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.

High ultraviolet-B sensitivity due to lower CPD photolyase activity is needed for biotic stress response to the rice blast fungus, Magnaporthe oryzae.

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.

Autophagy Contributes to the Quality Control of Leaf Mitochondria.

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 UVB 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 the 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.

Chloroplast Autophagy and Ubiquitination Combine to Manage Oxidative Damage and Starvation Responses.

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.

UV-B signalling in rice: Response identification, gene expression profiling and mutant isolation.

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.

222 nm ultraviolet radiation C causes more severe damage to guard cells and epidermal cells of Arabidopsis plants than does 254 nm ultraviolet radiation.

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.

SUPPRESSOR OF GAMMA RESPONSE 1 acts as a regulator coordinating crosstalk between DNA damage response and immune response in Arabidopsis thaliana.

Plants live in constantly changing and often unfavorable or stressful environments. Environmental changes induce biotic and abiotic stress, which, in turn, may cause genomic DNA damage. Hence, plants simultaneously suffer abiotic/biotic stress and DNA damage. However, little information is available on the signaling crosstalk that occurs between DNA damage and abiotic/biotic stresses. Arabidopsis thaliana SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) is a pivotal transcription factor that regulates thousands of genes in response to DNA double-strand break (DSB), and we recently reported that SOG1 has a role in immune responses. In the present study, the effects of SOG1 overexpression on the DNA damage and immune responses were examined. Results found that SOG1 overexpression enhances the regulation of numerous downstream genes. Relative to the wild type plants, then, DNA damage responses were observed to be strongly induced. SOG1 overexpression also upregulates chitin (a major components of fungal cell walls) responsive genes in the presence of DSBs, implying that pathogen defense response is activated by DNA damage via SOG1. Further, SOG1 overexpression enhances fungal resistance. These results suggest that SOG1 regulates crosstalk between DNA damage response and the immune response and that plants have evolved a sophisticated defense network to contend with environmental stress.

Entire Photodamaged Chloroplasts Are Transported to the Central Vacuole by Autophagy.

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.

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.

Selective Elimination of Membrane-Damaged Chloroplasts via Microautophagy.

Plant chloroplasts constantly accumulate damage caused by visible wavelengths of light during photosynthesis. Our previous study revealed that entire photodamaged chloroplasts are subjected to vacuolar digestion through an autophagy process termed chlorophagy; however, how this process is induced and executed remained poorly understood. In this study, we monitored intracellular induction of chlorophagy in Arabidopsis (Arabidopsis thaliana) leaves and found that mesophyll cells damaged by high visible light displayed abnormal chloroplasts with a swollen shape and 2.5 times the volume of normal chloroplasts. In wild-type plants, the activation of chlorophagy decreased the number of swollen chloroplasts. In the autophagy-deficient autophagy mutants, the swollen chloroplasts persisted, and dysfunctional chloroplasts that had lost chlorophyll fluorescence accumulated in the cytoplasm. Chloroplast swelling and subsequent induction of chlorophagy were suppressed by the application of exogenous mannitol to increase the osmotic pressure outside chloroplasts or by overexpression of VESICLE INDUCING PROTEIN IN PLASTID1, which maintains chloroplast envelope integrity. Microscopic observations of autophagy-related membranes showed that swollen chloroplasts were partly surrounded by autophagosomal structures and were engulfed directly by the tonoplast, as in microautophagy. Our results indicate that an elevation in osmotic potential inside the chloroplast due to high visible light-derived envelope damage results in chloroplast swelling and serves as an induction factor for chlorophagy, and this process mobilizes entire chloroplasts via tonoplast-mediated sequestering to avoid the cytosolic accumulation of dysfunctional chloroplasts.

Expression Profile of Cell Cycle-Related Genes in Human Fibroblasts Exposed Simultaneously to Radiation and Simulated Microgravity.

Multiple unique environmental factors such as space radiation and microgravity (µG) pose a serious threat to human gene stability during space travel. Recently, we reported that simultaneous exposure of human fibroblasts to simulated µG and radiation results in more chromosomal aberrations than in cells exposed to radiation alone. However, the mechanisms behind this remain unknown. The purpose of this study was thus to obtain comprehensive data on gene expression using a three-dimensional clinostat synchronized to a carbon (C)-ion or X-ray irradiation system. Human fibroblasts (1BR-hTERT) were maintained under standing or rotating conditions for 3 or 24 h after synchronized C-ion or X-ray irradiation at 1 Gy as part of a total culture time of 2 days. Among 57,773 genes analyzed with RNA sequencing, we focused particularly on the expression of 82 cell cycle-related genes after exposure to the radiation and simulated µG. The expression of cell cycle-suppressing genes (ABL1 and CDKN1A) decreased and that of cell cycle-promoting genes (CCNB1, CCND1, KPNA2, MCM4, MKI67, and STMN1) increased after C-ion irradiation under µG. The cell may pass through the G1/S and G2 checkpoints with DNA damage due to the combined effects of C-ions and µG, suggesting that increased genomic instability might occur in space.

Plant Nuclei Move to Escape Ultraviolet-Induced DNA Damage and Cell Death.

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.

Establishment of monitoring methods for autophagy in rice reveals autophagic recycling of chloroplasts and root plastids during energy limitation.

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.

Ultraviolet-B-induced DNA damage and ultraviolet-B tolerance mechanisms in species with different functional groups coexisting in subalpine moorlands.

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.

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.

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.

Overexpression of rice OsREX1-S, encoding a putative component of the core general transcription and DNA repair factor IIH, renders plant cells tolerant to cadmium- and UV-induced damage by enhancing DNA excision repair.

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.

Autophagy contributes to nighttime energy availability for growth in Arabidopsis.

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.

Is UV-induced DNA damage greater at higher elevation?

UNLABELLED: • 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.

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.