Tadashi Kumagai

UV Radiation–Sensitive Norin 1 Rice Contains Defective Cyclobutane Pyrimidine Dimer Photolyase

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.

Effects of supplemental UV-B radiation on the growth and yield of two cultivars of Japanese lowland rice (Oryza sativa L.) under the field in a cool rice-growing region of Japan

Abstract 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.

MYCOCHROME SYSTEM AND CONIDIAL DEVELOPMENT IN CERTAIN FUNGI IMPERFECTI

Abstract. A photoreceptor system, “mycochrome”, is involved in a blue and near UV reversible photo‐reaction which in turn plays an important role in the photocontrol of conidial development in Alternaria tomato, Botrytis cinerea and Helminthosporium oryzae. Conidial development was controlled by alternating exposures to blue and near UV light with the final response being determined by the final light received. When the final light was near UV a conidium developed; when it was blue, conidiation was inhibited and a “sterile” conidiophore was formed. The effects of the two lights were alternatively reversible.

PHOTOCONTROL OF FUNGAL DEVELOPMENT

The life of fungi is greatly modulated by light, as seen in the formation of reproductive structures, pigment biosynthesis, phototaxis, phototropism and so on. The development of reproductive structures such as conidia, perithecia and basidiocarps is ecologically very significant because it finally leads not only to enlargement of the habitat of the fungi but also to preservation of their own species. Two kinds of photoreactions have been found in the photocontrol of sporulation: a promotive one for spore development and an inhibitory one. Whether both or one of those photoreactions is involved in sporulation depends on the species. Some fungi sporulate and discharge spores at a definite time during the day, as if they were responding to photoperiodism. Most of the photomorphogenetic responses in fungi are controlled either by near-UV and blue light or by near-UV light alone. The responsible light receptor is referred to as a blue/UV-A light-absorbing pigment (or cryptochrome(s)) and as a UV-B lightabsorbing pigment (Senger, 1984). A few photoresponses, as seen in spore germination and spore discharge, have also been found to be influenced by yellow-red light (Ingold, 1971). In the past 4 years, however, there have been few reports on such red light effects in fungal development. Nevertheless, we should keep in mind that only about 100 species of fungi out of more than 50 000 on earth have been studied, and several species out of those have been studied in detail in the field of photobiolOgY. This review will focus mainly on advancements made during 19841987 in the study of photocontrol of fungal sporulation and in related studies. The following references should also be noted: photomovement (Hader, 1987), photophysiology of Phycomyces (Galland and Lipson, 1984; Cerda-Olmedo and Lipson, 1986), Photobiology of fungi (Furuya, 1986). blue light responses (Senger, 1984, 1987; Durand. 1985).

EFFECTS OF LIGHT ENVIRONMENT DURING CULTURE ON UV-INDUCED CYCLOBUTYL PYRIMIDINE DIMERS AND THEIR PHOTOREPAIR IN RICE (ORYZA SATIVA L.)

Abstract— We examined the effects of a light environment during culture of rice plants (Oryza sativa) on the steady‐state cyclobutyl pyrimidine dimer (CPD) level, CPD induction by challenge UVB exposure and the ability to photorepair CPD. The steady‐state CPD level in plants grown under visible radiation with supplemental UVB radiation in a growth chamber was several times higher than in plants grown without supplemental UVB radiation, whereas in outdoor‐grown plants, it was not enhanced by supplemental UVB radiation. The susceptibility to CPD induction by challenge UVB exposure was highest in dark‐grown plants and decreased with increasing irradiance of visible radiation at low and high levels and outdoors. Chronic UVB radiation reduced the susceptibility to UV‐induced CPD in plants grown both indoors and outdoors. There was a significant negative correlation between CPD levels induced by challenge UVB exposure and the content of UV‐absorbing compounds. The UV‐induced CPD could be reduced by subsequent blue radiation in all samples except in dark‐grown seedlings. The higher the irradiance of visible radiation in the culture, the greater the ability to photorepair CPD. Chronic UVB radiation did not increase the ability to photorepair CPD.

UVB-induced cyclobutyl pyrimidine dimer and photorepair with progress of growth and leaf age in rice

Abstract We have examined the susceptibility to cyclobutyl pyrimidine dimer (CPD) induction by UVB radiation, the ability to photorepair cyclobutyl pyrimidine dimers (CPDs), and the levels of ultraviolet (UV)-absorbing compounds in various developing stages of leaves in UVB-resistant (Sasanishiki) and -sensitive (Norin 1) rices (Oryza sativa L.). UVB produces more CPDs in the second, third and fourth fully expanded leaves than in the fifth and sixth fully expanded leaves. In leaves in which the levels of UV-absorbing compounds are lower, more CPDs are produced. The ability to photorepair CPDs in fully expanded leaves of different stages of each cultivar remains approximately constant, although the photorepair capacity in the fourth leaf rapidly increases from leaf emergence until full expansion. The photorepair capacity is lower in Norin 1 than that in Sasanishiki in all the leaf stages or different aged leaves. These results therefore suggest that rice plants could be more susceptible to CPD induction at the stage when the second, third or fourth leaves are developing, and UVB sensitivity correlates with low CPD photorepair.

qUVR-10 , a Major Quantitative Trait Locus for Ultraviolet-B Resistance in Rice, Encodes Cyclobutane Pyrimidine Dimer 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 F2 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(qUVR-10). 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.

BLUE AND NEAR ULTRAVIOLET REVERSIBLE PHOTOREACTION IN CONIDIAL DEVELOPMENT OF THE FUNGUS, ALTERNARIA TOMATO

In the sporulation of Alternaria tomato, conidiophores are induced by near ultraviolet irradiation but not by darkness, and the conidia develop only when the irradiation is followed by a period of darkness. Conidial development is suppressed by a short exposure to blue light at a definite time during the dark period following the inductive irradiation. The suppression of conidial development by blue light can be reversed by exposure to near ultraviolet light immediately following the blue light irradiation. This reversion is reversibly suppressed by a further exposure to blue light immediately following near ultraviolet irradiation.

A gene for a Class II DNA photolyase from Oryza sativa: cloning of the cDNA by dilution-amplification

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.

Mapping of quantitative trait loci associated with ultraviolet-B resistance in rice (Oryza sativa L.)

Abstract 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 BC1F5 (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.