendicularly to the NUV was more effective in counteracting the induction than was blue light polarized parallel to the NUV. Both the blue-absorbing component (presumably a flavo-protein) and the PNUV (NUV absorbing moiety) of the mycochrome system are membrane-bound. The transition moment associated with blue light absorption in the presumed flavoprotein forms an angle of at least 53.degree. with the transition moment associated with NUV absorption in PNUV.

Lemna paucicostata Hegelm. T-101, a short-day plant, flowers when plants preirradiated with red light (R) for 24 h are subjected to inductive darkness for 72 h followed by two short-day cycles (6 h R+ 18 h dark). However, flowering is inhibited by blue-or far-red-light pulses applied at the beginning of the inductive dark period. These inhibitory light effects are fully reversible by a R pulse. The action spectra for the inhibitory light effect and for its reversal show that the light pulses act exclusively through phytochrome. It is concluded that a low level of Pfr at the beginning of the inductive dark period prevents flowering.

Induction of conidiation in Alternaria tomato (Cke.) Weber and Helminthosporium oryzae V. Breda de Haan was repeatedly controlled by ulternating doses of near ultraviolet light and blue light. These fungi did not form conidia in total darkness. However, when dark-grown colonies were exposed to near altraviolet light, conidiophore formation was induced, and conidia were then produced upon return to darkness. Conidiation was inhibited by blue light applied immediately after near ultraviolet light. Furthermore, the final response depended on the last kind of light received. The action spectrum for induction of conidiation showed a peak near 300 nm with a shoulder between 310 nm and 330 nm, as is common in photoconidiation of various fungi. The action spectrum for the reversion of induction of conidiation showed a peak near 447 nm with a shoulder between 400 nm and 440 nm, one minor peak around 478 nm and another one near 385 nm. This is very similar to the spectrum for the ubiquitous blue light receptor (cryptochrome).

Induction of flowering of etiolated Lemna paucicostata Hegelm. T-101, a short-day plant, was inhibited by far-red (FR) or blue light (BL) applied at the beginning of a 72-h inductive dark period which was followed by two short days. In either case the inhibition was reversed by a subsequent exposure of the plants to near-ultraviolet radiation (NUV), with a peak of effectiveness near 380 nm. Inhibition by BL or FR and its reversion by NUV are repeatable, i.e., NUV is acting in these photoresponses like red light although with much lower effectiveness. Thus, it is considered that NUV acts through phytochrome and no specific BL and NUV photoreceptor is involved in photocontrol of floral induction on this plant.