Poaceae plants transfer cyclobutane pyrimidine dimer photolyase to chloroplasts for ultraviolet-B resistance.

Photoreactivation enzyme that repairs cyclobutane pyrimidine dimer (CPD) induced by ultraviolet-B radiation, commonly called CPD photolyase (PHR) is essential for plants living under sunlight. Rice (Oryza sativa) PHR (OsPHR) is a unique triple-targeting protein. The signal sequences required for its translocation to the nucleus or mitochondria are located in the C-terminal region but have yet to be identified for chloroplasts. Here, we identified sequences located in the N-terminal region, including the serine-phosphorylation site at position 7 of OsPHR, and found that OsPHR is transported/localized to chloroplasts via a vesicle transport system under the control of serine-phosphorylation. However, the sequence identified in this study is only conserved in some Poaceae species, and in many other plants, PHR is not localized to the chloroplasts. Therefore, we reasoned that Poaceae species need the ability to repair CPD in the chloroplast genome to survive under sunlight and have uniquely acquired this mechanism for PHR chloroplast translocation.

Increased mitochondrial Ca2+ contributes to health decline with age and Duchene muscular dystrophy in C. elegans.

Sarcopenia is a geriatric syndrome characterized by an age-related decline in skeletal muscle mass and strength. Here, we show that suppression of mitochondrial calcium uniporter (MCU)-mediated Ca2+ influx into mitochondria in the body wall muscles of the nematode Caenorhabditis elegans improved the sarcopenic phenotypes, blunting movement and mitochondrial structural and functional decline with age. We found that normally aged muscle cells exhibited elevated resting mitochondrial Ca2+ levels and increased mitophagy to eliminate damaged mitochondria. Similar to aging muscle, we found that suppressing MCU function in muscular dystrophy improved movement via reducing elevated resting mitochondrial Ca2+ levels. Taken together, our results reveal that elevated resting mitochondrial Ca2+ levels contribute to muscle decline with age and muscular dystrophy. Further, modulation of MCU activity may act as a potential pharmacological target in various conditions involving muscle loss.

Transgenic rice Oryza glaberrima with higher CPD photolyase activity alleviates UVB-caused growth inhibition.

The ultraviolet B (UVB) sensitivity of rice cultivated in Asia and Africa varies greatly, with African rice cultivars (Oryza glaberrima Steud. and O. barthii A. Chev.) being more sensitive to UVB because of their low cyclobutane pyrimidine dimer (CPD) photolyase activity, which is a CPD repair enzyme, relative to Asian rice cultivars (O. sativa L.). Hence, the production of UVB-resistant African rice with augmented CPD photolyase activity is of great importance, although difficulty in transforming the African rice cultivars to this end has been reported. Here, we successfully produced overexpressing transgenic African rice with higher CPD photolyase activity by modifying media conditions for callus induction and regeneration using the parental line (PL), UVB-sensitive African rice TOG12380 (O. glaberrima). The overexpressing transgenic African rice carried a single copy of the CPD photolyase enzyme, with a 4.4-fold higher level of CPD photolyase transcripts and 2.6-fold higher activity than its PL counterpart. When the plants were grown for 21 days in a growth chamber under visible radiation or with supplementary various UVB radiation, the overexpressing transgenic plants have a significantly increased UVB resistance index compared to PL plants. These results strongly suggest that CPD photolyase remains an essential factor for tolerating UVB radiation stress in African rice. As a result, African rice cultivars with overexpressed CPD photolyase may survive better in tropical areas more prone to UVB radiation stress, including Africa. Collectively, our results provide strong evidence that CPD photolyase is a useful biotechnological tool for reducing UVB-induced growth inhibition in African rice crops of O. glaberrima.

Histone deacetylase HDA-4-mediated epigenetic regulation in space-flown C. elegans.

Epigenetic changes during long-term spaceflight are beginning to be studied by NASA's twin astronauts and other model organisms. Here, we evaluate the epigenetic regulation of gene expression in space-flown C. elegans by comparing wild type and histone deacetylase (hda)-4 mutants. Expression levels of 39 genes were consistently upregulated in all four generations of adult hda-4 mutants grown under microgravity compared with artificial Earth-like gravity (1G). In contrast, in the wild type, microgravity-induced upregulation of these genes occurred a little. Among these genes, 11 contain the domain of unknown function 19 (DUF-19) and are located in a cluster on chromosome V. When compared with the 1G condition, histone H3 trimethylation at lysine 27 (H3K27me3) increased under microgravity in the DUF-19 containing genes T20D4.12 to 4.10 locus in wild-type adults. On the other hand, this increase was also observed in the hda-4 mutant, but the level was significantly reduced. The body length of wild-type adults decreased slightly but significantly when grown under microgravity. This decrease was even more pronounced with the hda-4 mutant. In ground-based experiments, one of the T20D4.11 overexpressing strains significantly reduced body length and also caused larval growth retardation and arrest. These results indicate that under microgravity, C. elegans activates histone deacetylase HDA-4 to suppress overregulation of several genes, including the DUF-19 family. In other words, the expression of certain genes, including negative regulators of growth and development, is epigenetically fine-tuned to adapt to the space microgravity.

SOG1, a plant-specific master regulator of DNA damage responses, originated from nonvascular land plants.

The suppressor of gamma response 1 (SOG1), a NAM, ATAF1, 2, and CUC2 (NAC)-type transcription factor found in seed plants, is a master regulator of DNA damage responses (DDRs). Upon DNA damage, SOG1 regulates the expression of downstream DDR genes. To know the origin of the DDR network in land plants, we searched for a homolog(s) of SOG1 in a moss Physcomitrium (Physcomitrella) patens and identified PpSOG1a and PpSOG1b. To assess if either or both of them function(s) in DDR, we knocked out the PpSOG1s using CRISPR/Cas9-mediated gene editing and analyzed the responses to DNA-damaging treatments. The double-knockout (KO) sog1a sog1b plants showed resistance to γ-rays, bleomycin, and ultraviolet B (UVB) treatments similarly seen in Arabidopsis sog1 plants. Next, we irradiated wild-type (WT) and KO plants with γ-rays and analyzed the whole transcriptome to examine the effect on the expression of DDR genes. The results revealed that many P. patens genes involved in the checkpoint, DNA repair, replication, and cell cycle-related genes were upregulated after γ-irradiation, which was not seen in sog1a sog1b plant. These results suggest that PpSOG1a and PpSOG1b work redundantly on DDR response in P. patens; in addition, plant-specific DDR systems had been established before the emergence of vascular plants.

Autophagy-deficient Arabidopsis mutant atg5, which shows ultraviolet-B sensitivity, cannot remove ultraviolet-B-induced fragmented mitochondria.

Mitochondria damaged by ultraviolet-B radiation (UV-B, 280-315 nm) are removed by mitophagy, a selective autophagic process. Recently, we demonstrated that autophagy-deficient Arabidopsis thaliana mutants exhibit a UV-B-sensitive phenotype like that of cyclobutane pyrimidine dimer (CPD)-specific photolyase (PHR1)-deficient mutants. To explore the relationship between UV-B sensitivity and autophagy in UV-B-damaged plants, we monitored mitochondrial dynamics and autophagy in wild-type Arabidopsis (ecotype Columbia); an autophagy-deficient mutant, atg5; a PHR1-deficient mutant, phr1; an atg5 phr1 double mutant; and AtPHR1-overexpressing (AtPHR1ox) plants following high-dose UV-B exposure (1.5 W m-2 for 1 h). At 10 h after exposure, the number of mitochondria per mesophyll leaf cell was increased and the volumes of individual mitochondria were decreased independently of UV-B-induced CPD accumulation in all genotypes. At 24 h after exposure, the mitochondrial number had recovered or almost recovered to pre-exposure levels in plants with functional autophagy (WT, phr1, and AtPHR1ox), but had increased even further in atg5. This suggested that the high dose of UV-B led to the inactivation and fragmentation of mitochondria, which were removed by mitophagy activated by UV-B. The UV-B-sensitive phenotype of the atg5 phr1 double mutant was more severe than that of atg5 or phr1. In wild-type, phr1, and AtPHR1ox plants, autophagy-related genes were strongly expressed following UV-B exposure independently of UV-B-induced CPD accumulation. Therefore, mitophagy might be one of the important repair mechanisms for UV-B-induced damage. The severe UV-B-sensitive phenotype of atg5 phr1 is likely an additive effect of deficiencies in independent machineries for UV-B protection, autophagy, and CPD photorepair.

Very high sensitivity of African rice to artificial ultraviolet-B radiation caused by genotype and quantity of cyclobutane pyrimidine dimer photolyase.

Ultraviolet-B (UVB) radiation damages plants and decreases their growth and productivity. We previously demonstrated that UVB sensitivity varies widely among Asian rice (Oryza sativa L.) cultivars and that the activity of cyclobutane pyrimidine dimer (CPD) photolyase, which repairs UVB-induced CPDs, determines UVB sensitivity. Unlike Asian rice, African rice (Oryza glaberrima Steud. and Oryza barthii A. Chev.) has mechanisms to adapt to African climates and to protect itself against biotic and abiotic stresses. However, information about the UVB sensitivity of African rice species is largely absent. We showed that most of the African rice cultivars examined in this study were UVB-hypersensitive or even UVB-super-hypersensitive in comparison with the UVB sensitivity of Asian O. sativa cultivars. The difference in UVB resistance correlated with the total CPD photolyase activity, which was determined by its activity and its cellular content. The UVB-super-hypersensitive cultivars had low enzyme activity caused by newly identified polymorphisms and low cellular CPD photolyase contents. The new polymorphisms were only found in cultivars from West Africa, particularly in those from countries believed to be centres of O. glaberrima domestication. This study provides new tools for improving both Asian and African rice productivity.

Increased hydrostatic pressure induces nuclear translocation of DAF-16/FOXO in C. elegans.

Mechanical stimulation is well known to be important for maintaining tissue and organ homeostasis. Here, we found that hydrostatic pressure induced nuclear translocation of a forkhead box O (FOXO) transcription factor DAF-16, in C. elegans within minutes, whereas the removal of this pressure resulted in immediate export of DAF-16 to the cytoplasm. We also monitored DAF-16-dependent transcriptional changes by exposure to 1 MPa pressure for 5 min, and found significant changes in collagen and other genes in a DAF-16 dependent manner. Lifespan was markedly prolonged with exposure to cyclic pressure treatment (1 MPa once a day for 5 min from L1 larvae until death). Furthermore, age-dependent decline in locomotor activity was suppressed by the treatment. In contrast, the nuclear translocation of the yes-associated protein YAP-1 was not induced under the same pressure conditions. Thus, moderate hydrostatic pressure improves ageing progression through activation of DAF-16/FOXO in C. elegans.

Divergent evolution of rice blast resistance Pi54 locus in the genus Oryza.

BACKGROUND: The rice blast resistance gene Pi54 was cloned from Oryza sativa ssp. indica cv. Tetep, which conferred broad-spectrum resistance against Magnaporthe oryzae. Pi54 allelic variants have been identified in not only domesticates but also wild rice species, but the majority of japonica and some indica cultivars lost the function. RESULTS: We here found that Pi54 (Os11g0639100) and its homolog Os11g0640600 (named as #11) were closely located on a 25 kbp region in japonica cv. Sasanishiki compared to a 99 kbp region in japonica cv. Nipponbare. Sasanishiki lost at least six genes containing one other R-gene cluster (Os11g0639600, Os11g0640000, and Os11g0640300). Eight AA-genome species including five wild rice species were classified into either Nipponbare or Sasanishiki type. The BB-genome wild rice species O. punctata was Sasanishiki type. The FF-genome wild rice species O. brachyantha (the basal lineage of Oryza) was neither, because Pi54 was absent and the orientation of the R-gene cluster was reversed in comparison with Nipponbare-type species. The phylogenetic analysis showed that #11gene of O. brachyantha was on the root of both Pi54 and #11 alleles. All Nipponbare-type Pi54 alleles were specifically disrupted by 143 and 37/44 bp insertions compared to Tetep and Sasanishiki type. In addition, Pi54 of japonica cv. Sasanishiki lost nucleotide-binding site and leucine-rich repeat (NBS-LRR) domains owing to additional mutations. CONCLUSIONS: These results suggest that Pi54 might be derived from a tandem duplication of the ancestor #11 gene in progenitor FF-genome species. Two divergent structures of Pi54 locus caused by a mobile unit containing the nearby R-gene cluster could be developed before domestication. This study provides a potential genetic resource of rice breeding for blast resistance in modern cultivars sustainability.

Yokukansan improves behavioral and psychological symptoms of dementia by suppressing dopaminergic function.

Although three drugs, risperidone, yokukansan, and fluvoxamine, have shown equal efficacy in treating behavioral and psychological symptoms of dementia (BPSD) in our previous study, their mechanisms of action are different from one another. Monoamines have attracted attention for their key roles in mediating several behavioral symptoms or psychological symptoms through synaptic signaling. We aimed to clarify the monoamines changed by treatment with each drug in patients with BPSD. The main purpose of this study was to determine whether plasma levels of catecholamine metabolites are correlated with pharmacological treatments. This was an 8-week, rater-blinded, randomized, flexible-dose, triple-group trial. In total, 90 subjects were recruited and subsequently three different drugs were allocated to 82 inpatients with BPSD. We examined BPSD data from patients who completed 8 weeks of treatment. Eventually, we analyzed 42 patients (yokukansan: 17; risperidone: 9; fluvoxamine: 16). Homovanillic acid, a metabolite of dopamine, and 3-methoxy-4-hydroxyphenylglycol, a metabolite of noradrenaline, in their plasma were analyzed by high-performance liquid chromatography with electrochemical detection. All three drugs showed equal significant efficacy between baseline and study endpoint. By contrast, biomarkers showed mutually different changes. Patients in the yokukansan group had significantly decreased plasma homovanillic acid levels from baseline. Conversely, patients in the risperidone and fluvoxamine groups exhibited no significant changes in plasma homovanillic acid levels from baseline. Yokukansan contains geissoschizine methyl ether, which is known to have a partial agonist effect on dopamine D2 receptors. An improvement in BPSD condition with the intake of yokukansan is suggested to occur through a suppressed dopaminergic function, which is similar to the effect of aripiprazole.

UV-B-Induced CPD Photolyase Gene Expression is Regulated by UVR8-Dependent and -Independent Pathways in Arabidopsis.

Plants have evolved various mechanisms that protect against the harmful effects of UV-B radiation (280-315 nm) on growth and development. Cyclobutane pyrimidine dimer (CPD) photolyase, the repair enzyme for UV-B-induced CPDs, is essential for protecting cells from UV-B radiation. Expression of the CPD photolyase gene (PHR) is controlled by light with various wavelengths including UV-B, but the mechanisms of this regulation remain poorly understood. In this study, we investigated the regulation of PHR expression by light with various wavelengths, in particular low-fluence UV-B radiation (280 nm, 0.2 µmol m(-2) s(-1)), in Arabidopsis thaliana seedlings grown under light-dark cycles for 7 d and then adapted to the dark for 3 d. Low-fluence UV-B radiation induced CPDs but not reactive oxygen species. AtPHR expression was effectively induced by UV-B, UV-A (375 nm) and blue light. Expression induced by UV-A and blue light was predominantly regulated by the cryptochrome-dependent pathway, whereas phytochromes A and B played a minor but noticeable role. Expression induced by UV-B was predominantly regulated by the UVR8-dependent pathway. AtPHR expression was also mediated by a UVR8-independent pathway, which is correlated with CPD accumulation induced by UV-B radiation. These results indicate that Arabidopsis has evolved diverse mechanisms to regulate CPD photolyase expression by multiple photoreceptor signaling pathways, including UVR8-dependent and -independent pathways, as protection against harmful effects of UV-B radiation.

Transport of rice cyclobutane pyrimidine dimer photolyase into mitochondria relies on a targeting sequence located in its C-terminal internal region.

The cyclobutane pyrimidine dimer (CPD), which represents a major type of DNA damage induced by ultraviolet-B (UVB) radiation, is a principal cause of UVB-induced growth inhibition in plants. CPD photolyase is the primary enzyme for repairing CPDs and is crucial for determining the sensitivity of Oryza sativa (rice) to UVB radiation. CPD photolyase is widely distributed among species ranging from eubacteria to eukaryotes, and is classified into class I or II based on its primary structure. We previously demonstrated that rice CPD photolyase (OsPHR), which belongs to class II and is encoded by a single-copy gene, is a unique nuclear/mitochondrial/chloroplast triple-targeting protein; however, the location and nature of the organellar targeting information contained within OsPHR are unknown. Here, the nuclear and mitochondrial targeting signal sequences of OsPHR were identified by systematic deletion analysis. The nuclear and mitochondrial targeting sequences are harbored within residues 487-489 and 391-401 in the C-terminal region of OsPHR (506 amino acid residues), respectively. The mitochondrial targeting signal represents a distinct topogenic sequence that differs structurally and functionally from classical N-terminal pre-sequences, and this region, in addition to its role in localization to the mitochondria, is essential for the proper functioning of the CPD photolyase. Furthermore, the mitochondrial targeting sequence, which is characteristic of class-II CPD photolyases, was acquired before the divergence of class-II CPD photolyases in eukaryotes. These results indicate that rice plants have evolved a CPD photolyase that functions in mitochondria to protect cells from the harmful effects of UVB radiation.

Efficacy and tolerability of risperidone, yokukansan, and fluvoxamine for the treatment of behavioral and psychological symptoms of dementia: a blinded, randomized trial.

The descriptive term behavioral and psychological symptoms of dementia (BPSD) is used to cover a range of noncognitive disturbances including anxiety, depression, irritability, aggression, agitation, eating disorders, and inappropriate social or sexual behaviors. Behavioral and psychological symptoms of dementia are seen in about 90% of patients with dementia. We aimed to compare the efficacy and tolerability of risperidone, yokukansan, and fluvoxamine used for BPSD in elderly patients with dementia. Ninety inpatients with dementia according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria were investigated in Sato Hospital, Koutokukai. We conducted an 8-week, rater-blinded, randomized trial, administering flexibly dosed risperidone, yokukansan, or fluvoxamine. Primary outcome measures were Neuropsychiatric Inventory in Nursing Home Version total score and its items. Secondary outcome measures were cognitive function measured by Mini-Mental State Examination and daily life function measured by Functional Independence Measure (FIM). Neurological adverse effects were measured by the Drug-Induced Extra-Pyramidal Symptoms Scale. At the end of the study, we analyzed 76 patients (92.7%). Mean Neuropsychiatric Inventory in Nursing Home Version total score decreased in all 3 drug groups, with no significant between-group differences. Mini-Mental State Examination and Functional Independence Measure scores did not change significantly. Drug-Induced Extra-Pyramidal Symptoms Scale scores did not change in the yokukansan and fluvoxamine groups, but increased significantly in the risperidone group. Risperidone, yokukansan, and fluvoxamine were equally effective in the treatment of BPSD in elderly patients. However, yokukansan or fluvoxamine for BPSD showed a more favorable profile in tolerability compared with risperidone. This trial is registered at UMIN Clinical Trials Registry (identifier: UMIN000006146).

Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams.

UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar 'Sasanishiki') that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined.

Identification of a phosphorylation site in cyclobutane pyrimidine dimer photolyase of rice.

Cyclobutane pyrimidine dimer (CPD) photolyase monomerises ultraviolet (UV) radiation-induced CPDs present in DNA, using energy from UVA and visible light. In plants, CPD photolyase activity is a crucial factor for determining UVB sensitivity. We previously demonstrated that native rice CPD photolyase is phosphorylated. To determine the phosphorylation site(s), the phosphorylation status of CPD photolyase was analyzed in rice varieties that have amino acid alterations at the potential phosphorylation sites. In wild-rice species, CPD photolyase was phosphorylated. In Poaceae species, CPD photolyase was phosphorylated in wheat but not in maize. Mutant CPD photolyase proteins, in which these putative phosphorylated residues were replaced with alanine residues, were synthesized using an insect cell-free translation system. A slow-migrating band disappeared when the serine residue at position 7 was mutated. A phospho-specific antibody was generated to determine whether this residue is phosphorylated in CPD photolyase. Only the slow-migrating band of native rice CPD photolyase was detected using this antibody, indicating that the serine residue at position 7 is a phosphorylation site in native rice CPD photolyase.

Augmentation of CPD photolyase activity in japonica and indica rice increases their UVB resistance but still leaves the difference in their sensitivities.

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.

Eukaryotic class II cyclobutane pyrimidine dimer photolyase structure reveals basis for improved ultraviolet tolerance in plants.

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 Å 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.

Cyclobutane pyrimidine dimer (CPD) photolyase repairs ultraviolet-B-induced CPDs in rice chloroplast and mitochondrial DNA.

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.

Light-induced activation of class II cyclobutane pyrimidine dimer photolyases.

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 from FAD(ox), and subsequently FADH(-) from FADH, 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.

Visualization of flavonol distribution in the abaxial epidermis of onion scales via detection of its autofluorescence in the absence of chemical processes.

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.