Ebook: Cell Biology and Instrumentation: UV Radiation, Nitric Oxide and Cell Death in Plants

The contributions from speakers and the poster presentations for the Advanced Research Workshop (ARW), “Cell Biology and Instrumentation: UV Radiation, Nitric Oxide and Cell Death in Plants” are archived in this volume. The ARW was held at the Hotel “MRIYA”, Yalta, Ukraine, from 8 to 11, September 2004. The ARW was organized to create links among scientists from NATO and Partner countries to stabilize and sustain their scientific communities. Contributions were received from the European Union, the Eastern European countries of the Former Soviet Union, and the USA.

The main aims of the Workshop were to introduce new theoretical developments and instrumentation for cell biology, update our understanding of the effects of UV radiation, and evaluate how plants use UV signals to protect against damage and enhance their productivity.

Cellular processes, signaled by UV radiation, contribute to the behavior of plants under various and different stresses in the environment. The importance of the free radical, nitric oxide (NO) was identified as a key early signal in this process. Stress-induced NO can be protective, produce physiological disorders, DNA damage, and programmed cell death (apoptosis).

This volume is divided into three parts: Instrumentation and Ecological Aspects, Effects of UV Radiation, Nitric oxide and Plant stress, and Plant Stress and Programmed Cell Death.

The Workshop:

(i) Evaluates case histories, and introduces new instruments for the non-invasive sensing and imaging of UV-stress-related damage in vegetation

(ii) Identifies the cell biological hazards of UV radiation coupled to other environmental stresses

(iii) Examines how UV light may relate to the production of NO by plants in terms of DNA damage, error-prone repair cell cycles, and the multiple mechanisms of programmed cell death

The oral and poster papers presented during the four-day meeting are included.

All attendees express their sincere gratitude to the NATO International Scientific Exchange Programme, whose financial support made the meeting possible. We also thank the agency ITEM (Intelligence, Technology, Materials) in Kyiv whose director Dr. L. Chernyshov professionally helped us to organise the workshop. We thank V. Stepanov, N. Sukach, and P. Karpov for their clerical and technical assistance, which ensured the conference and social arrangements ran smoothly.

Yaroslav Blume, Kyiv, Ukraine; Don Durzan, Davis, USA; Petro Smertenko, Kyiv, Ukraine

Selected properties of plant cell populations are useful but have limitations for the design of UV-monitoring instruments and their utility. Diagnostics based on nitric oxide bursts, dose-dependent DNA damage and cell death continue to evolve. Case histories are needed for the realistic modeling of local and global effects of UV radiation, environmental health and decision-making.

The main aspects of photobiological measurements and obtaining action spectra have been considered. It has been shown that the proper use of internationally defined quantities and units in scientific publications promotes the best international communication in the science of photobiology and photochemistry. The accuracy and precision of the applied action spectra, measurement techniques and consideration of exposure geometry are of importance. The factors which influence the quality of the original photobiological research, the possible sources of error and the levels of uncertainty in applying laboratory action spectra to human health risk assessment have to be taken into account.

Solar ultraviolet radiation is a significant factor in environmental biology. It can be measured spectrally by a spectroradiometer, or non-spectrally by a broadband radiometer. The characteristics of these instruments determine the reliability and suitability of the results. The radiation data should be accompanied by detailed documentation on the calibration and operating procedures, sufficient to assure the data user that the measurements are accurate enough for the intended purpose. A description of the observing site and conditions should also be supplied, so that the user can determine whether the observing conditions meet the requirements of the environmental investigations.

The intensity of solar UV radiation at the Earth's surface is highly variable. The most important parameters for cloudless conditions are in the sequence of their significance: solar zenith angle, total ozone content of the atmosphere, amount and type of aerosols, albedo of the surrounding and altitude above sea level. Based on measurements, the effects of these parameters are discussed individually. Furthermore, clouds usually attenuate the irradiance, only in exceptional cases they can lead to an increase over short time periods. The attenuation by clouds is less strong for UV radiation compared with radiation from the whole spectral range.

Earth observations from satellites located in deep space offer the exciting opportunity to look at the Earth in a bulk thermodynamic sense, particularly as an open system exchanging radiative energy with the Sun and space, in a way never done before – “the Earth as a whole planet”, astronomers would say. This is a fundamental scientific goal with very appealing prospects for Earth sciences. Climate research requires stable, accurate, long-term observations made with adequate spatial and temporal resolution in a synoptic context. From deep-space vantage points we can, with a single spacecraft, sample the outgoing energy from virtually an entire hemisphere of Earth at once with high temporal and spatial resolution. Measured spectral radiances will be transformed into data products (e.g., ozone; aerosols; cloud fraction, thickness, optical depth, and height; sulfur dioxide; precipitable water vapor; volcanic ash; and UV irradiance). At present this is only partially possible by combining data from low Earth orbit and geostationary orbit satellites into an asynoptic composite of hundreds of thousands of pixels - rather like assembling an enormous jigsaw puzzle. Another advantage of the deep-space perspective is that, because of the integral view of the planet's hemispheres, the observations will simultaneously overlap the observations of every LEO and GEO satellite in existence, making possible a unique synergy with great potential benefits for the Earth sciences.

The main consequence of the ozone depletion, which is apparent over much of the globe several yeas ago, is the increase of the biologically damaging UV irradiance at the ground level. Data for the airborne spectral observations of upward irradiance at the upper level (∼5 km) and downward irradiance at the down level (∼500m) are compared for finding the empirical dependence between them. Radiation experiments aver different surfaces (sand, water, snow) and in different conditions (clear or cloudy sky) are considered.

The solar UV radiation flux reaching the Earth is one of important factors to evaluate the planetary energy balance. The UV flux (200 – 400 nm) at the top of atmosphere is about 8% of the total flux from the Sun. But the amount of UV measured at ground level is lower, this because the UV, crossing the atmosphere, undergoes both strong spectral absorption and diffusion. The spectral part 200 – 280 is practically negligible at ground.

The normal irradiance, and the radiation on a horizontal surface, as global spectral irradiance is expressed by vertical component of direct radiation and the diffused one. They are obtained by instruments calibrated in physical units, or evaluated by models.

The work shows the problems linked to a correct understanding and interpretation of solar UV data sampled at ground, several practical and theoretical problems of measurement are shown. In particular, the comparison among different sites or instruments is analyzed, and the effects due to O₃ level, albedo and cloudiness are discerned. The impact of clouds and their position respect to the Sun on the observation carried out both by spectral and broad-band instruments are also examined by models.

The UV observations weighted for the plant action spectrum are rather limited. The activities in the UV ground-based measurements mainly focused on the human erythemal effects. Our objective is to find the long-term variations in the UV radiation weighted by the plant effects using the data (erythemal UV irradiance and total ozone measurements) collected at in the period of 1992–2003. The transfer function from the erythemal weighted UV radiation to the UV radiation convolved with the plant action spectrum is constructed using total ozone observations (from the Brewer spectrophotometer) and UV spectral and UV erythemal data (from the Brewer spectrophotometer and the broadband UV biometer SL 501 for the erythemal effects) taken during the intercomparison campaign of the spectrophotometers in Warsaw, May 2004. The long-term variations in the reconstructed data are discussed concerning the impact of total ozone and cloudiness fluctuations on the UV radiation for the period of 1992–2003 (Belsk [52N,21E], Poland).

ZnSe(Te) crystals can be used as components of “semiconductor-metal” structures with Schottky barrier that are photosensitive in the UV range. Basing on the n- ZnSe(Te,O)-Ni structures in combination with filters, selective photodetectors have been developed for UVA and UVB ranges, which were used in development of small-sized household and professional instruments.

New UV meters and software have been developed and manufactured for environmental monitoring in the Ukraine. The multifunctional meters offer low cost and provide one and four channels for biological, meteorological and medical applications. The main specifications, advantages, and design features of each device developed is presented.

Solar UV interacts with and alters a number of cellular components. Among these, DNA is perhaps the most critical and irreplaceable target. The majority of UV-induced damage takes the form of pyrimidine dimers. These lesions act both as blocks to the progression of both replicative DNA polymerases and the RNA polymerase holoenzyme. Expression of “dimer bypass” DNA polymerases may also lead to permanent changes (mutations) in DNA sequence, by inserting potentially incorrect bases opposite the lesion. Thus the excision or reversal of UV-induced damage is important both in the soma, as it is required for the maintenance of gene expression, and in the 'germline', where error-free repair pathways maintain genomic integrity. The basic mechanisms of photoreactivation and excision repair of UV-induced dimers are well understood in plants, although we would like to learn more about the tissue-specificity and environmental regulation of these important UV-protective mechanisms. Field experiments suggest that repair of UV-induced dimers is not essential to the survival of Arabidopsis plants, nor is the expression of the sinapic acid esters that act as natural sunscreens. However, plants that are defective in both repair and sunscreen production die within hours of exposure to natural light.

Progression through the cell cycle in the presence of unrepaired DNA damage products leads to a progressive deterioration of the genome. During S phase, persisting lesions are either miscopied or produce daughter strand gaps opposite dimers, which are difficult to repair in an error-free manner. For this reason, cells respond to persisting DNA damage by arresting the cell cycle in order to provide time prior to the initiation of S phase (G1/S arrest), the continuation of S phase (intra-S arrest) or progression into M (G2/M arrest). These DNA damage-induced cell cycle “checkpoints” are now being characterized in plants. Given the homologies between plant and mammalian damage checkpoint genes, it is likely that many aspects of cell cycle regulation by UV-induced damage are shared between plants and animals.

One UV-induced mammalian response to persisting DNA damage is the induction of programmed cell death. This actively induced apoptosis and necrosis of damaged cells leads to the inflammation observed in sunburn. The induction of cell death has two beneficial effects: the resulting bursts of radicals are thought to further stimulate the repair response of neighboring cells, and the suicide of damaged cells precludes their possible progression into cancer. Plant cells can also be killed by very high doses of UV light, in what might be a programmed response (as the genomic “laddering” characteristic of programmed cell death occurs), but it is not clear that such a response occurs under natural light. Given plants natural resistance to the lethal effects of cancer, it is possible that plants differ from mammals in this aspect of UV-response, and lack a sensitive apoptotic response to DNA damage.

This paper deals with UV-induced ds (ss)-DNA damage, placed in buffer solution and in wet, dry absorbed layer, using optical spectroscopy and conductivity methods. Mechanisms of UV induced ds (ss)-DNA damage are accounted of photochemical reactions of cyclobutane-pyrimidine dimer and (4-6) adduct formation. These models are based on: decreasing absorption intensity with maxima at 252 nm for ds-DNA and 256 nm for ss-DNA water solutions, and long-wave shifting of absorption maximum (252 nm) for ds-DNA water solution. The absorption spectral range of 235 < λ < 252 nm is defined by absorption on T and A bases. Photoluminescence spectra of wet ds-DNA layer have maxima at 432,440 and 454, 463 nm before and after UV irradiation of 337 nm and 365 nm for one hour respectively.

The photochemical reactions appear by decreasing dry absorbed ds-DNA molecular layer with networks conductivity under periodically switched UV irradiation. As a result the conductivity decreases after the first radiation reflecting the reducing of pyrimidine bases (that formed dimers) and the contribution to ds-DNA conductivity. The increasing of conductivity after UV irradiation could be caused by a particular reparation of ds-DNA under applied voltage of 1 V. But it's important to develop methods for the further ability to direct possible photochemical reactions in ds (ss)-DNA.

UV radiation damage DNA and can trigger Apoptosis in animal cells. Using A. thaliana we have shown that UV radiation can induce apoptotic-like changes at the cellular level and that an UV experimental system was relevant to the study of Programmed Cell Death (PCD) in plants. UV induction of PCD requires light and a protease cleaving the caspase substrate Asp-Glu-Val-Asp (DEVDase activity) is induced within 30 minutes and peaks at one hour. This DEVDase appears related to animal caspases at the biochemical level, being insensitive to broad-range cysteine protease inhibitors. In addition, caspase1, caspase-3 inhibitors and the pancaspase inhibitor p35 were able to suppress DNA fragmentation and cell death. These results suggest that UV radiation induce a PCD in plants that is apoptotic-like. This pathway appears however to have differences when compare to the animal pathway. In particular, there is no convincing evidence that plant cell activates PCD in response to DNA damage.

Plant secondary metabolites are involved in versatile functions on different levels in the plant organism. One of the roles is scavenging of free radicals and the protection against excess oxidation caused by UV irradiation, chemical oxidants or pathogen attack or other kinds of stress. Involved are phenolic compounds from different classes such as numerous phenol carboxylic acids, hydroxylated flavonoids such as flavones, flavonols, anthocyanins, procyanidins and isoflavonoids. Many of these substances have been isolated from plant species possessing valuable and intensively studied medicinal properties.

For assessment of the free radical scavenging and antioxidant capacity of phenolic complexes in plants the chemical in vitro (cell free) tests can be used for their relative simplicity and sometimes reasonable cost. Here, we will report the application of several antioxidant and anti-free radical spectrophotometric assays for testing the antioxidant abilities of some rarely studied plant species containing different classes of polyphenols. In addition, the antimutagenic bacterial assays were used to examine the in vivo genoprotective activity of these compounds against chemical mutagens. Among the investigated compounds there are lipophilic flavones from Scutellaria baicalensis and Iridaceae-type isoflavonoids from Belamcanda chinensis. Phenolic acids, procyanidins and flavonols containing Lamiaceae species such as Leonurus cardiaca, Lamium sp., Stachys betonica, Marrubium vulgaris, Galeopsis tetrahit have been also studied to comprise wider spectrum of different types of polyphenols. The antimutagenic activity of the extracted phenol complexes and isolated compounds correlates with free radical scavenging. In the Ames bacterial assays the direct mutagenesis by chemical mutagens can be distinguished from the mutagenesis induced by activation of pro-mutagen with cytochrome P-450 enzymatic microsomal fractions. Free radical scavenging by the low molecular weight compounds can play an important role as the last line of defense against oxidative damage of the cells for they are more stable than enzymatic antioxidant apparatus and can be easily accumulated in stress conditions (e.g. deposited in the cell wall or the vacuole). Superoxide scavenging can protect the cells against the production of deleterious peroxynitrite upon reaction of the relatively harmless superoxide with an important signaling molecule - nitric oxide. The in planta function of the antioxidant and antigenotoxic compounds should be further explored in order to obtain the complete insight into their role in protecting the plant cell.

The radiation amplification factor (RAF) and its dimensionless sensitivity provide a useful and general mathematical base. Dimensionless sensitivity (α) is represented by the formula α = d(lgy)/d(lgx). With such definition the ranges of constancy of the α(V) dependency correspond to the power behaviour (y^∼x^α). Processing of data by this approach permits us to compare different physical values. Use of the exponent RAF offers greater possibilities for comparing the effects and mechanisms of UV radiation.

Ultraviolet B radiations increase the activity of the pure hydrolase (alpha amylase, E.C.3.2.1.1.) and Merck peroxidase (E.C.1.11.1.7.), by means of free radicals generated from synthetic polymers walls of the experimental tubes. The activation is higher in UV-B than in UV-A. UV-B and UV-A increase the intensity determined on nude alga Tetraselmis suecica, on exponential phase of cultivated bacteria Escherichia coli O₁₅₇, Acinetobacter calcoaceticus, and on total germs, after the short time of exposure in the thermo-stated conditions, the cell structure destruction by means of free radicals of activated hydrolases.

The nude alga Tetraselmis suecica is more resistant and store starch and lipids. It has the skill to convert the energy of radiations in the chemical energy of synthesis products. Creating a thick inhomogeneous sub-silique, under which a new silique appears, encysts some individuals of Tetraselmis suecica. Other cells increase their glucide (intra-plastids starch granules) and lipid reserves of provisions (oleosoma appear in the central part of the cell and affect the tillacoide lamellar structure; plasto-globules appear as well).

Cultivated bacteria on poor specific media have a small development in 300 – 800 nm. If the bacteria are cultivated on reach media, which absorb UV (Martin medium) they are developed by n³ rule, instead n² in the first stage, after irradiations of bacteria culture bottle (transmittance 235 – 800 nm).

Reactive oxygen species can arise from normal metabolic activity such as organelle-based electron transport or be intermediates in signal transduction pathways activated by plant respiratory burst oxidase homologs (Rboh). Divergent stress including temperature drought and UV-B exposure yield overlapping transcriptome response profiles whose origin can be traced to the use of reactive oxygen signaling intermediates. However, ROS signaling is ubiquitous and contributes in pathways controlled by hormones and developmental cues as well. Scavenging systems and NO are likely to temper its signaling properties and help contribute to the specificity of particular responses.

Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. In animals, NO is implicated in a number of diverse physiological processes such as neurotransmission, vascular smooth muscle relaxation, and platelet inhibition. It may have beneficial effects, for example as a messenger in immune responses, but it's also potentially toxic when the antioxidant system is weak and an excess of reactive oxygen species (ROS) accumulates. During the last few years NO has been detected also in several plant species, and the increasing number of reports on its function in plants have implicated NO as an important effector of growth, development, and defense. The innate immune system of organisms as diverse as vertebrates, invertebrates, and plants shows several characteristics similar with respect to involvement of NO. In the mammalian immune system, NO cooperates with ROS to induce apoptosis of tumor cells and macrophage killing of bacteria. In plants a similar mechanism has evolved to prevent tissue invasion by pathogens. The rapid accumulation of ROS and NO through the activation of enzyme systems similar to neutrophil NADPH oxidase and nitric oxide synthase (NOS) is one of the earliest events in the resistance response. Both NO and ROS are necessary to trigger host cell death in order to delimit the infected zone and avoid the multiplication and spread of the pathogen. NO and ROS are also components of highly amplified and integrated defense system that triggers the local expression of resistance genes. NO also functions independently of ROS in the induction of various defense genes including pathogenesis-related proteins and enzymes of phenylpropanoid metabolism involved in the production of lignin, antibiotics and the secondary signal salicylic acid. NO signaling functions depend on its reactivity and ROS are key modulators of NO in triggering cell death, although through mechanisms different from those commonly observed in animals.

The key research questions must be directed to the effects of UV on NO generation and action in plants. Research programmes will require methods to assess accurately NO emissions from leaves and to determine NO concentrations in cells and sub-cellular microdomains; the use of mutants and transgenic plants altered in NO synthetic and scavenging capacities; analyses of the molecular and biochemical events required for activation of PCD by NO and UV; and the development of techniques to monitor simultaneously cell death, NO and ROS generation in the field during exposure to UV.

In the late 1990s, NO became an increasingly popular target of investigation in plants. As in mammals, NO fulfils a broad spectrum of signaling functions in pathophysiological processes in plants. Here, we summerize studies published in recent years that provide novel insights into the signaling functions of NO produced by plant cells exposed to abiotic stresses and biotic stress (pathogen-derived elicitors). Particularly, we report that NO emerges as a key messenger governing the overall control of Ca²⁺ homeostasis. Although the precise signaling functions of NO are poorly understood, its capacity to modulate Ca²⁺ homeostasis provides an extraordinary and remarkably effective way of conveying information.

Nitric oxide (NO) is a very important molecule taking part in various signaling pathways in plants including plant responses to different stress conditions. In this work we have investigated the effect of NO on the cytosolic free calcium concentration ([Ca²⁺]_cyt) under oxidative stress induced by hydrogen peroxide. Using seedlings of transformed Arabidopsis thaliana expressing Ca²⁺-reporter apoaequorin in cytosol, we have shown that H₂O₂ influences [Ca²⁺]_cyt in a dose-depended manner and induces two peaks of calcium-dependent chemiluminescence measured in plant seedlings. The NO donors sodium nitroprusside (SNP) and (±)-E-2-[(E)-Hydroxyimino]-6-methoxy-4-methyl-5-nitro-3-hexenamide (NOR-1) alone led to a transient increase in [Ca²⁺]_cyt, but simultaneously reduced the amplitude of the first peak of H₂O₂–induced [Ca²⁺]_cyt increase by 42%. The nitric oxide synthase (NOS) and nitrate reductase (NR) inhibitor analysis showed that the production of NO, participating in H₂O₂-induced Ca²⁺-response, occurred due to NOS-like plant enzyme to be sensitive to oxidative stress. In summary, our data indicate that NO is likely to have a protective effect under oxidative stress and reduced a calcium increase induced by reactive oxygen species like hydrogen peroxide.