UV-B-induced DNA damage and repair pathways in polar Pseudogymnoascus sp. from the Arctic and Antarctic regions and their effects on growth,pigmentation and conidiogenesis

Solar radiation regulates most biological activities on Earth. Prolonged exposure to solar UV radiation can cause deleterious effects by inducing two major types of DNA damage, namely, cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts. These lesions may be repaired by the photoreactivation (Phr) and nucleotide excision repair (NER) pathways; however, the principal UV-induced DNA repair pathway is not known in the fungal genus Pseudogymnoascus. In this study, we demonstrated that an unweighted UV-B dosage of 1.6 kJ m⁻² d⁻¹ significantly reduced fungal growth rates (by between 22% and 35%) and inhibited conidia production in a 10 d exposure. The comparison of two DNA repair conditions, light or dark, which respectively induced photoreactivation (Phr) and NER, showed that the UV-B-induced CPDs were repaired significantly more rapidly in light than in dark conditions. The expression levels of two DNA repair genes, RAD2 and PHR1 (encoding a protein in NER and Phr respectively), demonstrated that NER rather than Phr was primarily activated for repairing UV-B-induced DNA damage in these Pseudogymnoascus strains. In contrast, Phr was inhibited after exposure to UV-B radiation, suggesting that PHR1 may have other functional roles. We present the first study to examine the capability of the Arctic and Antarctic Pseudogymnoascus sp. to perform photoreactivation and/or NER via RT-qPCR approaches, and also clarify the effects of light on UV-B-induced DNA damage repair in vivo by quantifying cyclobutene pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts. Physiological response data, including relative growth rate, pigmentation and conidia production in these Pseudogymnoascus isolates exposed to UV-B radiation are also presented.     

A comparison of photolyase activity in three Australian tree frogs

Ultraviolet-B (UV-B) irradiation of DNA generates mutagenic photoproducts such as cyclobutane pyrimidine dimers (CPDs) which can affect the growth and development of amphibian embryos. Differential ability to repair UV-B-induced DNA damage may be␣responsible for differences in population stability between␣some amphibian species. Photoreactivation via the enzyme photolyase is a major mechanism used to remove CPDs from DNA. The aim of this study was to determine if photolyase activity differed in three sympatric Australian amphibian species, one of which has suffered marked population declines (Litoria aurea) and two whose populations do not appear to be in decline (L. dentata and L. peronii). The specific activity of photolyase was measured in each species and compared to the hatching success of their eggs under unfiltered summer sunlight. The mean specific activities of photolyase were 1.10 ± 0.18 × 10¹¹, 5.76 ± 1.01 × 10¹¹, and 2.66 ± 0.15 × 10¹¹ CPDs repaired per hour per microgram of egg protein extract, for L. aurea, L. dentata and L. peronii, respectively. When intrinsic differences in hatching success between species were controlled for, the relative percentage hatching success under unfiltered sunlight of L. aurea (77%) was lower than that of L.␣peronii (91%) and L. dentata (98%); however, these values did not differ significantly. L. aurea had the lowest photolyase activity of the three species and showed a non-significant trend of reduced hatching success under UV-B exposure. Received: 15 December 1997 / Accepted: 9 March 1998     

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.     

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.     

UV-B light induces an adaptive response to UV-C exposure via photoreactivation activity in Euglena gracilis.

Phytoplankton such as Euglena are constantly exposed to solar light which is used for photosynthesis. Although the solar ultraviolet (UV) induces DNA damage such as cyclobutane-pyrimidine dimers (CPDs), many kinds of living organisms can repair CPDs by photoreactivation (PR) utilizing the near-UV/blue light component in sunlight. Euglena cells are known to possess such PR activity. In the present paper, the formation of CPDs induced by UV-C exposure and the photoreactivation PR repair of these CPDs by UV-A are demonstrated. To clarify the adaptive responses prior UV-B irradiation on PR activity, cells were cultured in the dark or under UV-B light. When the cells were cultured in the dark for 3 d prior to UV-C exposure, PR activity decreased. When the cells were cultured under UV-B light, however, PR activity increased. These results suggest that exposing the cells to UV-B prior to exposure to UV-C induced an adaptive response towards DNA damage caused by UV-C exposure, and this UV-C induced damage was repaired through PR activity.     

Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation

DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.     

In situ Responses of Phytoplankton from the Subtropical Lake La Angostura (Tucumán, Argentina) in Relation to Solar Ultraviolet Radiation Exposure and Mixing Conditions

In situ experiments were conducted at various depths in the water column to determine the effects of solar ultraviolet radiation (UVR, 280–400 nm) on photosynthesis of natural phytoplankton assemblages from the subtropical Lake La Angostura (Argentina, 26°45′ S; 65°37° W, 1980 m asl.). Water samples were taken daily and incubated under three radiation treatments: (a) Samples exposed to UVR + Photosynthetic Available Radiation (PAR) – PAB treatment (280–700 nm); (b) Samples exposed to ultraviolet-A radiation (UV-A) + PAR – PA treatment (320–700 nm), and, (c) Samples exposed to PAR only – P treatment (400–700 nm). Additionally, depth profiles were done to determine different physical (i.e., temperature and underwater radiation field) and biological characteristics of the water column – photosynthetic pigments, UV-absorbing compounds, cell concentration, deoxyribonucleic acid (DNA) and cyclobutane pyrimidine dimers (CPDs). The effects of UVR on natural phytoplankton assemblages were significant only in the first 50 cm of the water column, causing a decrease in photosynthetic rates of 36 and 20% due to UV-A and ultraviolet-B radiation (UV-B), respectively; below this depth, however, there were no significant differences between radiation treatments. Concentration of CPDs per mega base of DNA in natural phytoplankton was low, <27 CPDs MB⁻¹ between 0 and 4 m. Data on net DNA damage, together with that on mixing conditions of the water column, suggest that mixing can favour phytoplankton by allowing cells to be transported to depths where active repair can take place. This mechanism to reduce UVR-induced DNA damage would be of great advantage for these assemblages dominated by small cyanobacteria and chlorophytes where UV-absorbing compounds that could act as sunscreens are virtually absent.     

UV-susceptibility of zoospores of the brown macroalga Laminaria digitata from Spitsbergen

The UV-susceptibility of zoospores of the lower sublittoral kelp Laminaria digitata was studied in the laboratory under varying fluence of spectral irradiance consisting of photosynthetically active radiation (PAR, 400–700 nm; = P), PAR + UV-A radiation (UV-A, 320–400 nm; = PA), and PAR + UV-A + UV-B radiation (UV-B, 280–320 nm; = PAB). In vivo absorption of phlorotannin, localisation of phlorotannin-containing physodes, structural changes, DNA damage and repair, photosynthesis and germination of zoospores were measured after exposure treatments and after 2–6 days of recovery in dim white light. Photodegradation of phlorotannins was observed after extended exposure to ultraviolet radiation (UVR). The UV-protective function of extra- and intracellular phlorotannins was, therefore, observed only after 8 h, but not after 16-h UVR exposure. The energetic cost of photoprotection may have caused the delay in ontogenic development of zoospores after 8-h exposure to PA and PAB treatment; longer exposure time corresponding to 16-h PA and PAB treatment eventually lead to cell degeneration at 6 days post-cultivation. The formation of cyclobutane–pyrimidine dimers (CPDs), as indicator of DNA damage, was not blocked by the UV-absorbing phlorotannins during the 16-h PAB exposure and the inability for DNA damage repair was likely responsible for low photosynthetic recovery and spore mortality. The higher sensitivity of L. digitata zoospores to UVR compared to other kelps such as Saccorhiza dermatodea and Alaria esculenta confirmed our hypothesis that the depth distribution of adult sporophytes in the field correlates to the sensitivity of their corresponding early life history stages to different stress factors in general and UVR in particular.     

Effect of low doses of UV-A and UV-B radiation on photosynthetic activities in<Emphasis Type="Italic">Phaseolus mungo</Emphasis> L.

The effect of low doses of UV-A (320–400 nm) and UV-B (280–320 nm) radiation on photosynthetic activities inPhaseolus mungo L. was investigated under field condition. Supplementation of UV-A enhanced the synthesis of chlorophyll and carotenoids than the UV-B supplemented plants. Significant increase was seen in the concentration of UV-B absorbing compounds of UV-B treated plants. Increase of PS 2 activity in UV-A treated plants was seen. Changes in photosynthetic activity were measured in terms of PS 2 mediated O₂ evolution and Chl a fluorescence.     

Tri-trophic consequences of UV-B exposure: plants,herbivores and parasitoids

In this paper we demonstrate a UV-B-mediated link between host plants, herbivores and their parasitoids, using a model system consisting of a host plant Brassica oleracea, a herbivore Plutella xylostella and its parasitoid Cotesia plutellae. Ultraviolet-B radiation (UV-B) is a potent elicitor of a variety of changes in the chemistry, morphology and physiology of plants and animals. Recent studies have demonstrated that common signals, such as jasmonic acid (JA), play important roles in the mechanisms by which plants respond to UV-B and to damage by herbivores. Plant responses elicited by UV-B radiation can affect the choices of ovipositing female insects and the fitness of their offspring. This leads to the prediction that, in plants, the changes induced as a consequence of UV damage will be similar to those elicited in response to insect damage, including knock-on effects upon the next trophic level, predators. In our trials female P. xylostella oviposited preferentially on host plants grown in depleted UV-B conditions, while their larvae preferred to feed on tissues from UV-depleted regimes over those from UV-supplemented ones. Larval feeding patterns on UV-supplemented tissues met the predictions of models which propose that induced defences in plants should disperse herbivory; feeding scars were significantly smaller and more numerous – though not significantly so – than those on host plant leaves grown in UV-depleted conditions. Most importantly, female parasitoids also showed a clear pattern of preference when given the choice between host plants and attendant larvae from the different UV regimes; however, in the case of the female parasitoids, the choice was in favour of potential hosts foraging on UV-supplemented tissues. This study demonstrates the potential for UV-B to elicit a variety of interactions between trophic levels, most likely mediated through effects upon host plant chemistry.     

Response of the estuarine cyanobacterium <Emphasis Type="Italic">Lyngbya aestuarii</Emphasis> to UV-B radiation

Growth response and changes in the spectral properties of methanolic extract of an estuarine cyanobacterium, Lyngbya aestuarii Agardh, to UV-B radiation were studied. Increase in growth accompanied by increase in chlorophyll a, protein and carbohydrate content was observed up to 12 h of UV-B irradiation followed by a decline with further increase in the duration of UV exposure. Carotenoid content progressively increased with the UV-B dose. The organism synthesized, to a significant extent, mycosporine amino acid-like substances (MAAs) upon UV-B exposure. The cells in the trichome became coiled followed by formation of small bundles as a response to UV-B radiation. SDS protein profile of the UV irradiated cells showed repression of 20 and 22 kDa proteins. However, irradiation with UV-B for 6–24 h led to overproduction of 84, 73, 60, 46, 40, 37 KDa proteins, possibly conferring protection to the organism from UV-B. UV irradiated cells cultured in florescent light for up to 7 days showed revival from UV damage of the pigments and macromolecular contents, suggesting existence of a repair mechanism in the organism.     

Marine Bacterial Isolates Display Diverse Responses to UV-B Radiation

The molecular and biological consequences of UV-B radiation were investigated by studying five species of marine bacteria and one enteric bacterium. Laboratory cultures were exposed to an artificial UV-B source and subjected to various post-UV irradiation treatments. Significant differences in survival subsequent to UV-B radiation were observed among the isolates, as measured by culturable counts. UV-B-induced DNA photodamage was investigated by using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers (CPDs). The CPDs determined following UV-B exposure were comparable for all of the organisms except Sphingomonas sp. strain RB2256, a facultatively oligotrophic ultramicrobacterium. This organism exhibited little DNA damage and a high level of UV-B resistance. Physiological conditioning by growth phase and starvation did not change the UV-B sensitivity of marine bacteria. The rates of photoreactivation following exposure to UV-B were investigated by using different light sources (UV-A and cool white light). The rates of photoreactivation were greatest during UV-A exposure, although diverse responses were observed. The differences in sensitivity to UV-B radiation between strains were reduced after photoreactivation. The survival and CPD data obtained for Vibrio natriegens when we used two UV-B exposure periods interrupted by a repair period (photoreactivation plus dark repair) suggested that photoadaptation could occur. Our results revealed that there are wide variations in marine bacteria in their responses to UV radiation and subsequent repair strategies, suggesting that UV-B radiation may affect the microbial community structure in surface water.     

Powerful skin cancer protection by a CPD-photolyase transgene

BACKGROUND: The high and steadily increasing incidence of ultraviolet-B (UV-B)-induced skin cancer is a problem recognized worldwide. UV introduces different types of damage into the DNA, notably cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs). If unrepaired, these photolesions can give rise to cell death, mutation induction, and onset of carcinogenic events, but the relative contribution of CPDs and 6-4PPs to these biological consequences of UV exposure is hardly known. Because placental mammals have undergone an evolutionary loss of photolyases, repair enzymes that directly split CPDs and 6-4PPs into the respective monomers in a light-dependent and lesion-specific manner, they can only repair UV-induced DNA damage by the elaborate nucleotide excision repair pathway. RESULTS: To assess the relative contribution of CPDs and 6-4PPs to the detrimental effects of UV light, we generated transgenic mice that ubiquitously express CPD-photolyase, 6-4PP-photolyase, or both, thereby allowing rapid light-dependent repair of CPDs and/or 6-4PPs in the skin. We show that the vast majority of (semi)acute responses in the UV-exposed skin (i.e., sunburn, apoptosis, hyperplasia, and mutation induction) can be ascribed to CPDs. Moreover, CPD-photolyase mice, in contrast to 6-4PP-photolyase mice, exhibit superior resistance to sunlight-induced tumorigenesis. CONCLUSIONS: Our data unequivocally identify CPDs as the principal cause of nonmelanoma skin cancer and provide genetic evidence that CPD-photolyase enzymes can be employed as effective tools to combat skin cancer.     

Genotoxic stress in plants: shedding light on DNA damage, repair and DNA repair helicases

Plant cells are constantly exposed to environmental agents and endogenous processes that inflict damage to DNA and cause genotoxic stress, which can reduce plant genome stability, growth and productivity. Plants are most affected by solar UV-B radiation, which damage the DNA by inducing the formation of two main UV photoproducts such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). Reactive oxygen species (ROS) are also generated extra- or intra-cellularly, which constitute yet another source of genotoxic stress. As a result of this stress, the cellular DNA-damage responses (DDR) are activated, which transiently arrest the cell cycle and allow cells to repair DNA before proceeding into mitosis. DDR requires the activation of Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) genes, which regulate the cell cycle and transmit the damage signals to downstream effectors of cell-cycle progression. Since genomic protection and stability are fundamental to ensure and sustain plant diversity and productivity, therefore, repair of DNA damages is essential. In plants the bulky DNA lesions, CPDs and 6-4PPs, are repaired by a simple and error-free mechanism: photoreactivation, which is a light-dependent mechanism and requires CPD or 6-4PP specific photolyases. In addition to this direct repair process, the plants also have sophisticated light-independent general repair mechanisms, such as the nucleotide excision repair (NER) and base excision repair (BER). The completed plant genome sequences reveal that most of the genes involved in NER and BER are present in higher plants, which suggests that the network of in-built DNA-damage repair mechanisms is conserved. This article describes the insight underlying the DNA damage and repair pathways in plants. The comet assay to measure the DNA damage and the role of DNA repair helicases such as XPD and XPB are also covered.     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)     

The effects of UV-B radiation on genetic and biochemical changes of Pelargonium graveolens L′Her

Ultraviolet radiation induces biochemical and genetic changes in plants. The aim of this study was to investigate the effects of UV-B radiation on genetic stability, phenolic compounds and antioxidant activity of Pelargonium graveolens L′Her. Plant cuttings were exposed to 0, 0.12. 0.26 and 0.38 W/m2 of UV-B radiation. Results indicated that by increasing the UV-B radiation intensity, total phenols, flavonoids and anthocyanin contents, Phenylalanine ammonia lyase activity and antioxidant capacity were increased. Analysis of four flavonols (quercetin, myricetin, kaempferol and rutin) contents of leaves extract by HPLC indicated that these four flavonols were enhanced in all treated plants and also the ratio of quercetin to kaempferol (Q/K) showed a significant increase (P ≤ 0.05) in UV-B treated plants in compare to control. To evaluate the genetic variation in treated plants, 10 ISSR primers were used. The highest level of percentage of polymorphism (P%), Shannon index (I), number of effective allele (Ne) and Nei’ genetic diversity (He), were observed at the highest UV-B radiation (0.38 W/m2). The AMOVA analysis also showed a significant genetic differentiation (P ≥ 0.001) among the studied groups, and confirmed the differentiation of groups obtained by the cluster analysis of molecular data. Overall, these results showed that biochemical changes in different intensities of UV-B were in line with genetic variations, so that the highest biochemical and genetic variations were observed in 0.38 W/m2 treatment.     

Stratospheric Ozone Depletion: High Arctic Tundra Plant Growth on Svalbard is not Affected by Enhanced UV-B after 7 years of UV-B Supplementation in the Field

The response of tundra plants to enhanced UV-B radiation simulating 15 and 30% ozone depletion was studied at two high arctic sites (Isdammen and Adventdalen, 78° N, Svalbard).The set-up of the UV-B supplementation systems is described, consisting of large and small UV lamp arrays, installed in 1996 and 2002. After 7 years of exposure to enhanced UV-B radiation, plant cover, density, morphological (leaf fresh and dry weight, leaf thickness, leaf area, reproductive and ecophysiological parameters leaf UV-B absorbance, leaf phenolic content, leaf water content) were not affected by enhanced UV-B radiation. DNA damage in the leaves was not increased with enhanced UV-B in Salix polaris and Cassiope tetragona. DNA damage in Salix polaris leaves was higher than in leaves of C. tetragona. The length of male gametophyte moss plants of Polytrichum hyperboreum was reduced with elevated UV-B as well as the number of Pedicularis hirsuta plants per plot, but the inflorescence length of Bistorta vivipara was not significantly affected. We discuss the possible causes of tolerance of tundra plants to UV-B (absence of response to enhanced UV-B) in terms of methodology (supplementation versus exclusion), ecophysiological adaptations to UV-B and the biogeographical history of polar plants