Shigemitsu Tano

An Ultraviolet-B-Resistant Mutant with Enhanced DNA Repair in Arabidopsis

An ultraviolet-B (UV-B)-resistant mutant,uvi1 (UV-B insensitive 1), of Arabidopsis was isolated from 1,280 M1 seeds that had been exposed to ion beam irradiation. The fresh weight ofuvi1 under high-UV-B exposure was more than twice that of the wild type. A root-bending assay indicated that root growth was less inhibited by UV-B exposure in uvi1 than in the wild type. When the seedlings were grown under white light, the UV-B dose required for 50% inhibition was about 6 kJ m−2for the wild type and 9 kJ m−2 for uvi1. When the seedlings were irradiated with UV-B in darkness, the dose required for 50% inhibition was about 1.5 kJ m−2 for the wild type and 4 kJ m−2 for uvi1. An enzyme-linked immunosorbent assay showed that the reduction in levels of cyclobutane pyrimidine dimers (CPDs) under white light and of (6-4) photoproducts in darkness occurred faster in uvi1 than in the wild type. These results indicate that uvi1 had increased photoreactivation of CPDs and dark repair of (6-4) photoproducts, leading to strong UV-B resistance. Furthermore, the transcript levels of PHR1 (CPD photolyase gene) were much higher inuvi1 than in the wild type both under white light and after UV-B exposure. Placing the plants in the dark before UV-B exposure decreases the early reduction of CPDs in the wild type but not inuvi1. Our results suggest that UVI1 is a negative regulator of two independent DNA repair systems.

Degradation of chloroplast DNA in second leaves of rice (Oryza sativa) before leaf yellowing

SummaryThe second leaf ofOryza sativa develops, grows and ages within the 10 days that follow imbibition under our controlled continuous-light conditions. Proplastids in the leaf cells develop, mature to become chloroplasts and then age and disintegrate. In an examination of this life process, we studied first the behavior and the number of copies of plastid DNA and levels of chlorophyll by epifluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified microscope photon-counting system (VIMPCS). The results indicated that the number of copies of the plastid DNA per plastid increased and reached to plateau value of approximately 100 at the time when the elongation of the mesophyll cells and the enlargement of chloroplasts ceased 96 h after imbibition. However, 24 h later, the number of copies of plastid DNA per chloroplast began to decrease and fell rapidly to approximately 30 copies within 168 h after imbibition. Our examination of the number of chloroplasts per mesophyll cell indicated that no division of chloroplasts occurred more than 72 h after imbibition. The results suggest that the decrease in number of copies of plastid DNA per chloroplast was not due to an increase in the number of chloroplasts, but that this decrease was caused by degradation by unidentified enzymes. Since visible senescence of leaves, which was characterized by development of a yellowish color, began 168 h after imbibition, the degradation of plastid DNA seemed to occur 48 h before the visible leaf senescence. When we tested the nucleolytic activities in the second leaves after imbibition by digestion of plasmids in vitro and DNA-SDS polyacrylamide gel electrophoresis, five Ca2+−, four Zn2+−, and four Mn2+−dependent nucleases were detected in the leaf blades, and one of the Ca2+−, two of the Zn2+−, and two of the Mn2+−dependent nucleases were also identified in a purified preparation of intact chloroplasts. When the activity of the Zn2+−dependent nucleases (51 kDa and 13 kDa) increased markedly, degradation of the plastid DNA occurred. These results suggest that the destruction of chloroplast DNA, which occurs approximately 48 h before leaf yellowing, could be due to the activation of some metallo-nucleases and, furthermore, this enzymatic degradation propels the leaf towards senescence.

Preferential digestion of chloroplast nuclei (nucleoids) during senescence of the coleoptile ofOryza sativa

SummaryThe coleoptile ofOryza sativa develops, grows and ages within 4 days that follow imbibition. It is, thus, a very useful system for experimental analysis of the life cycle of organelles, for example, the development, growth and aging of plastids in higher plants. We examined the behavior and levels of DNA and chlorophyll in the plastid by epifluorescence microscopy after staining with 4′-6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified-photon counting system (VIMPCS). The whitish yellow coleoptile appeared soon after imbibition and, between the first 24 and 60 h that followed imbibition, it grew markedly in a longitudinal direction, with concomitant elongation of the cells, and an increase in the volume of plastids and in the amount of DNA in the plastids. The chlorophyll content per plastid began to increase when the coleoptile turned green, 48 h after imbibition, and reached a plateau value when the coleoptile was 3.5 mm in length, 72 h after imbibition. More than 12 h later, the chlorophyll disappeared just before the breakdown of chloroplasts was initiated. Proplastids in young coleoptiles, contained a plastid nucleus which was located in the central area of the plastids and each nucleus consisted of approximately 6 copies of plastid DNA (ptDNA). The number of copies of ptDNA per plastid increased gradually, with a concomitant increase in the volume of the plastids after imbibition, and reached approximately 130 times the value in the young proplastids, 60 h after imbibition, when the plastid developed into a chloroplast. However, each plastid nucleus did not scatter throughout the entire interior region of each chloroplast. The disappearance of each plastid nucleus occurred more than 12 h before the degeneration of the chloroplasts. The number of plastids per cell increased from 10 to 15 in young coleoptiles within 12 h after imbibition. Yet the number remained constant throughout subsequent growth and aging of the coleoptile. Thus the preferential reduction in the amount of chloroplast DNA was not due to the division of the plastid but could, perhaps, be associated directly with the aging of the cells of the coleoptile which precedes senescence of the coleoptiles.

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

SummaryThe behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca2+-dependent nuclease and a 23 kDa Zn2+ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.

Repair of radiation-induced single-strand breaks in DNA of barley embryo

Repair of single-strand breaks induced by gamma-irradiation was studied in barley embryos. Nuclei were isolated from embryos at specified times after initiation of water absorption under different temperature conditions. From the shift of the peak of DNA in alkaline sucrose gradient, it was observed that clear repair of induced breaks occurred in the irradiated embryo during 2 h after soaking. However, this type of repair was not observed after 5 h up to 16 h, because natural degradation of DNA also occurred in the non-irradiated embryos. The estimated mol. wt. of DNA from the non-irradiated dry embryo was 0.93 X 10(-8) daltons; on the other hand, it was decreased to 0.15 X 10(-8) daltons by 30 kR of gamma-irradiation. This decreased mol. wt. recovered to 0.52 X 10(-8) daltons (64% recovery) after 2 h soaking at 25 degrees C. The repair observed in this experiment was not dependent on the regular synthesis of macromolecules, especially DNA.

Marker chromosomes commonly observed in the genus Glycine

SummarySoybean [Glycine max (L.) Merr.] chromosomes were analyzed using the chromosome image analyzing system, CHIAS, and seven groups, including subgroups, were identified based on morphological characteristics. Two pairs of chromosomes were conspicuous in their morphological traits. One pair of chromosomes, which had the largest arm ratio among all the chromosomes, was commonly observed in the species in all three subgenera of the genus Glycine. These chromosomes also displayed a unique pattern after N-banding and were detected as marker chromosomes. G. soja, which is considered to be the ancestor of G. max, has two types of marker chromosomes. The lines that carry the same type as G. max may be the ancestors of G. max among the lines of G. soja. The morphological differences of the marker chromosomes within the species in the subgenus Soja are discussed in relation to the domestication process of soybean.

Effects of low-dose irradiation from /sup 131/I on the induction of somatic mutations in Tradescantia

Low doses of 131I (5 to 100 nCi) were applied to the inflorescence of Tradescantia clone 02 and the mutational events were observed in the stamen hair cells. A linear dose-effect relationship was obtained over this very low dose range. However, environmental condition may cause fluctuation in mutation frequency; therefore, it is suggested that great caution has to be paid to elucidate the validity of this monitoring system.

Effects of several nitroso compounds on the induction of somatic mutations in Tradescantia with special regard to the dose response and threshold dose

Abstract Five kinds of chemical mutagens (EMS, NEU, NMU, NEDA and NDMA) were tested for the induction of somatic mutations using teh Tradescantia stamen hair system with special regard to the dose-response relationships and the minimum effective dose for the induction of mutations. Three types of dose-response relationship were obtained depending on the particular chemical. The minimum dose for the induction of mutations in this system was estimated to be 5 pg and 20 pg per flower for NMU and NEW, respectively and 100 pg per flower for EMS, NDEA and NDMA.

RFLP analysis of nuclear DNAs homologous with mitochondrial plasmid-like DNAs in cultivated rice.

SummaryB1 and B2 are small, circular, mitochondrial plasmid-like DNAs found in male-sterile cytoplasm (cms-Bo) of rice. In this study, nuclear sequences homologous to these DNAs were investigated among a number of rice cultivars. Several copies of nuclear B1-and B2-homologous sequences were detected in all examined cultivars, regardless of the presence or absence of the B1 and B2 DNAs in mitochondria, indicating that the existence of the B1- and B2-homologous sequences in the rice nuclear genome was widespread. A restriction fragment length polymorphism (RFLP) was detected for both sequences, and we propose that these DNAs could be useful RFLP markers for the rice nuclear genome. To analyze these nuclear homologues genetically, segregation analysis of the RFLP was carried out in the F2 progenies of an Indica-Japonica rice hybrid. Of the B1 homologues, there were two nonallelic fragments, one specific to the Indica parent and the other to the Japonica. These results indicate that the B1 and B2 homologues were dispersed in the nuclear genome. The integration of B1-homologous DNA into the nuclear DNA may have occurred independently after sexual isolation of the Indica and Japonica rice varietal groups, or a intranuclear transposition of these sequences took place during the process of rice differentiation into the varietal groups.

Chemical Nature of Radiation-Induced Single-Strand Breaks in the DNA of Dormant Barley Seeds in Vivo

Gamma irradiation of dormant barley seeds has led to the production of DNA single-strand breaks, the liberation of inorganic phosphate, and the formation of phosphomonoester groups. For each single-strnad break produced in DNA, 0.22 molecules of inorganic phosphate were liberated and 1.1 molecules of phosphomonoester groups were formed. Of the phosphomonoester groups formed, 40% were 5′-phosphomonoesters. Only 8.6% of the 5′-termini produced by radiation were 5′-hydroxyl termini. The chemical mechanisms involved in the induction of single-strand breaks in the DNA of dormant barley seeds are discussed.