日出間 純

Abstract 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.

Plants distribute many nutrients to chloroplasts during leaf development and maturation. When leaves senesce or experience sugar starvation, the autophagy machinery degrades chloroplast proteins to facilitate efficient nutrient reuse. Here, we report on the intracellular dynamics of an autophagy pathway responsible for piecemeal degradation of chloroplast components. Through live-cell monitoring of chloroplast morphology, we observed the formation of chloroplast budding structures in sugar-starved leaves. The buds were then released and incorporated into the vacuolar lumen as an autophagic cargo termed a Rubisco-containing body. These budding structures did not accumulate in mutants of core autophagy machinery, suggesting that autophagosome creation is required for forming chloroplast protrusions. Simultaneous tracking of chloroplast morphology and autophagosome development revealed that the isolation membranes of autophagosomes tightly interact with part of the chloroplast surface before forming chloroplast buds. Chloroplasts then protrude at the site associated with the isolation membranes, which divide synchronously with autophagosome maturation. This autophagy-related division does not require DYNAMIN-RELATED PROTEIN 5B (DRP5B), which constitutes the division ring for chloroplast proliferation in growing leaves. An unidentified division machinery may thus fragment chloroplasts for degradation in coordination with the development of the chloroplast-associated isolation membrane.

Far-ultraviolet radiation C light (far-UVC; 222 nm wavelength) has received attention as a safer light for killing pathogenic bacteria and viruses, as no or little DNA damage is observed after irradiation in mammalian skin models. Far-UVC does not penetrate deeply into tissues; therefore, it cannot reach the underlying critical basal cells. However, it was unclear whether far-UVC (222-UVC) irradiation could cause more biological damage at shallower depths than the 254 nm UVC irradiation (254-UVC), which penetrates more deeply. This study investigated the biological effects of 222- and 254-UVC on the small and transparent model organism Caenorhabditis elegans. At the same energy level of irradiation, 222-UVC introduced slightly less cyclobutane pyrimidine dimer damage to naked DNA in solution than 254-UVC. The survival of eggs laid during 0-4 h after irradiation showed a marked decrease with 254-UVC but not 222-UVC. In addition, defect of chromosomal condensation was observed in a full-grown oocyte by 254-UVC irradiation. In contrast, 222-UVC had a significant effect on the loss of motility of C. elegans. The sensory nervous system, which includes dopamine CEP and PVD neurons on the body surface, was severely damaged by 222-UVC, but not by the same dose of 254-UVC. Interestingly, increasing 254-UVC irradiation by about 10-fold causes similar damage to CEP neurons. These results suggest that 222-UVC is less penetrating, so energy transfer occurs more effectively in tissues near the surface, causing more severe damage than 254-UVC.

Sensitivity to ultraviolet-B (UVB, 280–315 nm) radiation varies widely among rice (Oryza sativa) cultivars due to differences in the activity of cyclobutane pyrimidines dimer (CPD) photolyase. Interestingly, cultivars with high UVB sensitivity and low CPD photolyase activity have been domesticated in tropical areas with high UVB radiation. Here, we investigated how differences in CPD photolyase activity affect plant resistance to the rice blast fungus, Magnaporthe oryzae, which is one of the other major stresses. We used Asian and African rice cultivars and transgenic lines with different CPD photolyase activities to evaluate the interaction effects of CPD photolyase activity on resistance to M. oryzae. In UVB-resistant rice plants overexpressing CPD photolyase, 12 h of low-dose UVB (0.4 W m−2) pretreatment enhanced sensitivity to M. oryzae. In contrast, UVB-sensitive rice (transgenic rice with antisense CPD photolyase, A-S; and rice cultivars with low CPD photolyase activity) showed resistance to M. oryzae. Several defense-related genes were upregulated in UVB-sensitive rice compared to UVB-resistant rice. UVB-pretreated A-S plants showed decreased multicellular infection and robust accumulation of reactive oxygen species. High UVB-induced CPD accumulation promoted defense responses and cross-protection mechanisms against rice blast disease. This may indicate a trade-off between high UVB sensitivity and biotic stress tolerance in tropical rice cultivars. Graphical Abstract: [Figure not available: see fulltext.].

第36回宇宙環境利用シンポジウム (2022年1月18日-19日. オンライン開催) Space Utilization Research (January 18-19, 2022. Online Meeting) 資料番号: SA6000168015 F-03

Abstract Lamps that emit 222 nm short-wavelength ultraviolet (UV) radiation can be safely used for sterilization without harming human health. However, there are few studies on the effects of 222 nm UVC (222-UVC) radiation exposure on plants compared with the effects of germicidal lamps emitting primarily 254 nm UVC (254-UVC) radiation. We investigated the growth inhibition and cell damage caused by 222-UVC exposure to Arabidopsis plants, especially mitochondrial dynamics, which is an index of damage caused by UVB radiation. Growth inhibition resulted from 254-UVC or 222-UVC exposure depending on the dose of UVC radiation. However, with respect to the phenotype of 222-UVC-irradiated plants, the leaves curled under 1 kJ m⁻² and were markedly bleached under 10 kJ m⁻² compared with those of plants irradiated with 254-UVC. The cellular state, especially the mitochondrial dynamics, of epidermal and mesophyll cells of Arabidopsis leaves exposed to 254-UVC or 222-UVC radiation was investigated using Arabidopsis plants expressing mitochondrial matrix-targeted yellow fluorescent protein (MT-YFP) under the control of Pro35S to visualize the mitochondria. 222-UVC (1 or 5 kJ m⁻²) severely damaged the guard cells within the epidermis, and YFP signals and chloroplast autofluorescence in guard cells within the epidermis exposed to 222-UVC (1 or 5 kJ m⁻²) were not detected compared with those in cells exposed to 254-UVC radiation. In addition, 222-UVC irradiation led to mitochondrial fragmentation in mesophyll cells, similar to the effects of 254-UVC exposure. These results suggest that 222-UVC severely damages guard cells and epidermal cells and that such damage might have resulted in growth inhibition.

Responses of rice seedlings to UV-B radiation (UV-B) were investigated, aiming to establish rice as a model plant for UV-B signalling studies. The growth of japonica rice coleoptiles, grown under red light, was inhibited by brief irradiation with UV-B, but not with blue light. The effective UV-B fluences (10-1 -103 μmol m-2 ) were much lower than those reported in Arabidopsis. The response was much less in indica rice cultivars and its extent varied among Oryza species. We next identified UV-B-specific anthocyanin accumulation in the first leaf of purple rice and used this visible phenotype to isolate mutants. Some isolated mutants were further characterized, and one was found to have a defect in the growth response. Using microarrays, we identified a number of genes that are regulated by low-fluence-rate UV-B in japonica coleoptiles. Some up-regulated genes were analysed by real-time PCR for UV-B specificity and the difference between japonica and indica. More than 70% of UV-B-regulated rice genes had no homologs in UV-B-regulated Arabidopsis genes. Many UV-B-regulated rice genes are related to plant hormones and especially to jasmonate biosynthetic and responsive genes in apparent agreement with the growth response. Possible involvement of two rice homologs of UVR8, a UV-B photoreceptor, is discussed.

There has been significant interest in the photosensitivity, or photo-resistance, of Japanese rice cultivars, which synthesize tocols (Vitamin E), a class of phytochemicals including tocol derivatives tocopherol (T) and tocotrienol (T3). In the present study, the distribution of tocols in the leaves, seeds, stems, and roots of six Japanese rice cultivars was investigated. The relationship between the different tocols in cultivars and their ultraviolet B sensitivity index (USB-SI) was analyzed. The leaves contained the highest average total amount of tocols at 230 μg.fresh-g-1, followed by seeds, stems, and roots. In leaves and stems, the most abundant component was α-T which was more than 85%. On the other hand, the tocols in seeds were 38% δ-T3, 32% α-T, and 20% α-T3. The tocols in roots were 55% α-T, 14% γ-T, and 13% δ-T3. The total tocol content in four plant parts exhibited a negative correlation (P < 0.05) in stem and root, and a negative relationship (r < -0.70) with the UVB-SI of the cultivars, suggesting that the total tocol contents were closely related to the resistance to UVB in Japanese rice plants.

In autophagy, cytoplasmic components of eukaryotic cells are transported to lysosomes or the vacuole for degradation. Autophagy is involved in plant tolerance to the photooxidative stress caused by ultraviolet B (UVB) radiation, but its roles in plant adaptation to UVB damage have not been fully elucidated. Here, we characterized organellar behavior in UVB-damaged Arabidopsis (Arabidopsis thaliana) leaves and observed the occurrence of autophagic elimination of dysfunctional mitochondria, a process termed mitophagy. Notably, Arabidopsis plants blocked in autophagy displayed increased leaf chlorosis after a 1-h UVB exposure compared to wild-type plants. We visualized autophagosomes by labeling with a fluorescent protein-tagged autophagosome marker, AUTOPHAGY8 (ATG8), and found that a 1-h UV-B treatment led to increased formation of autophagosomes and the active transport of mitochondria into the central vacuole. In atg mutant plants, the mitochondrial population increased in UVB-damaged leaves due to cytoplasmic accumulation of fragmented, depolarized mitochondria. Furthermore, we observed that autophagy was involved in the removal of depolarized mitochondria when mitochondrial function was disrupted by mutation of the FRIENDLY gene, which is required for proper mitochondrial distribution. Therefore, autophagy of mitochondria functions in response to mitochondrion-specific dysfunction as well as UVB damage. Together, these results indicate that autophagy is centrally involved in mitochondrial quality control in Arabidopsis leaves.

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.

Deep space exploration by humans has become more realistic, with planned returns to the Moon, travel to Mars, and beyond. Space radiation with a low dose rate would be a constant risk for space travelers. The combined effects of space radiation and partial gravity such as on the Moon and Mars are unknown. The difficulty for such research is that there are no good simulating systems on the ground to investigate these combined effects. To address this knowledge gap, we developed the Simulator of the environments on the Moon and Mars with Neutron irradiation and Gravity change (SwiNG) for in vitro experiments using disposable closed cell culture chambers. The device simulates partial gravity using a centrifuge in a three-dimensional clinostat. Six samples are exposed at once to neutrons at a low dose rate (1 mGy/day) using Californium-252 in the center of the centrifuge. The system is compact including two SwiNG devices in the incubator, one with and one without radiation source, with a cooling function. This simulator is highly convenient for ground-based biological experiments because of limited access to spaceflight experiments. SwiNG can contribute significantly to research on the combined effects of space radiation and partial gravity.

Autophagy and the ubiquitin-proteasome system are the major degradation processes for intracellular components in eukaryotes. Although ubiquitination acts as a signal inducing organelle-targeting autophagy, the interaction between ubiquitination and autophagy in chloroplast turnover has not been addressed. In this study, we found that two chloroplast-associated E3 enzymes, SUPPRESSOR OF PPI1 LOCUS1 and PLANT U-BOX4 (PUB4), are not necessary for the induction of either piecemeal autophagy of chloroplast stroma or chlorophagy of whole damaged chloroplasts in Arabidopsis (Arabidopsis thaliana). Double mutations of an autophagy gene and PUB4 caused synergistic phenotypes relative to single mutations. The double mutants developed accelerated leaf chlorosis linked to the overaccumulation of reactive oxygen species during senescence and had reduced seed production. Biochemical detection of ubiquitinated proteins indicated that both autophagy and PUB4-associated ubiquitination contributed to protein degradation in the senescing leaves. Furthermore, the double mutants had enhanced susceptibility to carbon or nitrogen starvation relative to single mutants. Together, these results indicate that autophagy and chloroplast-associated E3s cooperate for protein turnover, management of reactive oxygen species accumulation, and adaptation to starvation.

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.

Outer space is an environment characterized by microgravity and space radiation, including high-energy charged particles. Astronauts are constantly exposed to both microgravity and radiation during long-term stays in space. However, many aspects of the biological effects of combined microgravity and space radiation remain unclear. We developed a new three-dimensional (3D) clinostat synchronized heavy-ion irradiation system for use in ground-based studies of the combined exposures. Our new system uses a particle accelerator and a respiratory gating system from heavy-ion radiotherapy to irradiate samples being rotated in the 3D clinostat with carbon-ion beams only when the samples are in the horizontal position. A Peltier module and special sample holder were loaded on a static stage (standing condition) and the 3D clinostat (rotation condition) to maintain a suitable temperature under atmospheric conditions. The performance of the new device was investigated with normal human fibroblasts 1BR-hTERT in a disposable closed cell culture chamber. Live imaging revealed that cellular adhesion and growth were almost the same for the standing control sample and rotation sample over 48 h. Dose flatness and symmetry were judged according to the relative density of Gafchromic films along the X-axis and Y-axis of the positions of the irradiated sample to confirm irradiation accuracy. Doses calculated using the carbon-ion calibration curve were almost the same for standing and rotation conditions, with the difference being less than 5% at 1 Gy carbon-ion irradiation. Our new device can accurately synchronize carbon-ion irradiation and simulated microgravity while maintaining the temperature under atmospheric conditions at ground level.

Turnover of dysfunctional organelles is vital to maintain homeostasis in eukaryotic cells. As photosynthetic organelles, plant chloroplasts can suffer sunlight-induced damage. However, the process for turnover of entire damaged chloroplasts remains unclear. Here, we demonstrate that autophagy is responsible for the elimination of sunlight-damaged, collapsed chloroplasts in Arabidopsis thaliana. We found that vacuolar transport of entire chloroplasts, termed chlorophagy, was induced by UV-B damage to the chloroplast apparatus. This transport did not occur in autophagy-defective atg mutants, which exhibited UV-B-sensitive phenotypes and accumulated collapsed chloroplasts. Use of a fluorescent protein marker of the autophagosomal membrane allowed us to image autophagosome-mediated transport of entire chloroplasts to the central vacuole. In contrast to sugar starvation, which preferentially induced distinct type of chloroplast-targeted autophagy that transports a part of stroma via the Rubisco-containing body (RCB) pathway, photooxidative damage induced chlorophagy without prior activation of RCB production. We further showed that chlorophagy is induced by chloroplast damage caused by either artificial visible light or natural sunlight. Thus, this report establishes that an autophagic process eliminates entire chloroplasts in response to light-induced damage.

Space radiation is one of the major hazards for human health in space. Space radiation consists of various kinds of radiation including high energy heavy (HZE) ions; these complex radiation fields cannot be produced on the ground. In the International Space Station (ISS), exposure doses are approximately 200 times higher than on the Earth's surface. On the Moon and Mars, the radiation level is high, due to HZE ions. While the ISS is in free fall, the Moon has 1/6, Mars 1/3 of Earth's gravity. Many aspects of the biological effect of the combination of the lower gravity environment and space radiation remain unclear. But the future mission is now being planned to go to the Moon and Mars. It is necessary to clarify the problem of biological effect and physical dosimetry and then to resolve them as soon as possible. With this thought, here we try to present a scenario of space radiation research for next decades.

Green plants produce carbohydrate as an energy for all organisms by photosynthesis. It is therefore considered that plant cultivation is necessary for life support not only on Earth but also in space. To inhabit the space for a long duration, human needs to be closed in the life support system in which plants provide them with foods and a stress-relief circumstance. During evolution, on the other hand, plants developed various strategies to survive terrestrial environment on Earth because of their sessile nature. Plant responses to gravity and lights are examples of such strategy to avoid or mitigate stressful environment they come across. Now, space environment is available for biological studies to understand how plants respond to gravity and how plants are influenced by microgravity and/or space radiation. We extend such studies to understand the effects of space environment on plant growth and development in the seed-to-seed or the generation-to-generation experiments. To explore the deeper space or inhabit planets such as the moon or Mars, we next need to establish a sustainable recycling-oriented life support system with plant cultivation and environmental control facilities. Here, we show our research scenario of the space-utilizing plant science to achieve such objective, which is important to efficiently cultivate plants and develop the life support system in space. We believe our approach, in cooperation with various communities of the related fields, enables us to further reveal the biological systems required for not only colonizing to space but also conserving or improving the living Earth.

Understanding the mechanisms of adaptation of alpine aquatic organisms to the alpine environment, one of the ecosystems most vulnerable to climate change, would help us predict how those organisms would respond to the upcoming climate change. Here, we examined how one of Japan's endemic crustaceans, Daphnia tanakai, which has been found only in alpine lakes and pools in high mountain moors, is adapted to the alpine environment and what restricts its distribution only to the alpine environment. Lab experiments on UVB tolerance and growth rate under different pH and temperature based on the field survey for pools in mountain moors revealed that D. tanakai was differentiated from two low-altitude species (Daphnia dentifera and Daphnia pulicaria) by its tolerance to low pH such as 4.5, which is equal to the mean pH of pools in the high mountain moors occupied by D. tanakai. The growth rate at pH 4.5 was, however, significantly lower than that at higher pH, suggesting that low pH was not necessarily an optimal condition, and that other factors restrict the distribution to alpine lakes. Vulnerability to invertebrate predators such as Chaoborus was suggested as such a factor by additional experiments on swimming activity and predation and the fact that D. tanakai has only been found in pools without Chaoborus. Because it is known that the pH of alpine lakes is tightly linked with temperature, global warming may diminish the advantage of D. tanakai and allow invasion of alpine lakes by unwelcome predators and competitors from lower altitudes.

Understanding the mechanisms of adaptation of alpine aquatic organisms to the alpine environment, one of the ecosystems most vulnerable to climate change, would help us predict how those organisms would respond to the upcoming climate change. Here, we examined how one of Japan's endemic crustaceans, Daphnia tanakai, which has been found only in alpine lakes and pools in high mountain moors, is adapted to the alpine environment and what restricts its distribution only to the alpine environment. Lab experiments on UVB tolerance and growth rate under different pH and temperature based on the field survey for pools in mountain moors revealed that D. tanakai was differentiated from two low-altitude species (Daphnia dentifera and Daphnia pulicaria) by its tolerance to low pH such as 4.5, which is equal to the mean pH of pools in the high mountain moors occupied by D. tanakai. The growth rate at pH 4.5 was, however, significantly lower than that at higher pH, suggesting that low pH was not necessarily an optimal condition, and that other factors restrict the distribution to alpine lakes. Vulnerability to invertebrate predators such as Chaoborus was suggested as such a factor by additional experiments on swimming activity and predation and the fact that D. tanakai has only been found in pools without Chaoborus. Because it is known that the pH of alpine lakes is tightly linked with temperature, global warming may diminish the advantage of D. tanakai and allow invasion of alpine lakes by unwelcome predators and competitors from lower altitudes.

Ultraviolet-B (UV-B; 280-315 nm) radiation has been shown to be more stressful for plants at higher elevations. Species inhabiting different origins may have evolutionarily altered UV tolerance to match their phenotypes to local conditions. However, little is known about UV adaptation patterns between high- and lowland. Here, we evaluated UV damage to DNA and growth in ecotypes of two species of Arabidopsis from different elevations, four ecotypes of A. thaliana and three ecotypes of A. hailed subsp. gemmifera under supplemental UV-B. Harvests were done before UV-B treatment and at early and late stages after the exposure to enhanced UV-B irradiation. The accumulation level of cyclobutane pyrimidine dimer (CPD) was determined as a measure of UV damage to DNA. At the early stage, lowland ecotypes of two species exhibited a higher CPD level and greater inhibition in biomass production, indicating that lowland ecotypes were more sensitive to increased UV-B than highland ecotypes. In contrast, at the later stage, CPD level and growth inhibition became similar or even lower in lowland ecotypes. These results suggest that the response to UV stress was constitutive in highland ecotypes but more inducible in lowland ecotypes. The relative growth rate was negatively related to CPD level. These ecotypic differentiations were common to two Arabidopsis species, suggesting that local adaptation occurred in parallel under a constraint of tradeoff between growth and UV tolerance. (C) 2016 Elsevier B.V. All rights reserved.

Ultraviolet-B (UV-B; 280-315 nm) radiation has been shown to be more stressful for plants at higher elevations. Species inhabiting different origins may have evolutionarily altered UV tolerance to match their phenotypes to local conditions. However, little is known about UV adaptation patterns between high- and lowland. Here, we evaluated UV damage to DNA and growth in ecotypes of two species of Arabidopsis from different elevations, four ecotypes of A. thaliana and three ecotypes of A. hailed subsp. gemmifera under supplemental UV-B. Harvests were done before UV-B treatment and at early and late stages after the exposure to enhanced UV-B irradiation. The accumulation level of cyclobutane pyrimidine dimer (CPD) was determined as a measure of UV damage to DNA. At the early stage, lowland ecotypes of two species exhibited a higher CPD level and greater inhibition in biomass production, indicating that lowland ecotypes were more sensitive to increased UV-B than highland ecotypes. In contrast, at the later stage, CPD level and growth inhibition became similar or even lower in lowland ecotypes. These results suggest that the response to UV stress was constitutive in highland ecotypes but more inducible in lowland ecotypes. The relative growth rate was negatively related to CPD level. These ecotypic differentiations were common to two Arabidopsis species, suggesting that local adaptation occurred in parallel under a constraint of tradeoff between growth and UV tolerance. (C) 2016 Elsevier B.V. All rights reserved.

High doses of ultraviolet-B (UV-B; 280-315 nm) radiation can have detrimental effects on plants, and especially damage their DNA. Plants have DNA repair and protection mechanisms to prevent UV-B damage. However, it remains unclear how DNA damage and tolerance mechanisms vary among field species. We studied DNA damage and tolerance mechanisms in 26 species with different functional groups coexisting in two moorlands at two elevations. We collected current-year leaves in July and August, and determined accumulation of cyclobutane pyrimidine dimer (CPD) as UV-B damage and photorepair activity (PRA) and concentrations of UV-absorbing compounds (UACs) and carotenoids (CARs) as UV-B tolerance mechanisms. DNA damage was greater in dicot than in monocot species, and higher in herbaceous than in woody species. Evergreen species accumulated more CPDs than deciduous species. PRA was higher in Poaceae than in species of other families. UACs were significantly higher in woody than in herbaceous species. The CPD level was not explained by the mechanisms across species, but was significantly related to PRA and UACs when we ignored species with low CPD, PRA and UACs, implying the presence of another effective tolerance mechanism. UACs were correlated negatively with PRA and positively with CARs. Our results revealed that UV-induced DNA damage significantly varies among native species, and this variation is related to functional groups. DNA repair, rather than UV-B protection, dominates in UV-B tolerance in the field. Our findings also suggest that UV-B tolerance mechanisms vary among species under evolutionary trade-off and synergism.

A striking feature of plant nuclei is their light-dependent movement. In Arabidopsis (Arabidopsis thaliana) leaf mesophyll cells, the nuclei move to the side walls of cells within 1 to 3 h after blue-light reception, although the reason is unknown. Here, we show that the nuclear movement is a rapid and effective strategy to avoid ultraviolet B (UVB)-induced damages. Mesophyll nuclei were positioned on the cell bottom in the dark, but sudden exposure of these cells to UVB caused severe DNA damage and cell death. The damage was remarkably reduced in both blue-light-treated leaves and mutant leaves defective in the actin cytoskeleton. Intriguingly, in plants grown under high-light conditions, the mesophyll nuclei remained on the side walls even in the dark. These results suggest that plants have two strategies for reducing UVB exposure: rapid nuclear movement against acute exposure and nuclear anchoring against chronic exposure.

Autophagy is an evolutionarily conserved process leading to the degradation of intracellular components in eukaryotes, which is important for nutrient recycling especially in response to starvation conditions. Nutrient recycling is an essential process that underpins productivity in crop plants, such that remobilized nitrogen derived from older organs supports the formation of new organs or grain-filling within a plant. We extended our understanding of autophagy in a model plant, Arabidopsis thaliana, to an important cereal, rice (Oryza sativa). Through analysis of transgenic rice plants stably expressing fluorescent marker proteins for autophagy or chloroplast stroma, we revealed that chloroplast proteins are partially degraded in the vacuole via Rubisco-containing bodies (RCBs), a type of autophagosomes containing stroma. We further reported evidence that the RCB pathway functions during natural leaf senescence to facilitate subsequent nitrogen remobilization into newly expanding leaves. Thus, our recent studies establish the importance of autophagy in biomass production of cereals.

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 mu mol 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.

Autophagy is an intracellular process leading to vacuolar or lysosomal degradation of cytoplasmic components in eukaryotes. Establishment of proper methods to monitor autophagy was a key step in uncovering its role in organisms, such as yeast (Saccharomyces cerevisiae), mammals, and Arabidopsis (Arabidopsis thaliana), in which chloroplastic proteins were found to be recycled by autophagy. Chloroplast recycling has been predicted to function in nutrient remobilization for growing organs or grain filling in cereal crops. Here, to develop our understanding of autophagy in cereals, we established monitoring methods for chloroplast autophagy in rice (Oryza sativa). We generated transgenic rice-expressing fluorescent protein (FP) OsAuTophaGy8 (OsATG8) fusions as autophagy markers. FP-ATG8 signals were delivered into the vacuolar lumen in living cells of roots and leaves mainly as vesicles corresponding to autophagic bodies. This phenomenon was not observed upon the addition of wortmannin, an inhibitor of autophagy, or in an ATG7 knockout mutant. Markers for the chloroplast stroma, stromal FP, and FP-labeled Rubisco were delivered by a type of autophagic body called the Rubisco-containing body (RCB) in the same manner. RCB production in excised leaves was suppressed by supply of external sucrose or light. The release of free FP caused by autophagy-dependent breakdown of FP-labeled Rubisco was induced during accelerated senescence in individually darkened leaves. In roots, nongreen plastids underwent both RCB-mediated and entire organelle types of autophagy. Therefore, our newly developed methods to monitor autophagy directly showed autophagic degradation of leaf chloroplasts and root plastids in rice plants and its induction during energy limitation.

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 classI or II based on its primary structure. We previously demonstrated that rice CPD photolyase (OsPHR), which belongs to classII 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.

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 classI or II based on its primary structure. We previously demonstrated that rice CPD photolyase (OsPHR), which belongs to classII 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.

Premise of the study: Although ultraviolet radiation (UV) is known to have negative effects on plant growth, there has been no direct evidence that plants growing at higher elevations are more severely affected by ultraviolet-B (UV-B) radiation, which is known to increase with elevation. We examined damage to DNA, a primary target of UV-B, in the widespread species Polygonum sachalinense (Fallopia sachalinensis) and Plantago asiatica at two elevations. Methods: We sampled leaves of both species at 300 and 1700 m above sea level every 2 h for 11 d across the growing season and determined the level of cyclobutane pyrimidine dimer (CPD), a major product of UV damage to DNA. Key results: The CPD level was significantly influenced by the time of day, date, elevation, and their interactions in both species. The CPD level tended to be higher at noon or on sunny days. DNA damage was more severe at 1700 m than at 300 m: on average, 8.7% greater at high elevation in P. asiatica and 7.8% greater in P. sachalinense. Stepwise multiple regression analysis indicated that the CPD level was explained mainly by UV-B and had no significant relationship with other environmental factors such as temperature and photosynthetically active radiation. Conclusions: UV-induced DNA damage in plants is greater at higher elevations.

Screening of 40,000 Arabidopsis FOX (Full-length cDNA Over-eXpressor gene hunting system) lines expressing rice full-length cDNAs brings us to identify four cadmium (Cd)-tolerant lines, one of which carried OsREX1-S as a transgene. OsREX1-S shows the highest levels of identity to Chlamydomonas reinhardtii REX1-S (referred to as CrREX1-S, in which REX denotes Required for Excision) and to yeast and human TFB5s (RNA polymerase II transcription factor B5), both of which are components of the general transcription and DNA repair factor, TFIIH. Transient expression of OsREX1-S consistently localized the protein to the nucleus of onion cells. The newly generated transgenic Arabidopsis plants expressing OsREX1-S reproducibly displayed enhanced Cd tolerance, confirming that the Cd-tolerance of the initial identified line was conferred solely by OsREX1-S expression. Furthermore, transgenic Arabidopsis plants expressing OsREX1-S exhibited ultraviolet-B (UVB) tolerance by reducing the amounts of cyclobutane pyrimidine dimers produced by UVB radiation. Moreover, those transgenic OsREX1-S Arabidopsis plants became resistant to bleomycin (an inducer of DNA strand break) and mitomycin C (DNA intercalating activity), compared to wild type. Our results indicate that OsREX1-S renders host plants tolerant to Cd, UVB radiation, bleomycin and mitomycin C through the enhanced DNA excision repair.

To assess the mutational effects of radiation on vigorously proliferating plant tissue, the mutation spectrum was analyzed with Arabidopsis seedlings using the plasmid-rescue method. Transgenic plants containing the Escherichia coli rpsL gene were irradiated with gamma-rays and carbon ion beams (320-MeV C-12(6+)), and mutations in the rpsL gene were analyzed. Mutant frequency increased significantly following irradiation by gamma-rays, but not by 320-MeV C-12(6+). Mutation spectra showed that both radiations increased the frequency of frameshifts and other mutations, including deletions and insertions, but only gamma-rays increased the frequency of total base substitutions. These results suggest that the type of DNA lesions which cause base substitutions were less often induced by 320-MeV C-12(6+) than by gamma-rays in Arabidopsis seedlings. Furthermore, gamma-rays never increased the frequencies of G:C to T:A or A:T to C:G transversions, which are caused by oxidized guanine; 320-MeV C-12(6+), however, produced a slight increase in both transversions. Instead, gamma-rays produced a significant increase in the frequency of G:C to A:T transitions. These results suggest that 8-oxoguanine has little effect on mutagenesis in Arabidopsis cells.

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 M-1 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.

Autophagy is an intracellular process leading to the vacuolar degradation of cytoplasmic components. Autophagic degradation of chloroplasts is particularly activated in leaves under conditions of low sugar availability. Here, we investigated the importance of autophagy in the energy availability and growth of Arabidopsis (Arabidopsis thaliana). autophagy-deficient (atg) mutants showed reduced growth under short-day conditions. This growth inhibition was largely relieved under continuous light or under short-day conditions combined with feeding of exogenous sucrose, suggesting that autophagy is involved in energy production at night for growth. Arabidopsis accumulates starch during the day and degrades it for respiration at night. Nighttime energy availability is perturbed in starchless mutants, in which a lack of starch accumulation causes a transient sugar deficit at night. We generated starchless and atg double mutants and grew them under different photoperiods. The double mutants showed more severe phenotypes than did atg or starchless single mutants: reduced growth and early cell death in leaves were observed when plants were grown under 10-h photoperiods. Transcript analysis of dark-inducible genes revealed that the sugar starvation symptoms observed in starchless mutants became more severe in starchless atg double mutants. The contents of free amino acids (AAs) increased, and transcript levels of several genes involved in AA catabolism were elevated in starchless mutant leaves. The increases in branched-chain AA and aromatic AA contents were partially compromised in starchless atg double mutants. We conclude that autophagy can contribute to energy availability at night by providing a supply of alternative energy sources such as AAs.

In this study, we examined the physiological mechanisms in the responses of Arabidopsis mutant sensitive to ABA and drought 2-1 (sad2-1) to ultraviolet-B radiation (UVB) treatments. The effects of enhanced UVB radiation on plant growth, concentration of UV-absorbing compounds, photosynthesis, endogenous ABA and antioxidant system were investigated in two types of Arabidopsis thaliana the mutant sad2-1 and the wild type (WT, C24). Results indicated that, under UVB radiation, mutant sad2-1 showed a higher resistance than C24 through accumulating more UV absorption materials, maintaining bio-membrane balance and photosynthesis efficiency, enhancing endogenous ABA content and activating ROS scavenging enzymes. It can be postulated that ABA might participate in a complex signal crosstalk in increasing the tolerance of UVB. (C) 2012 SAAB. Published by Elsevier B.V. All rights reserved.

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. © 2012 Elsevier Masson SAS.