OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG.

Optogenetics' advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl ß-d-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. Here, we harnessed the light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as a tool for light control and engineered LacI into two light-responsive variants, OptoLacIL and OptoLacID. These variants exhibit direct responsiveness to light and darkness, respectively, eliminating the need for IPTG. Building upon OptoLacI, we constructed two light-controlled E. coli gene expression systems, OptoE.coliLight system and OptoE.coliDark system. These systems enable bifunctional gene expression regulation in E. coli through light manipulation and show superior controllability compared to IPTG-induced systems. We applied the OptoE.coliDark system to protein production and metabolic flux control. Protein production levels are comparable to those induced by IPTG. Notably, the titers of dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded 110% and 60% of those induced by IPTG, respectively. The development of OptoLacI will contribute to the advancement of the field of optogenetic protein engineering, holding substantial potential applications across various fields.

Mitigating salt stress by conditioning seeds with ultraviolet light (UV-C) in white oats (Avena sativa L.).

Seed conditioning with ultraviolet light (UV-C) might (1) improve crop yield and quality, (2) reduce the use of agrochemicals during cultivation, and (3) increase plant survival in high salinity environments. The aim of this study was to examine the effects of UV-C conditioning of white oat seeds at two doses (0.85 and 3.42 kJ m-2) under salinity stress (100 mM NaCl). Seeds were sown on germination paper and kept in a germination chamber at 20°C. Germination and seedling growth parameters were evaluated after 5 and 10 days. Data demonstrated that excess salt reduced germination and initial growth of white oat seedlings. In all the variables analyzed, exposure of seeds to UV-C under salt stress exerted a positive effect compared to non-irradiated control. The attenuating influence of UV-C in germination was greater at 0.85 than at 3.42 kJ m-2. Thus, data indicate that conditioning white oat seeds in UV-C light produced greater tolerance to salt stress. These findings suggest that UV-C conditioning of white oat seeds may be considered as a simple and economical strategy to alleviate salt-induced stress.

Comparison of Conventional and Microwave Assisted Heating on Carbohydrate Content, Antioxidant Capacity and Postprandial Glycemic Response in Oat Meals.

Minimally processed cereal breakfast products from whole grain entered the market due to consumer demand of more nutritional food with more controlled sugar release. However, the subsequent processing of such products with different cooking methods in the consumer's kitchen may lead to significant differentiation of their nutritional value. Therefore, the evaluation of the impact of frequently used cooking methods on a final quality of breakfast cereals meal is needed. The present study investigates how the two different methods of heating, conventional and microwave (MW) assisted, affect the carbohydrate content, profile and resulting glycemic index of so prepared food as well as the antioxidant activity of meals. Two products available on the market-oat bran and flakes-were used. The highest starch content in fluid phase of oatmeal was detected in samples heated for 3 min with microwaves, regardless the type. The lowest starch content was obtained for 5 min MW heated flakes sample. The total content of glucose was about 1.5 times lower in bran vs. flakes oatmeal. The highest ß-glucan content in fluid fraction was also observed for bran meal but its release was independent of applied conditions.

The impact of short-term UV irradiation on grains of sensitive and tolerant cereal genotypes studied by EPR.

BACKGROUND: UV irradiation has ionisation character and leads to the generation of reactive oxygen species (ROS). The destructive character of ROS was observed among others during interaction of cereal grains with ozone and was caused by changes in structures of biomolecules leading to the formation of stable organic radicals. That effect was more evident for stress sensitive genotypes. In this study we investigated the influence of UV irradiation on cereal grains originating from genotypes with different tolerance to oxidative stress. RESULTS: Grains and their parts (endosperm, embryo and seed coat) of barley, wheat and oat were subjected to short-term UV irradiation. It was found that UV caused the appearance of various kinds of reactive species (O2-• , H2 O2 ) and stable radicals (semiquinone, phenoxyl and carbon-centred). Simultaneously, lipid peroxidation occurred and the organic structure of Mn(II) and Fe(III) complexes become disturbed. CONCLUSIONS: UV irradiation causes damage of main biochemical structures of plant tissues, the effect is more significant in sensitive genotypes. In comparison with ozone treatment, UV irradiation leads to stronger destruction of biomolecules in grains and their parts. It is caused by the high energy of UV light, facilitating easier breakage of molecular bonds in biochemical compounds. © 2017 Society of Chemical Industry.

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain.

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the ß-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.

Chlorophyll fluorescence, photochemical reflective index and normalized difference vegetative index during plant senescence.

The relationship between the Photochemical Reflectance Index (PRI), Normalized Difference Vegetation Index (NDVI) and chlorophyll fluorescence along senescence was investigated in this work. Reflectance and radiance measurements were performed at canopy level in grass species presenting different photosynthetic metabolism: Avena sativa (C3) and Setaria italica (C4), at different stages of the natural senescence process. Sun induced-chlorophyll fluorescence at 760nm (SIF760) and the apparent fluorescence yield (SIF760/a, with a=irradiance at time of measurement) were extracted from the radiance spectra of canopies using the Fraunhofer Line Discrimination-method. The photosynthetic parameters derived from Kautsky kinetics and pigment content were also calculated at leaf level. Whilst stand level NDVI patterns were related to changes in the structure of canopies and not in pigment content, stand level PRI patterns suggested changes both in terms of canopy and of pigment content in leaves. Both SIF760/a and ΦPSII decreased progressively along senescence in both species. A strong increment in NPQ was evident in A. sativa while in S. italica NPQ values were lower. Our most important finding was that two chlorophyll fluorescence signals, ΦPSII and SIF760/a, correlated with the canopy PRI values in the two grasses assessed, even when tissues at different ontogenic stages were present. Even though significant changes occurred in the Total Chlr/Car ratio along senescence in both studied species, significant correlations between PRI and chlorophyll fluorescence signals might indicate the usefulness of this reflectance index as a proxy of photosynthetic RUE, at least under the conditions of this study. The relationships between stand level PRI and the fluorescence estimators (ΦPSII and SIF760/a) were positive in both cases. Therefore, an increase in PRI values as in the fluorescence parameters would indicate higher RUE.

A metabolomic study in oats (Avena sativa) highlights a drought tolerance mechanism based upon salicylate signalling pathways and the modulation of carbon, antioxidant and photo-oxidative metabolism.

Although a wealth of information is available on the induction of one or several drought-related responses in different species, little is known of how their timing, modulation and crucially integration influence drought tolerance. Based upon metabolomic changes in oat (Avena sativa L.), we have defined key processes involved in drought tolerance. During a time course of increasing water deficit, metabolites from leaf samples were profiled using direct infusion-electrospray mass spectroscopy (DI-ESI-MS) and high-performance liquid chromatography (HPLC) ESI-MS/MS and analysed using principal component analysis (PCA) and discriminant function analysis (DFA). The involvement of metabolite pathways was confirmed through targeted assays of key metabolites and physiological experiments. We demonstrate an early accumulation of salicylic acid (SA) influencing stomatal opening, photorespiration and antioxidant defences before any change in the relative water content. These changes are likely to maintain plant water status, with any photoinhibitory effect being counteracted by an efficient antioxidant capacity, thereby representing an integrated mechanism of drought tolerance in oats. We also discuss these changes in relation to those engaged at later points, consequence of the different water status in susceptible and resistant genotypes.

Increasing genetic variability in black oats using gamma irradiation.

The black oat (Avena strigosa Schreb) is commonly used for forage, soil cover, and green manure. Despite its importance, little improvement has been made to this species, leading to high levels of genotypic disuniformity within commercial cultivars. The objective of this study was to evaluate the efficiency of different doses of gamma rays [(60)Co] applied to black oat seeds on the increase of genetic variability of agronomic traits. We applied doses of 0, 10, 50, 100, and 200 Gy to the genotype ALPHA 94087 through exposure to [(60)Co]. Two experiments were conducted in the winter of 2008. The first aimed to test forage trait measurements such as plant height, dry matter yield, number of surviving tillers, and seedling stand. The second test assessed seed traits, such as yield and dormancy levels. Gamma irradiation seems not to increase seed yield in black oats, but it was effective in generating variability for the other traits. Tiller number and plant height are important selection traits to increase dry matter yield. Selection in advanced generations of mutant populations can increase the probability of identifying superior genotypes.

Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

Effects of long-term chronic exposure to radionuclides in plant populations.

The results of field studies carried out on different plant species (winter rye and wheat, spring barley, oats, Scots pine, wild vetch, crested hairgrass) in various radioecological situations (nuclear weapon testing, the Chernobyl accident, uranium and radium processing) to investigate the effects of long-term chronic exposure to radionuclides are discussed. Plant populations growing in areas with relatively low levels of pollution are characterized by an increased level of both cytogenetic disturbances and genetic diversity. Although ionizing radiation causes primary damage at the molecular level, there are emergent effects at the level of populations, non-predictable from the knowledge of elementary mechanisms of cellular effects formation. Accumulation of cellular alterations may afterward influence biological parameters important for populations such as health and reproduction. Presented data provide evidence that in plant populations inhabiting heavily contaminated territories cytogenetic damage could be accompanied by a decrease in reproductive capacity. However, in less contaminated sites, because of the scarcity of data available, a steady relationship between cytogenetic effects and reproductive capacity was not revealed. Under radioactive contamination of the plant's environment, a population's resistance to exposure may increase. However, there are radioecological situations where an enhanced radioresistance has not evolved or has not persisted.

Individual maize chromosomes in the C(3) plant oat can increase bundle sheath cell size and vein density.

C(4) photosynthesis has evolved in at least 66 lineages within the angiosperms and involves alterations to the biochemistry, cell biology, and development of leaves. The characteristic "Kranz" anatomy of most C(4) leaves was discovered in the 1890s, but the genetic basis of these traits remains poorly defined. Oat × maize addition lines allow the effects of individual maize (Zea mays; C(4)) chromosomes to be investigated in an oat (Avena sativa; C(3)) genetic background. Here, we have determined the extent to which maize chromosomes can introduce C(4) characteristics into oat and have associated any C(4)-like changes with specific maize chromosomes. While there is no indication of a simultaneous change to C(4) biochemistry, leaf anatomy, and ultrastructure in any of the oat × maize addition lines, the C(3) oat leaf can be modified at multiple levels. Maize genes encoding phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the 2'-oxoglutarate/malate transporter are expressed in oat and generate transcripts of the correct size. Three maize chromosomes independently cause increases in vein density, and maize chromosome 3 results in larger bundle sheath cells with increased cell wall lipid deposition in oat leaves. These data provide proof of principle that aspects of C(4) biology could be integrated into leaves of C(3) crops.

Excited-state dynamics of protochlorophyllide revealed by subpicosecond infrared spectroscopy.

To gain a better understanding of the light-induced reduction of protochlorophyllide (PChlide) to chlorophyllide as a key regulatory step in chlorophyll synthesis, we performed transient infrared absorption measurements on PChlide in d4-methanol. Excitation in the Q-band at 630 nm initiates dynamics characterized by three time constants: τ1 = 3.6 ± 0.2, τ2 = 38 ± 2, and τ3 = 215 ± 8 ps. As indicated by the C13'=O carbonyl stretching mode in the electronic ground state at 1686 cm⁻¹, showing partial ground-state recovery, and in the excited electronic state at 1625 cm⁻¹, showing excited-state decay, τ2 describes the formation of a state with a strong change in electronic structure, and τ3 represents the partial recovery of the PChlide electronic ground state. Furthermore, τ1 corresponds with vibrational energy relaxation. The observed kinetics strongly suggest a branched reaction scheme with a branching ratio of 0.5 for the path leading to the PChlide ground state on the 200 ps timescale and the path leading to a long-lived state (>>700 ps). The results clearly support a branched reaction scheme, as proposed previously, featuring the formation of an intramolecular charge transfer state with ∼25 ps, its decay into the PChlide ground state with 200 ps, and a parallel reaction path to the long-lived PChlide triplet state.

[Effect of strengthening solar ultraviolet B band irradiation on oat (Avena sativa L. ) yield and its components in Qing Tibetan Plateau].

Stratospheric ozone depletion occurs mainly over polar regions during the spring when the solar Ultraviolet B-band (280-315 nm, UV-B) radiation is most intense in a year, but over the Qing Tibetan Plateau region, the highest intensity is from June to September when the amount of UV-B radiation reaching the regions is more than that in the adjacent areas lying in the same latitude by 10%. From June to September is just the time of plant's germination, development, and reproduction in the alpine region. UV-B radiation may alter the reproduction of the forage plant, oat (Avena sativa.), which plays the vital role in developing indigenous herdsman's animal husbandry industry. The responses of oat yield and its components to the enhanced ultraviolet B band irradiation under the field condition were surveyed. The effect shows that the grain yield is decreased significantly by strengthened UV-B irradiation, and at the same time the main consequence is the decrease in both the number of ears per square meter and the number of grains per ear, but the weight of 1 000 grains appears not significantly different. Compared with the same respective location in a spikelet, the grain weight is decreased significantly under the treated condition, mostly because of the decreases in the number of the third and forth floret grain and the grain weight at those respective positions, and the percentage of the first and second floret grain and their weight are evidently approved on the contrary.

[Effects of enhanced solar UV-B radiation on the effective photosynthetic leaf area and milking phase of oat under natural field condition in Qing Tibetan plateau].

Stratospheric ozone depletion occurs mainly over polar regions during the spring when the solar ultraviolet B-band (280-315 nm, UV-B) radiation is most intense in a year, but over the Qing Tibetan Plateau region, the intensity is highest from June to September when the amount of UV-B radiation reaching the regions is more than that in the adjacent areas lying in the same latitude by 10%. From June to September is just the time of plant's germination, development, and reproduction in the alpine region. The enhanced UV-B radiation may alter the rate of senescence in the forage plant, oats (Avena sativa), which plays the vital role in developing indigenous herdsman's animal husbandry industry. In the trial the authors enhanced the UV-B radiation by using lamp bank of florescent lights to increase the ambient levels of UV-B radiation in the field, we treated 3 groups under nature solar radiation, solar+0.50 W x m2, and solar+1.00 W x m2 respectively, and surveyed the rate of senescence by measuring the rate of lessening in the effective photosynthetic leaf area, the concentration of the chlorophyll, and the milking phase of oat phenology. The results proved that the effect of the enhanced UV-B radiation on the rate of senescence of oats is caused by reducing the effective photosynthetic leaf area per plant by 21.5%, accelerating the rate of the loss of the chlorophyll compared with that of the controlled by an average of 7.6% and shortening the milking phase by an average of 2 days in the treated groups compared with the rate in the controlled, but there were not statistically significant differences from those of the nontreated group in these index of oat's senescence. All the results evidenced that the enhanced solar UV-B radiation has no significantly ominous consequence on the oat ecological characteristics concerning annual plant reproduction in Qing Tibetan plateau.

Solar ultraviolet-B radiation increases phenolic content and ferric reducing antioxidant power in Avena sativa.

We examined the influence of solar ultraviolet-B radiation (UV-B; 280-320 nm) on the maximum photochemical efficiency of photosystem II (F(v)/F(m)), bulk-soluble phenolic concentrations, ferric-reducing antioxidant power (FRAP) and growth of Avena sativa. Treatments involved placing filters on frames over potted plants that reduced levels of biologically effective UV-B by either 71% (reduced UV-B) or by 19% (near-ambient UV-B) over the 52 day experiment (04 July-25 August 2002). Plants growing under near-ambient UV-B had 38% less total biomass than those under reduced UV-B. The reduction in biomass was mainly the result of a 24% lower leaf elongation rate, resulting in shorter leaves and less total leaf area than plants under reduced UV-B. In addition, plants growing under near-ambient UV-B had up to 17% lower F(v)/F(m) values early in the experiment, and this effect declined with plant age. Concentrations of bulk-soluble phenolics and FRAP values were 17 and 24% higher under near-ambient UV-B than under reduced UV-B, respectively. There was a positive relationship between bulk-soluble phenolic concentrations and FRAP values. There were no UV-B effects on concentrations of carotenoids (carotenes + xanthophylls).

Red/far-red light modulates phospholipase D activity in oat seedlings: relation of enzyme photosensitivity to photosynthesis.

Phospholipase D (PLD) activity was found to be higher in etiolated oat seedlings than in green seedlings. White and red (R) light exposure inhibited PLD activity in etiolated seedlings. Far-red light eliminated R-light-induced decrease in PLD activity, indicating phytochrome participation in observed photomodulation. Inhibitor of electron transport in chloroplast 3-(3,4-dichlorophenyl)-1,1-dimethylurea stimulated and glucose suppressed PLD activity in green and etiolated oat seedlings, respectively. These results suggest that PLD activity in oat seedling is regulated by light with involvement of phytochrome photoreceptor, and associated with photosynthesis process.

All-optical switching in plant blue light photoreceptor phototropin.

We theoretically analyze all-optical switching in the recently characterized LOV2 domain from Avena sativa (oat) phot1 phototropin, a blue-light plant photoreceptor, based on nonlinear intensity-induced excited-state absorption. The transmission of a cw probe laser beam at 660 nm corresponding to the peak absorption of the first excited L-state, through the LOV2 sample, is switched by a pulsed pump laser beam at 442 nm that corresponds to the maximum initial D state absorption. The switching characteristics have been analyzed using the rate equation approach, considering all the three intermediate states and transitions in the LOV2 photocycle. It is shown that for a given pump pulse intensity, there is an optimum pump pulsewidth for which the switching contrast is maximum. It is shown that the probe laser beam can be completely switched off (100% modulation) by the pump laser beam at 50 kW/cm2 for a concentration of 1 mM with sample thickness of 5.5 mm. The switching characteristics are sensitive to various parameters such as concentration, rate constant of L-state, peak pump intensity and pump pulse width. At typical values, the switch-off and switch-on time is 1.6 and 22.3 micros, respectively. The switching characteristics have also been used to design all-optical NOT and the universal NOR and NAND logic gates.

Stimulation of chlororespiration by heat and high light intensity in oat plants.

High irradiance and moderate heat inhibit the activity of the photosynthetic apparatus of oat (Avena sativa L.) leaves. The incubation of oat leaves under high light intensity in conjunction with high temperatures strongly decreased the maximal quantum yield of photosystem (PS) II, indicating the close synergistic effect of both stress factors on PS II inhibition and the subsequent irreversible damage to the photosynthetic apparatus. The PS I A/B protein levels remained similar to control values in leaves incubated under high light intensity or moderate heat, and decreased only when both stress factors were simultaneously applied. Immunoblot analysis of thylakoid membranes using specific antibodies raised against the NDH-K subunit of the thylakoidal NADH dehydrogenase complex (NADH DH) and against plastid terminal oxidase (PTOX) revealed an increase in the amount of both proteins in response to high light intensity and/or heat treatments. In addition, these stress treatments were seen to stimulate the activity of electron donation by NADPH and ferredoxin to plastoquinone, the PTOX activity in plastoquinone oxidation and the NADH DH activity in thylakoid membranes. Incubation with n-propyl gallate (an inhibitor of PTOX) inhibited the increase of NDH-K and PTOX levels under high light intensity and heat, and slightly stimulated the activity of electron donation by NADPH and ferredoxin to plastoquinone. Antimycin A (an inhibitor of cyclic electron flow) increased the NADH DH activity and preserved the levels of NDH-K and PTOX in thylakoid membranes from leaves incubated under high light intensity and heat. The up-regulation of the PTOX and the thylakoidal NADH DH complex under these stress conditions supports a role for chlororespiration in the protection against high irradiance and moderate heat.

Time-resolved detection of conformational changes in oat phytochrome A: time-dependent diffusion.

Conformational changes in oat phytochrome A (phy) in solution after photoexcitation of the red-absorbing form (Pr) were studied in time-domain by the pulsed laser-induced transient grating technique. It was found that the diffusion coefficient (D) of far-red-absorbing form (Pfr) of large phy (1.3 x 10(-11) m(2) s(-1)) is markedly reduced compared with that of Pr (5.8 x 10(-11) m(2) s(-1)). This large reduction indicates that the conformation of Pfr is significantly changed from that of Pr, so that the intermolecular interaction with water molecules increases. This change completes within 1 ms after the photoexcitation. On the other hand, D of Pr of intact phy (4.1 x 10(-11) m(2) s(-1)) first decreases upon photoexcitation to 0.89 x 10(-11) m(2) s(-1) within 1 ms and then gradually increases with a time constant of 100 ms to the value of Pfr, 1.7 x 10(-11) m(2) s(-1). This slower phase suggests that the conformation of the N-terminal region changes with 100 ms to decrease the intermolecular interaction with water after a global change in the large phy region. The increase of D was interpreted in terms of alpha-helix formation in the Pfr form from the random coil structure in the Pr form.

Plant responses to current solar ultraviolet-B radiation and to supplemented solar ultraviolet-B radiation simulating ozone depletion: an experimental comparison.

Field experiments assessing UV-B effects on plants have been conducted using two contrasting techniques: supplementation of solar UV-B with radiation from fluorescent UV lamps and the exclusion of solar UV-B with filters. We compared these two approaches by growing lettuce and oat simultaneously under three conditions: UV-B exclusion, near-ambient UV-B (control) and UV-B supplementation (simulating a 30% ozone depletion). This permitted computation of "solar UV-B" and "supplemental UV-B" effects. Microclimate and photosynthetically active radiation were the same under the two treatments and the control. Excluding UV-B changed total UV-B radiation more than did supplementing UV-B, but the UV-B supplementation contained more "biologically effective" shortwave radiation. For oat, solar UV-B had a greater effect than supplemental UV-B on main shoot leaf area and main shoot mass, but supplemental UV-B had a greater effect on leaf and tiller number and UV-B-absorbing compounds. For lettuce, growth and stomatal density generally responded similarly to both solar UV-B and supplemented UV-B radiation, but UV-absorbing compounds responded more to supplemental UV-B, as in oat. Because of the marked spectral differences between the techniques, experiments using UV-B exclusion are most suited to assessing effects of present-day UV-B radiation, whereas UV-B supplementation experiments are most appropriate for addressing the ozone depletion issue.