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.     

Increased DNA repair in <Emphasis Type="Italic">Arabidopsis</Emphasis> plants overexpressing CPD photolyase

Ultraviolet-B (UV-B, 280–320 nm) radiation may have severe negative effects on plants including damage to their genetic information. UV protection and DNA-repair mechanisms have evolved to either avoid or repair such damage. Since autotrophic plants are dependent on sunlight for their energy supply, an increase in the amount of UV-B reaching the earth’s surface may affect the integrity of their genetic information if DNA damage is not repaired efficiently and rapidly. Here we show that overexpression of cyclobutane pyrimidine dimer (CPD) photolyase (EC 4.1.99.3) in Arabidopsis thaliana (L.), which catalyses the reversion of the major UV-B photoproduct in DNA (CPDs), strongly enhances the repair of CPDs and results in a moderate increase of biomass production under elevated UV-B.     

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.     

Climate and growth form: the consequences for genome size in plants

The adaptive significance of nuclear DNA variation in angiosperms is still widely debated. The discussion mainly revolves round the causative factors influencing genome size and the adaptive consequences to an organism according to its growth form and environmental conditions. Nuclear DNA values are now known for 3874 angiosperm species (including 773 woody species) from over 219 families (out of a total of 500) and 181 species of woody gymnosperms, representing all the families. Therefore, comparisons have been made on not only angiosperms, taken as a whole, but also on the subsets of data based on taxonomic groups, growth forms, and environment. Nuclear DNA amounts in woody angiosperms are restricted to less than 23.54 % of the total range of herbaceous angiosperms; this range is further reduced to 6.8 % when woody and herbaceous species of temperate angiosperms are compared. Similarly, the tropical woody dicots are restricted to less than 50.5 % of the total range of tropical herbaceous dicots, while temperate woody dicots are restricted to less than 10.96 % of the total range of temperate herbaceous dicots. In the family Fabaceae woody species account for less than 14.1 % of herbaceous species. Therefore, in the total angiosperm sample and in subsets of data, woody growth form is characterized by a smaller genome size compared with the herbaceous growth form. Comparisons between angiosperm species growing in tropical and temperate regions show highly significant differences in DNA amount and genome size in the total angiosperm sample. However, when only herbaceous angiosperms were considered, significant differences were obtained in DNA amount, while genome size showed a non-significant difference. An atypical result was obtained in the case of woody angiosperms where mean DNA amount of tropical species was almost 25.04 % higher than that of temperate species, which is because of the inclusion of 85 species of woody monocots in the tropical sample. The difference becomes insignificant when genome size is compared. Comparison of tropical and temperate species among dicots and monocots and herbaceous monocots taken separately showed significant differences both in DNA amount and genome size. In herbaceous dicots, while DNA amount showed significant differences the genome size varies insignificantly. There was a non-significant difference among tropical and temperate woody dicots. In three families, i.e., Poaceae, Asteraceae, and Fabaceae the temperate species have significantly higher DNA amount and genome size than the tropical ones. Woody gymnosperms had significantly more DNA amount and genome size than woody angiosperms, woody eudicots, and woody monocots. Woody monocots also had significantly more DNA amount and genome size than woody eudicots. Lastly, there was no significant difference between deciduous and evergreen hardwoods. The significance of these results in relation to present knowledge on the evolution of genome size is discussed.     

NaCI胁迫对UV-B辐射诱导的绿豆环丁烷嘧啶二聚体和紫外吸收物质含量变化的影响

将2个对UV-B敏感性不同的绿豆品种‘秦豆-20’和‘中绿-1’幼苗放在培养室内,进行0.4W/m~2 UV-B辐射和0.4%NaCl胁迫的单独或复合处理,研究了NaCl胁迫对UV-B辐射诱导的DNA伤害和修复的影响。结果显示:在NaCl胁迫下,(1)在光下抗UV-B的品种‘中绿-1’的环丁烷嘧啶二聚体(CPD)累积量降低,而敏感品种‘秦豆-20’的CPD累积量未发生变化;(2)两品种CPD形成量均比无NaCl胁迫时低;(3)抗UV-B品种DNA的光、暗修复能力均比无NaCl胁迫时高:(4)而敏感品种DNA的光修复能力比无NaCl胁迫时低、暗修复能力未发生变化。另外,CPD形成量与紫外吸收物含量间具有明显的负相关性。说明NaCl胁迫不仅影响2个绿豆品种幼苗的CPD形成量,而且影响DNA的光、暗修复能力,进而导致了CPD累积量发生变化,由此影响了幼苗的UV-B敏感性。结果也暗示CPD形成量的变化是由于紫外吸收物质含量的不同所导致的。     

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.     

Reviewing the Technical Designs for Experiments with Ultraviolet-B Radiation and Impact on Photosynthesis, DNA and Secondary Metabolism

The ultraviolet-B (UV-B) portion of sunlight has received much attention in the last three decades, because radiation from this spectral region increases due to the stratospheric ozone depletion, which results from increases of chlorofluorocarbons in the atmosphere. Plant responses to UV-B exposure vary greatly and the interpretation of and comparison between studies is hindered, mainly by the contrasting experimental conditions used and interactive factors such as low light levels and possible artifacts due to the artificial experimental conditions. It seems likely that increases in solar UV-B radiation of the magnitude anticipated under current stratospheric ozone projections will not significantly inhibit photosynthesis and cause DNA damage in plants. This is in part due to the well-evolved protection mechanisms present in most plant species. One of the significant plant responses to UV-B is changes in foliar secondary chemistry, which could be translated into significant effects at higher trophic levels through plant-herbivore interactions and decomposition. Enhanced UV-B radiation due to stratospheric ozone depletion could also cause morphological changes that would affect competitive interactions, especially if contrasting UV-B sensitivity exists among the competitors.     

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.     

温度对UV-B诱导的烟草叶圆片DNA损伤的影响

环丁烷嘧啶二聚体(CPD)和6-4光产物(6-4PP)是两种主要的UV-B诱导的DNA光损伤产物。利用单克隆抗体酶联免疫吸附分析法(ELISA),研究了温度对UV-B诱导的烟草叶圆片DNA损伤的影响。室温(24℃)条件下,UV-B处理引起了烟草叶圆片DNA中CPD和6-4PP的积累。0℃条件下,UV-B处理的烟草叶圆片DNA中CPD和6-4PP的积累比室温下分别降低了9.8%和12%。UV-B诱导的DNA损伤曾被认为是纯粹的光化学过程而与不受温度影响,而本实验结果表明,UV-B诱导的烟草叶圆片DNA形成CPD和6-4PP的过程具有温度依赖性。这一特性有利于植物对全球变化的适应,因而具有重要的生态学意义。     

Molecular mechanisms of the repair of UV-induced DNA damages in plants

Solar UV-B radiation is an environmental factor which damage and destabilize genomes. UV-B-induced DNA lesions have cytotoxic and genotoxic effects on the cells of pro- and eucaryotes including plants. In addition, such lesions can cause gene mutations in plants. The products of the damages of cellular DNA caused by UV are examined in the present review and plant reparative pathways including photoreactivation, base excision repair and nucleotide excision repair are analyzed. The review deals as well with the mechanisms of plant DNA damage tolerance which allow to reduce the toxic effects of UV-B radiation.     

Response of plant species and life form diversity to variable fire histories and biomass in the jarrah forest of south‐west Australia

Frequent fires reduce the abundance of woody plant species and favour herbaceous species. Plant species richness also tends to increase with decreasing vegetation biomass and cover due to reduced competition for light. We assessed the influence of variable fire histories and site biomass on the following diversity measures: woody and herbaceous species richness, overall species richness and evenness, and life form evenness (i.e. the relative abundance or dominance among six herbaceous and six woody plant life forms), across 16 mixed jarrah (Eucalyptus marginata) and marri (Corymbia calophylla) forest stands in south‐west Australia. Fire frequency was defined as the total number of fires over a 30‐year period. Overall species richness and species evenness did not vary with fire frequency or biomass. However, there were more herbaceous species (particularly rushes, geophytes and herbs) where there were fewer shrubs and low biomass, suggesting that more herbaceous species coexist where dominance by shrubs is low. Frequently burnt plots also had lower number and abundance of shrub species. Life form evenness was also higher at both high fire frequency and low biomass sites. These results suggest that the impact of fire frequency and biomass on vegetation composition is mediated by local interactions among different life forms rather than among individual species. Our results demonstrate that measuring the variation in the relative diversity of different woody and herbaceous life forms is crucial to understanding the compositional response of forests and other structurally complex vegetation communities to changes in disturbance regime such as increased fire frequency.     

Amphibian defenses against ultraviolet-B radiation

As part of an overall decline in biodiversity, amphibian populations throughout the world are disappearing. There are a number of potential causes for these declines, including those related to environmental changes such as increasing ultraviolet-B (UV-B) radiation due to stratospheric ozone depletion. UV-B radiation can kill amphibian embryos or can cause sublethal effects that can harm amphibians in later life stages. However, amphibians have defenses against UV-B damage that can limit damage or repair it after exposure to UV-B radiation. These include behavioral, physiological, and molecular defenses. These defenses differ interspecifically, with some species more able to cope with exposure to UV-B than others. Unfortunately, the defense mechanisms of many species may not be effective against increasing persistent levels of UV-B radiation that have only been present for the past several decades due to human-induced environmental damage. Moreover, we predict that persistent UV-B-induced mortality and sublethal damage in species without adequate defenses could lead to changes in community structure. In this article we review the effects of UV-B radiation on amphibians and the defenses they use to avoid solar radiation and make some predictions regarding community structure in light of interspecific differences in UV-B tolerance.     

基于FvCB模型的几种草本和木本植物光合生理生化特性

为研究不同生活型植物的光合能力及其叶片光合机构,采用直角双曲线修正模型和C3植物FvCB模型对7种木本植物和4种草本植物的CO₂响应曲线进行拟合,并对不同木本植物、不同草本植物和2种生活型植物的最大净光合速率(P_{n max})、Rubisco酶最大羧化速率(V_{c max})、最大电子传递速率(J_max)、光合暗呼吸速率(R_d)和叶肉阻力(r_m)等参数进行比较.结果表明: 7种木本植物P_{n max}大小顺序为乌桕、苎麻>润楠、海桐>青冈、苦槠、娜塔栎;乌桕、苎麻、润楠和海桐的V_{c max}显著大于青冈和苦槠;J_max大小顺序为乌桕>苎麻、海桐>娜塔栎、苦槠和青冈;润楠和苦槠的r_m显著大于乌桕、海桐和苎麻.商陆的P_{n max}显著大于藿香蓟和土牛膝;4种草本植物的V_{c max}无显著差异;商陆的J_max显著大于藿香蓟;龙葵和土牛膝的r_m显著大于藿香蓟;商陆的R_d显著大于藿香蓟和土牛膝.木本植物的P_{n max}、V_{c max}、J_max和r_m光合参数均显著大于草本植物,但二者的R_d无显著差异.不同物种之间以及2种生活型植物光合能力的差异主要是由叶片内部Rubisco酶羧化能力、电子传递能力和叶肉阻力等差异引起的.     

UV-B resistance as a criterion for the selection of desert microalgae to be utilized for inoculating desert soils

The adaption capability of microalgae species to intense UV-B radiation is an important feature for their survival under the harsh growth conditions they have to face when used for inoculating unconsolidated sand soils in desert areas. In this study, the responses of photosynthetic activity, reactive oxygen species (ROS) generation, and DNA strand breaks to UV-B radiation in four microalgae isolated from artificially induced biological soil crusts were investigated. It was found that low UV-B doses easily inhibited the photosynthetic activity and induced serious DNA damage in Chlorella vulgaris. Microcoleus vaginatus showed the capability to withstand only moderate doses of UV-B, while Nostoc was capable of facing high doses of UV-B due to its lower generation of ROS and higher capability to repair photosystem II (PSII) and DNA damages. On the other hand, Scytonema javanicum showed additional strategies to survive UV-B irradiance, namely the closure of PSII when ROS generation increased rapidly, in addition to a high repair ability of PSII and DNA damage. The results obtained point out different resistance and defense mechanisms of the four microalgae in response to UV-B irradiance and suggest that the strain of Nostoc sp. tested is the most suitable for surviving under the high UV irradiation levels typical of desertified areas.     

DNA barcoding herbaceous and woody plant species at a subalpine forest dynamics plot in Southwest China

Although DNA barcoding has been widely used to identify plant species composition in temperate and tropical ecosystems, relatively few studies have used DNA barcodes to document both herbaceous and woody components of forest plot. A total of 201 species (72 woody species and 129 herbaceous species) representing 135 genera distributed across 64 families of seed plants were collected in a 25 ha CForBio subalpine forest dynamics plot. In total, 491 specimens were screened for three DNA regions of the chloroplast genome (rbcL, matK, and trnH‐psbA) as well as the internal transcribed spacers (ITS) of nuclear ribosomal DNA. We quantified species resolution for each barcode separately or in combination using a ML tree‐based method. Amplification and sequencing success were highest for rbcL, followed by trnH‐psbA, which performed better than ITS and matK. The rbcL + ITS barcode had slightly higher species resolution rates (88.60%) compared with rbcL + matK (86.60%) and rbcL + trnH‐psbA (86.01%). The addition of trnH‐psbA or ITS to the rbcL + matK barcode only marginally increased species resolution rates, although in combination the four barcodes had the highest discriminatory power (90.21%). The situations where DNA barcodes did not discriminate among species were typically associated with higher numbers of co‐occurring con‐generic species. In addition, herbaceous species were much better resolved than woody species. Our study represents one of the first applications of DNA barcodes in a subalpine forest dynamics plot and contributes to our understanding of patterns of genetic divergence among woody and herbaceous plant species.     

Variation in species‐level plant functional traits over wetland indicator status categories

Wetland indicator status (WIS ) describes the habitat affinity of plant species and is used in wetland delineations and resource inventories. Understanding how species‐level functional traits vary across WIS categories may improve designations, elucidate mechanisms of adaptation, and explain habitat optima and niche. We investigated differences in species‐level traits of riparian flora across WIS categories, extending their application to indicate hydrologic habitat. We measured or compiled data on specific leaf area (SLA ), stem specific gravity (SSG ), seed mass, and mature height of 110 plant species that occur along the Colorado River in Grand Canyon, Arizona. Additionally, we measured leaf δ¹³C, δ¹⁵N, % carbon, % nitrogen, and C/N ratio of 56 species with C3 photosynthesis. We asked the following: (i) How do species‐level traits vary over WIS categories? (ii) Does the pattern differ between herbaceous and woody species? (iii) How well do multivariate traits define WIS categories? (iv) Which traits are correlated? The largest trait differences among WIS categories for herbaceous species occurred for SSG , seed mass, % leaf carbon and height, and for woody species occurred for height, SSG , and δ¹³C. SSG increased and height decreased with habitat aridity for both woody and herbaceous species. The δ¹³C and hence water use efficiency of woody species increased with habitat aridity. Water use efficiency of herbaceous species increased with habitat aridity via greater occurrence of C4 grasses. Multivariate trait assemblages differed among WIS categories. Over all species, SLA was correlated with height, δ¹³C, % leaf N, and C/N; height was correlated with SSG and % leaf C; SSG was correlated with % leaf C. Adaptations of both herbaceous and woody riparian species to wet, frequently inundated habitats include low‐density stem tissue. Adaptations to drier habitats in the riparian zone include short, high‐density cavitation‐resistant stem tissue, and high water use efficiency. The results enhance understanding about using traits to describe plant habitat in riparian systems.     

Contribution of phenolic compounds to the UV-B screening capacity of developing barley primary leaves in relation to DNA damage and repair under elevated UV-B levels

Epidermally located UV-absorbing hydroxycinnamic acid conjugates and flavonoid glycosides are known to be efficient UV-B protectants in higher plants, although important biological molecules are not always fully protected. However, repair mechanisms also exist, such as repair of damaged DNA by photolyases. To distinguish between the relative importance of the phenolic compounds and of DNA repair, developing primary leaves of two barley lines, mutant ant 30-310, deficient in flavonoids, and its parent line Ca 33787, were grown under relatively high visible light (650-700 micromol m(-2) s(-1) max for 6 h in a 13 h photoperiod) and supplemented with (+ UV-B) or without (-UV-B) 12 kJ m(-2) UV-B(BE) for 6 h daily. UV-B screening capacity of the leaf phenolics was determined at 315 nm during leaf development and compared with thymine dimers (TD) accumulation, as an indicator of UV-B-induced DNA damage and potential subsequent repair. The degree of damage was related to the phenolic contents of the leaves. UV-B screening capacity was increased ca. 4-fold in the parent line (+ UV-B), mainly due to UV-induced flavonoid (saponarin, lutonarin) accumulation in epidermal and subepidermal mesophyll tissue, relative to the flavonoid-deficient mutant. Nevertheless, in the parent line an 8-fold increase in TD levels occurred over the growth period of 18 days, whereas the mutant accumulated additional DNA damage, with 6- to 9-fold higher TD amounts. Surprisingly, under the high UV-B irradiation, growth and development of the primary leaves in both lines were only slightly reduced.