Perception of volatiles produced by UVC-irradiated plants alters the response to viral infection in na?ve neighboring plants

Interplant communication of stress via volatile signals is a well-known phenomenon. It has been shown that plants undergoing stress caused by pathogenic bacteria or insects generate volatile signals that elicit defense response in neighboring naïve plants.¹ Similarly, we have recently shown that naïve plants sharing the same gaseous environment with UVC-exposed plants exhibit similar changes in genome instability as UVC-exposed plants.² We found that methyl salicylate (MeSA) and methyl jasmonate (MeJA) serve as volatile signals communicating genome instability (as measured by an increase in the homologous recombination frequency). UVC-exposed plants produce high levels of MeSA and MeJA, a response that is missing in an npr1 mutant. Concomitantly, npr1 mutants are impaired in communicating the signal leading to genome instability, presumably because this mutant does not develop new necrotic lesion after UVC irradiation as observed in wt plants.² To analyze the potential biological significance of such plant-plant communication, we have now determined whether bystander plants that receive volatile signals from UVC-irradiated plants, become more resistant to UVC irradiation or infection with oilseed rape mosaic virus (ORMV). Specifically, we analyzed the number of UVC-elicited necrotic lesions, the level of anthocyanin pigments, and the mRNA levels corresponding to ORMV coat protein and the NPR1-regulated pathogenesis-related protein PR1 in the irradiated or virus-infected bystander plants that have been previously exposed to volatiles produced by UVC-irradiated plants. These experiments showed that the bystander plants responded similarly to control plants following UVC irradiation. Interestingly, however, the bystander plants appeared to be more susceptible to ORMV infection, even though PR1 mRNA levels in systemic tissue were significantly higher than in the control plants, which indicates that bystander plants could be primed to strongly respond to bacterial infection.     

Systemic plant signal triggers genome instability

Previously, we have shown that infection of tobacco plants with a viral pathogen triggers local and systemic induction of homologous recombination (HR). Here, we have tested the hypothesis of whether free radicals are potentially involved in the induction of the systemic effect. We report a significant induction of HR in tobacco plants treated with radical-generating agents, UVC or rose Bengal (RB). Importantly, the recombination increase was observed in local (treated) as well as systemic (non-treated) tissue. The systemic increase in recombination implies the existence of a signal that is transmitted to non-treated tissue. Several sets of grafting experiments proved the generation of said signal by both RB and UVC exposure. A statistically significant increase in HR was observed in tissue that received a systemic signal via a grafted leaf. Similar data were obtained from transgenic plants naphthalene degrading salicylate 1-hydroxylase (NahG) unable to accumulate salicylic acid (SA). Interestingly, pre-treatment of plants with the radical-scavenging compound N-acetyl-l-cysteine (NAC) led to a significantly lower recombination increase upon grafting after treatment with UVC and RB. Moreover, leaves taken for grafting from NAC-pre-treated plants exhibited a lower level of oxidized organic compounds. Our data suggest the involvement of free radical production in either generation or maintenance of the recombination signal. We discuss potential mechanisms for generation of the signal and possible adaptive advantages of enhanced genomic flexibility following exposure to DNA-damaging agents.     

Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants

In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.     

Changes in homologous recombination frequency in Arabidopsis thaliana plants exposed to stress depend on time of exposure during development and on duration of stress exposure

In the past, we showed that exposure to abiotic and biotic stresses changes the homologous recombination frequency (HRF) in somatic tissue and in the progeny. In current work we planned to answer the following question: do stress intensity/duration and time during exposure influence changes in somatic HRF and transgenerational changes in HRF? Here, we tested the effects of exposure to UV-C, cold and heat on HRF at 7, 14, 21 and 28 days post germination (dpg). We found that exposure at 14 and 21 dpg resulted in a higher increase in HRF as compared to exposure at 7 dpg; longer exposure to UV-C resulted in a higher frequency of HR, whereas prolonged exposure to cold or heat, especially at later developmental stages, had almost no effect on somatic HRF. Exposure at 7 dpg had a positive effect on somatic growth of plants; plants exposed to stress at this age had larger leaves. The analysis of HRF in the progeny showed that the progeny of plants exposed to stress at 7 dpg had an increase in somatic HRF and showed larger sizes of recombination spots on leaves. The progeny of plants exposed to UV-C at 7 dpg and the progeny of plants exposed to cold or heat at 28 dpg had larger leaves as compared to control plants. To summarize, our experiments showed that changes in somatic and transgenerational HRF depend on the type of stress plants are exposed to, time of exposure during development and the duration of exposure.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-013-0197-z) contains supplementary material, which is available to authorized users.     

Induction of bystander effects by UVA,UVB, and UVC radiation in human fibroblasts and the implication of reactive oxygen species

Radiation-induced bystander effects are various types of responses displayed by nonirradiated cells induced by signals transmitted from neighboring irradiated cells. This phenomenon has been well studied after ionizing radiation, but data on bystander effects after UV radiation are limited and so far have been reported mainly after UVA and UVB radiation. The studies described here were aimed at comparing the responses of human dermal fibroblasts exposed directly to UV (A, B, or C wavelength range) and searching for bystander effects induced in unexposed cells using a transwell co-incubation system. Cell survival and apoptosis were used as a measure of radiation effects. Additionally, induction of senescence in UV-exposed and bystander cells was evaluated. Reactive oxygen species (ROS), superoxide radical anions, and nitric oxide inside the cells and secretion of interleukins 6 and 8 (IL-6 and IL-8) into the medium were assayed and evaluated as potential mediators of bystander effects. All three regions of ultraviolet radiation induced bystander effects in unexposed cells, as shown by a diminution of survival and an increase in apoptosis, but the pattern of response to direct exposure and the bystander effects differed depending on the UV spectrum. Although UVA and UVB were more effective than UVC in generation of apoptosis in bystander cells, UVC induced senescence both in irradiated cells and in neighbors. The level of cellular ROS increased significantly shortly after UVA and UVB exposure, suggesting that the bystander effects may be mediated by ROS generated in cells by UV radiation. Interestingly, UVC was more effective at generation of ROS in bystanders than in directly exposed cells and induced a high yield of superoxide in exposed and bystander cells, which, however, was only weakly associated with impairment of mitochondrial membrane potential. Increasing concentration of IL-6 but not IL-8 after exposure to each of the three bands of UV points to its role as a mediator in the bystander effect. Nitric oxide appeared to play a minor role as a mediator of bystander effects in our experiments. The results demonstrating an increase in intracellular oxidation, not only in directly UV-exposed but also in neighboring cells, and generation of proinflammatory cytokines, processes entailing cell damage (decreased viability, apoptosis, senescence), suggest that all bands of UV radiation carry a potential hazard for human health, not only due to direct mechanisms, but also due to bystander effects.     

Chromosome-type exchange aberrations are induced by inhibiting repair of UVC-induced DNA lesions in quiescent human lymphocytes

Human lymphocytes in the quiescent stage were UVC-irradiated and then incubated for 90 min in the presence of the DNA-repair inhibitor ara-C. The cells were then cultured and analyzed for chromosome aberrations. A single treatment with UVC or ara-C gives rise to a very low yield of dicentrics, whereas the combined treatment can induce a high frequency of these chromosome-type aberrations. The yield in the combined treatment is approximately proportional to the square of the UVC fluence in the range 1-3 J/m2. In addition, the experiments demonstrate that synergistic effects arise when cells are treated with UVC + ara-C and then exposed to X-rays. The results can be explained on the assumption that the UVC + ara-C treatment induces DNA double-strand breaks which, to the first approximation, are randomly distributed over the chromosomes. These breaks are able to interact with each other as well as with X-ray-induced DNA double-strand breaks to form a chromosome-type exchange aberration.     

Correction of the ultraviolet light induced DNA-repair defect in xeroderma pigmentosum cells by electroporation of a normal human endonuclease

Cells from patients with xeroderma pigmentosum, complementation group A (XPA), are known to be defective in repair of pyrimidine dimers and other forms of damage produced by 254-nm ultraviolet (UVC) radiation. We have isolated a DNA endonuclease, pI 7.6, from the chromatin of normal human lymphoblastoid cells which recognizes damage produced by UVC light, and have introduced this endonuclease into UVC-irradiated XPA cells in culture to determine whether it can restore their markedly deficient DNA repair-related unscheduled DNA synthesis (UDS). Introduction of the normal endonuclease, which recognizes predominantly pyrimidine dimers, but not the corresponding XPA endonuclease into UVC-irradiated XPA cells restored their levels of UDS to approximately 80% of normal values. Electroporation of both the normal and the XPA endonuclease into normal human cells increases UDS in normal cells to higher than normal values. These results indicate that the normal endonuclease can restore UDS in UVC-irradiated XPA cells. They also indicate that XPA cells have an endonuclease capable of increasing the efficiency of repair of UVC damage in normal cells.     

Recent advances in understanding of the DNA double-strand break repair machinery of plants

Living cells suffer numerous and varied alterations of their genetic material. Of these, the DNA double-strand break (DSB) is both particularly threatening and common. Double-strand breaks arise from exposure to DNA damaging agents, but also from cell metabolism-in a fortuitous manner during DNA replication or repair of other kinds of lesions and in a programmed manner, for example during meiosis or V(D)J gene rearrangement. Cells possess several overlapping repair pathways to deal with these breaks, generally designated as genetic recombination. Genetic and biochemical studies have provided considerable amounts of data about the proteins involved in recombination processes and their functions within these processes. Although they have long played a key role in building understanding of genetics, relatively little is known at the molecular level of the genetic recombination processes in plants. The use of reverse genetic approaches and the public availability of sequence tagged mutants in Arabidopsis thaliana have led to increasingly rapid progress in this field over recent years. The rapid progress of studies of recombination in plants is obviously not limited to the DSB repair machinery as such and we ask readers to understand that in order to maintain the focus and to rest within a reasonable length, we present only limited discussion of the exciting advances in the of plant meiosis field, which require a full review in their own right . We thus present here an update on recent advances in understanding of the DSB repair machinery of plants, focussing on Arabidopsis and making a particular effort to place these in the context of more general of understanding of these processes.     

Plants experiencing chronic internal exposure to ionizing radiation exhibit higher frequency of homologous recombination than acutely irradiated plants

Ionizing radiation (IR) is a known mutagen responsible for causing DNA strand breaks in all living organisms. Strand breaks thus created can be repaired by different mechanisms, including homologous recombination (HR), one of the key mechanisms maintaining genome stability [A. Britt, DNA damage and repair in plants, Annu. Rev. Plant. Phys. Plant Mol. Biol., 45 (1996) 75-100; H. Puchta, B. Hohn, From centiMorgans to basepairs: homologous recombination in plants, Trends Plant Sci., 1 (1996) 340-348.]. Acute or chronic exposure to IR may have different influences on the genome integrity. Although in a radioactively contaminated environment plants are mostly exposed to chronic pollution, evaluation of both kinds of influences is important. Estimation of the frequency of HR in the exposed plants may serve as an indication of genome stability.We used previously generated Arabidopsis thaliana and Nicotiana tabacum plants, transgenic for non-active versions of the beta-glucoronidase gene (uidA) [P. Swoboda, S. Gal, B. Hohn, H. Puchta, Intrachromosomal homologous recombination in whole plants, EMBO J., 13 (1994) 484-489; H. Puchta, P. Swoboda, B. Hohn, Induction of homologous DNA recombination in whole plants, Plant, 7 (1995) 203-210.] serving as a recombination substrate, to study the influence of acute and chronic exposure to IR on the level of HR as example of genome stability in plants. Exposure of seeds and seedlings to 0.1 to 10.0 Gy 60Co resulted in increased HR frequency, although the effect was more pronounced in seedlings. For the study of the influence of chronic exposure to IR, plants were grown on two chemically different types of soils, each artificially contaminated with equal amounts of 137Cs. We observed a strong and significant correlation between the frequency of HR in plants, the radioactivity of the soil samples and the doses of radiation absorbed by plants (in all cases r0.9, n=6, P<0.05). In addition, we noted that plants grown in soils with different chemical composition, but equal radioactivity, exhibited different levels of HR, dependent upon the absorbed dose of radiation. Remarkably, we observed a much higher frequency of HR in plants exposed to chronic irradiation when compared to acutely irradiated plants. Although acute application of 0.1-0.5 Gy did not lead to an increase of frequency of HR, the chronic exposure of the plants to several orders of magnitude lower dose of 200 muGy led to a 5-6-fold induction of the frequency of HR as compared to the control.     

Transposition of chromosome loci in bystander cells upon exposure to an adaptive dose of ionizing radiation

During the process of the realization of the bystander effect the trans of the Signal from irradiated cells to the intact cell (bystander cells) happens. So both type of cells (irradiated and intact cells) have the same damages and reactions. There are new data about bystander effect as the transduction mechanism of the adaptive response and we have investigated this phenomenon. There are an incubation of the intact (bystander cells) and the exposed (X-radiation of 10 cGy) human lymphocytes and we analyze the location of the centromeric loci of the first chromosome. We observed hat for the first time that after X-ray exposition of the adaptive doses the transposition of the chromosome loci from the peripheral to the central parts of the nucleus in intact (bystander cell) G0-lymphocytes which were incubated in the growth environment cells with irradiated cells removal. We support that the starting states of the adaptive response is the loci extrication of the matrix, the transposition and the approach homologous chromosomes. This process is necessary for the DNA double strand breaks reparation (in the case of injured dose X-radiation) with the participation of the homologous recombination.     

Double-strand break repair in plants is developmentally regulated

In this study, we analyzed double-strand break (DSB) repair in Arabidopsis (Arabidopsis thaliana) at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on nonfunctional beta-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-d intervals from 2 to 31 d post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31 dpg (2.77 x 10(-8)) being only 30% of the recombination rate at 2 dpg (9.14 x 10(-8)). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31 dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70 revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants. These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.     

In Vitro and In Vivo Characterization of Ultraviolet Light C-Irradiated Human Platelets in a 2 Event Mouse Model of Transfusion

UV-based pathogen reduction technologies have been developed in recent years to inactivate pathogens and contaminating leukocytes in platelet transfusion products in order to prevent transfusion-transmitted infections and alloimmunization. UVC-based technology differs from UVA or UVB-based technologies in that it uses a specific wavelength at 254 nm without the addition of any photosensitizers. Previously, it was reported that UVC irradiation induces platelet aggregation and activation. To understand if UVC-induced changes of platelet quality correlate with potential adverse events when these platelets are transfused into animals, we used a 2-event SCID mouse model in which the predisposing event was LPS treatment and the second event was infusion of UVC-irradiated platelets. We analyzed lung platelet accumulation, protein content in bronchoalveolar lavage fluid as an indication of lung injury, and macrophage inflammatory protein-2 (MIP-2) release in mice received UVC-irradiated or untreated control platelets. Our results showed UVC-irradiated platelets accumulated in lungs of the mice in a dose-dependent manner. High-doses of UVC-irradiated platelets were sequestered in the lungs to a similar level as we previously reported for UVB-irradiated platelets. Unlike UVB-platelets, UVC-platelets did not lead to lung injury or induce MIP-2 release. This could potentially be explained by our observation that although UVC treatment activated platelet surface αIIbβ3, it failed to activate platelet cells. It also suggests lung platelet accumulation and subsequent lung damage are due to different and separate mechanisms which require further investigation.     

Pathogen-induced systemic DNA rearrangement in plants

Infection of tobacco plants with tobacco mosaic virus and oilseed rape mosaic virus was shown to induce a threefold increase in homologous DNA recombination in non-infected tissues. Grafting experiments by Igor Kovalchuk et al. demonstrated that this increase was mediated by a systemic recombination signal that traveled ahead of the virus. A similar increase in DNA recombination was also observed in the progeny of the infected plants, indicating that pathogen-induced recombination can lead to heritable adaptations to environmental stresses.     

UV-C-irradiated Arabidopsis and tobacco emit volatiles that trigger genomic instability in neighboring plants

We have previously shown that local exposure of plants to stress results in a systemic increase in genome instability. Here, we show that UV-C-irradiated plants produce a volatile signal that triggers an increase in genome instability in neighboring nonirradiated Arabidopsis thaliana plants. This volatile signal is interspecific, as UV-C-irradiated Arabidopsis plants transmit genome destabilization to naive tobacco (Nicotiana tabacum) plants and vice versa. We report that plants exposed to the volatile hormones methyl salicylate (MeSA) or methyl jasmonate (MeJA) exhibit a similar level of genome destabilization as UV-C-irradiated plants. We also found that irradiated Arabidopsis plants produce MeSA and MeJA. The analysis of mutants impaired in the synthesis and/or response to salicylic acid (SA) and/or jasmonic acid showed that at least one other volatile compound besides MeSA and MeJA can communicate interplant genome instability. The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (npr1) mutant, defective in SA signaling, is impaired in both the production and the perception of the volatile signals, demonstrating a key role for NPR1 as a central regulator of genome stability. Finally, various forms of stress resulting in the formation of necrotic lesions also generate a volatile signal that leads to genomic instability.     

Novel features of radiation-induced bystander signaling in Arabidopsis thaliana demonstrated using root micro-grafting

Radiation-induced bystander effects (RIBE) have been well demonstrated in whole organisms, as well as in single-cell culture models in vitro and multi-cellular tissues models in vitro, however, the underlying mechanisms remain unclear, including the temporal and spatial course of bystander signaling. The RIBE in vivo has been shown to exist in the model plant Arabidopsis thaliana (A. thaliana). Importantly, the unique plant grafting provides a delicate approach for studying the temporal and spatial course of bystander signaling in the context of whole plants. In our previous study, the time course of bystander signaling in plants has been well demonstrated using the root micro-grafting technique. In this study, we further investigated the temporal cooperation pattern of multiple bystander signals, the directionality of bystander signaling, and the effect of bystander tissues on the bystander signaling. The results showed that the bystander response could also be induced efficiently when the asynchronously generated bystander signals reached the bystander tissues in the same period, but not when they entered into the bystander tissues in an inversed sequence. The absence of bystander response in root-inversed grafting indicated that the bystander signaling along roots might be of directionality. The bystander signaling was shown to be independent of the bystander tissues.     

Induction of replication protein A in bystander cells

The bystander effect is a biological phenomenon whereby cells not directly targeted by DNA-damaging agents elicit a response similar to that of targeted cells. Understanding the mechanisms underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for radiotherapy of tumors. Identification of DNA repair and signal transduction proteins that are induced specifically in bystander cells may help in deducing the molecular mechanism(s) responsible for this complex phenomenon. With this objective, we have studied the expression of replication protein A (RPA), which is involved in various DNA metabolic activities such as replication, repair and recombination. We analyzed RPA expression by immunofluorescence and Western blot techniques in both gamma-irradiated primary human fibroblast cells and bystander cells that were recipients of conditioned growth medium harvested from gamma-irradiated cell cultures. A two- to threefold induction of RPA was observed in bystander MRC5 cells treated with conditioned medium collected from gamma-irradiated WI38 or MRC5 cells. Lack of induction of RPA in sham-manipulated MRC5 cells treated with irradiated medium alone (without cells) indicates that the signal elicited from the irradiated cells is responsible for induction of RPA in bystander cells. RPA was induced more effectively in bystander cells than in irradiated cells at the earliest time analyzed (30 min), and the RPA level declined to that of sham-treated control cells by 24 h after treatment. In addition to RPA, apurinic/apyrimidinic endonuclease (APE, a key enzyme of the base excision repair pathway) also showed enhanced expression in bystander cells. Our findings suggest that the induction of RPA and APE is due to a combination of DNA strand breaks and oxidized base lesions in the genomic DNA of bystander cells.     

Induction of homologous recombination following in utero exposure to DNA-damaging agents

Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures.     

Ultraviolet-light exposure induces a heritable sensitivity to the induction of SCE by mitomycin-C

The dose-response relationship for mitomycin-C (MMC)-induced sister-chromatid exchange (SCE) has been determined in the progeny of Chinese hamster lung fibroblasts (V79) exposed to 5.0 J/m2 ultraviolet light-C (UVC, 254 nm) and in the progeny of non-UVC-irradiated controls. Progeny of UVC-irradiated cultures exhibited sensitivity to MMC-induced SCE at doses of MMC that were not detectably lethal. This sensitivity was manifest as an increase in SCE per cell in a large proportion of the cells derived from UVC-exposed cultures and thus appears not to result from the expression of a rare event such as mutation.