Molecular response to climate change: temperature dependence of UV-induced DNA damage and repair in the freshwater crustacean Daphnia pulicaria

In temperate lakes, asynchronous cycles in surface water temperatures and incident ultraviolet (UV) radiation expose aquatic organisms to damaging UV radiation at different temperatures. The enzyme systems that repair UV‐induced DNA damage are temperature dependent, and thus potentially less effective at repairing DNA damage at lower temperatures. This hypothesis was tested by examining the levels of UV‐induced DNA damage in the freshwater crustacean Daphnia pulicaria in the presence and absence of longer‐wavelength photoreactivating radiation (PRR) that induces photoenzymatic repair (PER) of DNA damage. By exposing both live and dead (freeze‐killed) Daphnia as well as raw DNA to UV‐B in the presence and absence of PRR, we were able to estimate the relative importance and temperature dependence of PER (light repair), nucleotide excision repair (NER, dark repair), and photoprotection (PP). Total DNA damage increased with increasing temperature. However, the even greater increase in DNA repair rates at higher temperatures led net DNA damage (total DNA damage minus repair) to be greater at lower temperatures. Photoprotection accounted for a much greater proportion of the reduction in DNA damage than did repair. Experiments that looked at survival rates following UV exposure demonstrated that PER increased survival rates. The important implication is that aquatic organisms that depend heavily on DNA repair processes may be less able to survive high UV exposure in low temperature environments. Photoprotection may be more effective under the low temperature, high UV conditions such as are found in early spring or at high elevations.     

Evolutionary history of alpine and subalpine Daphnia in western North America

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Effects of Temperature and Photorepair Radiation on a Marine Ciliate Exposed to UVB Radiation

The influences of intensity and repeated exposure to ultraviolet‐B radiation (UVB), photoreactivating repair radiation (PRR), and temperature on the scuticociliate Parauronema acutum were explored under laboratory conditions. Population growth was negatively affected after exposure to the equivalent of one sunny summer day of ambient UVB, especially in the absence of PRR. Repeated daily exposure to UVB severely compromised ciliate survival. UVB‐exposed treatments without PRR recovered slower and reached lower final abundances than treatments receiving PRR. Reducing the daily UVB exposure approximately 25% improved ciliate recovery after exposure. In the single exposure treatments, temperature effects were not consistent, except that growth was slowest for control and treatments at the lowest temperature (15 °C). These data suggest that dark repair and/or photoprotection are present in P. acutum, but photoenzymatic repair was the more effective mechanism in reversing UVB damage. Repeated exposure treatments without PRR had zero or declining growth at all temperatures (15, 20 and 25 °C), as did those with PRR at 15 °C. Significant temperature/dose differences were identified in the repeated exposure treatments; ciliates subjected to the higher UVB intensity with PRR survived only at 25 °C, while ciliate populations under reduced UVB increased at 20 and 25 °C.     

Dimer/monomer status and in vivo function of salt‐bridge mutants of the plant UV‐B photoreceptor UVR8

UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor for ultraviolet‐B (UV‐B) light that initiates photomorphogenic responses in plants. UV‐B photoreception causes rapid dissociation of dimeric UVR8 into monomers that interact with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) to initiate signal transduction. Experiments with purified UVR8 show that the dimer is maintained by salt‐bridge interactions between specific charged amino acids across the dimer interface. However, little is known about the importance of these charged amino acids in determining dimer/monomer status and UVR8 function in plants. Here we evaluate the use of different methods to examine dimer/monomer status of UVR8 and show that mutations of several salt‐bridge amino acids affect dimer/monomer status, interaction with COP1 and photoreceptor function of UVR8 in vivo. In particular, the salt‐bridges formed between arginine 286 and aspartates 96 and 107 are key to dimer formation. Mutation of arginine 286 to alanine impairs dimer formation, interaction with COP1 and function in vivo, whereas mutation to lysine gives a weakened dimer that is functional in vivo, indicating the importance of the positive charge of the arginine/lysine residue for dimer formation. Notably, a UVR8 mutant in which aspartates 96 and 107 are conservatively mutated to asparagine is strongly impaired in dimer formation but mediates UV‐B responses in vivo with a similar dose–response relationship to wild‐type. The UV‐B responsiveness of this mutant does not correlate with dimer formation and monomerisation, indicating that monomeric UVR8 has the potential for UV‐B photoreception, initiating signal transduction and responses in plants.     

Water- and temperature-dependence of DNA damage and repair in the fruticose lichen Cladonia arbuscula ssp. mitis exposed to UV-B radiation

The induction of cyclobutane pyrimidine dimers (CPDs) by ultraviolet‐B radiation (UV‐B, 280–315 nm) and repair mechanisms were studied in the lichen Cladonia arbuscula ssp. mitis exposed to different temperatures and water status conditions. In addition, the development and repair of CPDs were studied in relation to the different developmental stages of the lichen thallus podetial branches. Air‐dried lichen thalli exposed to UV‐B radiation combined with relatively high visible light (HL, 800 μmol m^?2 s^?1; 400–700 nm) for 7 days showed a progressive increase of CPDs with no substantial repair, although HL was present during and after irradiation with UV‐B. Fully hydrated lichen thalli, that had not been previously exposed to UV‐B radiation for 7 days, were given short‐term UV‐B radiation treatment at 25°C, and accumulated DNA lesions in the form of CPDs, with repair occurring when they were exposed to photoreactivating conditions (2 h of 300 μmol m^?2 s^?1, 400–700 nm). A different pattern was observed when fully hydrated thalli were exposed to short‐term UV‐B radiation at 2°C, in comparison with exposure at 25°C. High levels of CPDs were induced at 2°C under UV‐B irradiation, without significant repair under subsequent photoreactivating light. Likewise, when PAR (300 μmol m^?2 s^?1) and UV‐B radiation were given simultaneously, the CPD levels were not lowered. Throughout all experiments the youngest, less differentiated parts of the lichen thallus – namely ‘tips’, according to our arbitrary subdivision – were the parts showing the highest levels of CPD accumulation and the lowest levels of repair in comparison with the older thallus tissue (‘stems’). Thus the experiments showed that Cladonia arbuscula ssp. mitis is sensitive to UV‐B irradiation in the air‐dried state and is not able to completely repair the damage caused by the radiation. Furthermore, temperature plays a role in the DNA damage repairing capacity of this lichen, since even when fully hydrated, C. arbuscula ssp. mitis did not repair DNA damage at the low temperatures.     

UV hyper‐resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase

Exposure to solar radiation can cause mortality in natural communities of pico‐phytoplankton, both at the surface and to a depth of at least 30 m. DNA damage is a significant cause of death, mainly due to cyclobutane pyrimidine dimer formation, which can be lethal if not repaired. While developing a UV mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated a UV‐hyper‐resistant variant of high light‐adapted strain MED4. The hyper‐resistant strain was constitutively upregulated for expression of the mutT‐phrB operon, encoding nudix hydrolase and photolyase, both of which are involved in repair of DNA damage that can be caused by UV light. Photolyase (PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy from visible light in the process known as photoreactivation. Nudix hydrolase (MutT) hydrolyses 8‐oxo‐dGTP, an aberrant form of GTP that results from oxidizing conditions, including UV radiation, thus impeding mispairing and mutagenesis by preventing incorporation of the aberrant form into DNA. These processes are error‐free, in contrast to error‐prone SOS dark repair systems that are widespread in bacteria. The UV‐hyper‐resistant strain contained only a single mutation: a 1 bp deletion in the intergenic region directly upstream of the mutT‐phrB operon. Two subsequent enrichments for MED4 UV‐hyper‐resistant strains from MED4 wild‐type cultures gave rise to strains containing this same 1 bp deletion, affirming its connection to the hyper‐resistant phenotype. These results have implications for Prochlorococcus DNA repair mechanisms, genome stability and possibly lysogeny.     

Melanin content and MC1R function independently affect UVR‐induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)‐induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR‐induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR‐induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss‐of‐function MC1R, regardless of their MC or EC, sustained more UVR‐induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR‐induced DNA damage in melanocytes.     

Photosynthetic performance,DNA damage and repair in gametes of the endemic Antarctic brown alga Ascoseira mirabilis exposed to ultraviolet radiation

Abstract Stress physiology on the reproductive cells of Antarctic macroalgae remained unstudied. Ascoseira mirabilis is endemic to the Antarctic region, an isolated ecosystem exposed to extreme environmental conditions. Moreover, stratospheric ozone depletion leads to increasing ultraviolet radiation (280–400 nm) at the earth's surface, thus it is necessary to investigate the capacity of reproductive cells to cope with different UV irradiances. This study is aimed to investigate the impact of exposure to different spectral irradiance on the photosynthetic performance, DNA damage and gamete morphology of the A. mirabilis. Gametangia, gametes and zygotes of the upper sublittoral brown alga A. mirabilis were exposed to photosynthetically active radiation (PAR = P; 400–700 nm), P + UV‐A radiation (UV‐A, 320–400 nm) and P + UV‐A + UV‐B radiation (UV‐B, 280–320 nm). Rapid photosynthesis versus irradiance curves of freshly released propagules were measured. Photosynthetic efficiencies and DNA damage (in terms of cyclobutane pyrimidine dimers) were determined after 1, 2, 4 and 8 h exposure as well as after 2 days of recovery in dim white light. Saturation irradiance (I_k) in freshly released propagules was 52 μmol photons m⁻² s⁻¹. Exposure for 1 h under 22 μmol photons m⁻² s⁻¹ of PAR significantly reduced the optimum quantum yield (F_v/F_m), suggesting that propagules are low light adapted. Furthermore, UVR significantly contributed to the photoinhibition of photosynthesis. Increasing dose as a function of exposure time additionally exacerbated the effects of different light treatments. The amount of DNA damage increased with the UV‐B dose but an efficient repair mechanism was observed in gametes pre‐exposed to a dose lower than 5.8 × 10³ J m⁻² of UV‐B. The results of this study demonstrate the negative impact of UV‐B radiation. However, gametes of A. mirabilis are capable of photosynthetic recovery and DNA repair when the stress factor is removed. This capacity was observed to be dependent on the fitness of the parental sporophyte.     

Rapid evolution of antioxidant defence in a natural population of Daphnia magna

Natural populations can cope with rapid changes in stressors by relying on sets of physiological defence mechanisms. Little is known onto what extent these physiological responses reflect plasticity and/or genetic adaptation, evolve in the same direction and result in an increased defence ability. Using resurrection ecology, we studied how a natural Daphnia magna population adjusted its antioxidant defence to ultraviolet radiation (UVR) during a period with increasing incident UVR reaching the water surface. We demonstrate a rapid evolution of the induction patterns of key antioxidant enzymes under UVR exposure in the laboratory. Notably, evolutionary changes strongly differed among enzymes and mainly involved the evolution of UV‐induced plasticity. Whereas D. magna evolved a strong plastic up‐regulation of glutathione peroxidase under UVR, it evolved a lower plastic up‐regulation of glutathione S‐transferase and superoxide dismutase and a plastic down‐regulation of catalase. The differentially evolved antioxidant strategies were collectively equally effective in dealing with oxidative stress because they resulted in the same high levels of oxidative damage (to lipids, proteins and DNA) and lowered fitness (intrinsic growth rate) under UVR exposure. The lack of better protection against UVR may suggest that the UVR exposure did not increase between both periods. Predator‐induced evolution to migrate to lower depths that occurred during the same period may have contributed to the evolved defence strategy. Our results highlight the need for a multiple trait approach when focusing on the evolution of defence mechanisms.     

INTERACTIONS BETWEEN UV‐B EXPOSURE AND PHOSPHORUS NUTRITION. II. EFFECTS ON RATES OF DAMAGE AND REPAIR1

The interactive effects of P starvation and exposure to UV radiation (UVR) on rates of damage ( k ) and repair ( r ), modeled from exposure response curves (ERCs), in the chlorophyte microalga Dunaliella tertiolecta Butcher were investigated. When nutrient‐replete cells were exposed to the UVR during growth, k and r both increased by approximately 62% and 100%, respectively. However, when cells were starved of phosphorus, k increased by a similar amount as observed in replete cells, but r decreased by about 70%, explaining the increased susceptibility of cells to UVR‐induced inhibition of photosynthesis under P starvation. Although not specifically investigated in this study, it is argued that the decreased repair capacity under P starvation is due to a decline in nucleotides such as ATP and GTP, which are necessary for protein repair.     

COMBINED EFFECTS OF ULTRAVIOLET RADIATION AND TEMPERATURE ON MORPHOLOGY,PHOTOSYNTHESIS, AND DNA OF ARTHROSPIRA (SPIRULINA) PLATENSIS (CYANOPHYTA)1

Natural levels of solar UVR were shown to break and alter the spiral structure of Arthrospira (Spirulina) platensis (Nordst.) Gomont during winter. However, this phenomenon was not observed during summer at temperatures of ～30°C. Since little has been documented on the interactive effects of solar UV radiation (UVR; 280–400 nm) and temperature on cyanobacteria, the morphology, photosynthesis, and DNA damage of A. platensis were examined using two radiation treatments (PAR [400–700 nm] and PAB [PAR + UV‐A + UV‐B: 280–700]), three temperatures (15, 22, and 30°C), and three biomass concentrations (100, 160, and 240 mg dwt [dry weight] · L^?1). UVR caused a breakage of the spiral structure at 15°C and 22°C, but not at 30°C. High PAR levels also induced a significant breakage at 15°C and 22°C, but only at low biomass densities, and to lesser extent when compared with the PAB treatment. A. platensis was able to alter its spiral structure by increasing helix tightness at the highest temperature tested. The photochemical efficiency was depressed to undetectable levels at 15°C but was relatively high at 30°C even under the treatment with UVR in 8 h. At 30°C, UVR led to 93%–97% less DNA damage when compared with 15°C after 8 h of exposure. UV‐absorbing compounds were determined as negligible at all light and temperature combinations. The possible mechanisms for the temperature‐dependent effects of UVR on this organism are discussed in this paper.     

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.     

RESPONSE OF LAMINARIA SACCHARINA (PHAEOPHYTA) GROWTH AND PHOTOSYNTHESIS TO SIMULTANEOUS ULTRAVIOLET RADIATION AND NITROGEN LIMITATION1

The brown macroalga Laminaria saccharina (L.) J. V. Lamour. was grown in large outdoor tanks at 50% ambient solar radiation for 3–4 weeks in July and August of 2000, 2001, and 2002, in either ambient or nitrogen (N)–enriched seawater and in either ambient light [PAR + ultraviolet radiation (UVR)] or ambient light minus UVR. Growth, N‐content, photosynthetic pigments, and RUBISCO content increased in N‐enriched seawater, indicating N‐limitation. UVR inhibited growth, but this inhibition was ameliorated by N‐enrichment. The response of growth to UVR could not be explained by changes in respiration and photosynthesis. Gross light‐saturated photosynthesis (P_max) remained unaffected by UVR but was significantly higher under N‐enrichment, as was dark respiration (R_d). UVR had no effect on pigments or N content. However, RUBISCO contents were low in the presence of UVR, reflecting the overall change in soluble cellular protein. Overall, our data indicate that the response to UVR in L. saccharina depends on other environmental factors, such as N, and these effects need to be considered when evaluating the response of macroalgae to increased UVR.     

Effectivity of bacterial repair systems in near UV radiation-induced damage

Inactivation of seven strains derived fromEscherichia coli B differing in their capacity to repair damage to their DNA (exc, pol, rec) after irradiation with far (254 nm) and middle and near (300 to 380 and 320–400 nm) UV light was investigated. The same bacterial strains were also used as hosts for the UV-irradiated pliage T7. The damage induced in bacteria and the phage by the near UV radiation was repaired only to a lesser extent by the investigated repair mechanisms or was not repaired at all.     

UV light-induced DNA damage detection in the unicellular green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Exposure of cells to ultraviolet radiation (UVR) is one of the best studied and most used model system for the examination of the biological effects of DNA damage, its repair and tolerance. The major product after UVR treatment is cyclobutane pyrimidine dimer (TT, TC, CC). Pyrimidine dimers are repaired by a direct reversal called photoreactivation or by excision of damage in a process of nucleotide excision repair. Several methods have been developed for the detection and quantification of pyrimidine dimers in DNA. The technique of Small and Greimann, in which DNA is incubated with the pyrimidine dimer-specific endonuclease, was used for the analysis of mutant strains with impaired excision repair system of the unicellular green alga Chlamydomonas reinhardtii. Another method is based on the binding of specific monoclonal antibodies to pyrimidine dimers. The aim of our work was to compare these two techniques with the use of mutant strains of C. reinhardtii — uvsX1 and uvsX2 which are assumed to be deficient in DNA damage recognition. One of their traits was sensitivity to UVR which could be caused by breakdown of the excision repair pathway. The results suggest that the immuno-approach is suitable for the detection of DNA damage induced by UVR. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

The Impact of UV Radiation on Paramecium Populations from Alpine Lakes

Paramecium populations from a clear and a glacier‐fed turbid alpine lake were exposed to solar simulated ultraviolet (UVR) and photosynthetically active radiation (PAR) at 8 and 15 °C. The ciliates were tested for DNA damage (comet assay), behavioral changes, and mortality after UVR + PAR exposure. High DNA damage levels (~58% tail DNA) and abnormal swimming behavior were observed, although no significant changes in cell numbers were found irrespective of the lake origin (clear, turbid), and temperatures. We conclude that environmental stressors such as UVR and their effects may influence the adaptation of ciliates living in alpine lakes.