Alteration of Extracellular Enzymes in Pinto Bean Leaves upon Exposure to Air Pollutants, Ozone and Sulfur Dioxide

Diamine oxidase and peroxidase, associated with the wall in pinto bean (Phaseolus vulgaris L. var Pinto) leaves, can be washed out by vacuum infiltration and assayed without grinding the leaf. The diamine oxidase activity is inhibited in vivo by exposure of the plants to ozone (dose of 0.6 microliters per liter x hour), whereas the peroxidase activity associated with the wall space is stimulated. This dose does not cause obvious necrosis or chlorosis of the leaf. These alterations are greater when the dose of ozone exposure is given as a triangular pulse (a slow rise to a peak of 0.24 microliters per liter followed by a slow fall) compared to that given as a constant square wave pulse of 0.15 microliters per liter for the same 4 hour period. Exposure of the plants to sulfur dioxide (at a concentration of 0.4 microliters per liter for 4 hours) does not result in any change in the diamine oxidase or peroxidase activities, yet the total sulfhydryl content of the leaf is increased, demonstrating the entry of sulfur dioxide. These two pollutants, with different chemical reactivities, affect the activities of the extracellular enzymes in different manners. In the case of ozone exposure, the inhibition of extracellular diamine oxidase could profoundly alter the movements of polyamines from cell to cell.     

Fusicoccin and Air Pollutant Injury to Plants : Evidence for Enhancement of SO(2) but Not O(3) Injury

Garden peas (Pisum sativum L. cv Alsweet) and a tomato mutant (Lycopersicon esculentum Mill. var flacca) were sprayed with fusicoccin, a fungal toxin affecting membrane transport properties, before exposure to SO₂ or O₃. Tomatoes treated with 10 micromolar fusicoccin and exposed to SO₂ (0.6 microliter per liter for 2 hours) exhibited twice as much foliar necrosis as untreated plants exposed to SO₂. Peas treated with fusicoccin and exposed to SO₂ (0.7 to 1.0 microliter per liter for 2 hours) exhibited 2 to 6 times more injury than untreated plants exposed to SO₂. Peas treated with fusicoccin and exposed to O₃ had less injury than untreated plants exposed to O₃ (0.1 to 0.3 microliter per liter for 2 hours). Several lines of evidence suggested that the fusicoccin enhancement of SO₂ injury is not the result of increased gas exchange, i.e. the tomato mutant has permanently open stomata under all conditions, and in peas fusicoccin had no effect on SO₂ or H₂O flux in plants exposed to 0.12 microliter per liter SO₂. However, a 21% greater leaf conductance in fusicoccin treated versus untreated plants indicated the possibility of some differences in gas exchange for peas exposed to 1.0 microliter per liter SO₂.     

Water Stress Reduces Ozone Injury via a Stomatal Mechanism

Various studies have shown that water-stressed plants are more tolerant of ozone exposures than are unstressed plants. Two probable explanations for this tolerance are (a) stomatal closure which reduces ozone uptake and (b) biochemical or anatomical changes within the leaves. Phaseolus vulgaris cv Pinto bean plants were established and transferred to membrane systems which controlled the osmotic potential around the roots at −35 or −80 kilopascals for 5 days prior to ozone treatment (0 or 1.0 microliters per liter for 2 hours). Both water-stressed and unstressed plants were sprayed with various concentrations of abscisic acid to close the stomata or with fusicoccin to induce stomata opening. The abaxial stomatal resistances of primary and trifoliate leaves were measured just prior to ozone exposure. Plant response to ozone was determined by stress ethylene production and chlorophyll loss. Both water stress and abscisic acid induced stomatal closure and reduced ozone injury. In water-stressed plants, fusicoccin induced stomatal opening and those plants were as sensitive to ozone as were the non-water-stressed plants. These data suggest that water stress protects plants from ozone injury mainly through its influence on stomatal aperture rather than through biochemical or anatomical changes.     

Lipids,amino acids,sugars, hardiness and growth of Festuca arundinacea

Tall fescue (Festuca arundinacea Schreb. cv S.170) plants were grown in environments differing only in temperature: 6/4, 16/14 or 21/19°. The content of total and individual sugars and amino acids in leaf laminae and roots did not relate closely to the hardiness of the organ. The unsaturation of lipid fatty acids alone was clearly unrelated to hardiness because the difference in unsaturation was greater in the neutral lipids, glycolipids and phospholipids from roots than from leaves but only the latter differed in hardiness. Total amounts of lipids could have been related to hardiness but phospholipids and glycolipids in the roots were not. At least some of these changes may be related to adaptation of growth to temperature.     

Abscission: the phytogerontological effects of ethylene

The role of ethylene in the aging of bean (Phaseolus vulgaris L. cv. Red Kidney) petiole abscission zone explants was examined. The data indicate that ethylene does accelerate aging in addition to inducing changes in break strength. Application of ethylene during the aging stage (stage 1) promoted abscission when followed by a second ethylene treatment during the cell separating stage (stage 2). The half-maximal effective concentration of ethylene to induce aging was around 0.3 microliter per liter; 10 microliters per liter was a saturating dose. CO₂ reversal of ethylene action during stage 1 was incomplete and gave ambiguous results. CO₂ (10%) reversed the effect of 10 microliters per liter ethylene but not 1 microliter per liter ethylene. The possibility that ethylene not only accelerated aging but was also a requirement for it was tested, and experimental evidence in favor of this idea was obtained. It was concluded that ethylene plays a dual role in the abscission of bean petiole explants: a phytogerontological effect and a cellulase-inducing effect.     

Differences in lipid and in fatty acid composition of taproots and lateral roots of faba bean plants (Vicia faba L.) grown in saline media

Abstract

The content and composition of lipids and fatty acids of taproots and lateral roots of Vicia faba were investigated with plants grown under saline (80 mM NaCl) and non-saline conditions. Lipids of both types of faba bean roots are constituted of ～80% phospholipids, of ～15% glycolipids and of some 5% of wax-esters. Phosphatidylethanolamine (PE) and phosphatidylcholine are the main constituents of both root types. Di-phosphatyidylglycerol and phosphatidylinositol are present in medium concentrations, whereas phosphatidylglycerol and phosphatidylserine are present only in trace amounts. The content of sulpholipids was much lower in salt-treated roots as compared with that of the controls. The content of cardiolipins and PE was higher in lateral roots than in the taproots. Roots of salt-treated plants had some 18% lower ether-soluble lipid content in the lateral roots, and approximately 28% lower than the taproots. Less than 25% of the fatty acids of the extracted phospholipids were saturated, with palmitic acid as the main constituent (13 – 18%). Linoleic acid comprised 65 – 70% of the unsaturated acids. Differences in the composition of some fatty acids were found between taproots and lateral roots of salt-treated plants. The observed differences in composition between root types suggests that the reported physiological differences between such roots could be based, at least in part, on structural or compositional differences in their lipids.     

Thigmomorphogenesis: membrane lipid and protein changes in bean plants as affected by mechanical perturbation and Ethrel

When bean plants (Phaseolus vulgaris L. cv. Red Cherokee Bush) are mechanically perturbed by rubbing, their stem elongation is inhibited and the stem thickness increases. The decrease in cell elongation and the increase in lateral cell divisions, which are partially responsible for this syndrome, were correlated with a decrease in the tree fatty acids and in the phospholipids of the membranes of microsomal fractions of first and second internodes of mechanically stimulated plants. This was true even though only the first internode was mechanically stimulated. Of the microsomal free fatty acids, mechanical perturbation induced an increase in myristic acid and large decreases in stearic, oleic, linoleic and linolenic acids. It also reduced the unsaturated:saturated ratio of the fatty acids. It induced a decrease in phosphatidyl choline but an increase in phosphatidyl ethanolamine. When the fatty acids were cleaved from the microsomal phospholipids, mechanical perturbation caused only a slight decrease in the unsaturated:saturated ratio and no significant changes in the double bond index. Mechanical perturbation induced an increase in the total microsomal protein and of membrane-associated latent IDPase. However, the activity of membrane-associated KCN-insensitive NADPH cytochrome-c reductase was decreased by mechanical perturbation. Treatment of the first internode with exogenous Ethrel produced results that were very similar in all respects to those obtained by mechanical perturbation. The factors inducing hardening against frost and drought, as achieved by mechanical perturbation and Ethrel treatment, are not only related to sterols or the polar head-groups of phospholipids but may also be related to the protein components, and all may have an effect on the fluidity of a bilayer membrane model. These data support the hypothesis that ethylene mediates thigmomorphogenesis and that mechanical perturbation of the first internode results in the acropetal transport of a translocatable thigmomorphogenetic factor.     

Lipid profiles of Aspergillus niger and its unsaturated fatty acid auxotroph, UFA2

A comparative study of the mycelial lipid composition of a wild strain (V35) and one unsaturated fatty acid auxotroph (UFA2) of Aspergillus niger has been performed. The lipid composition of both strains are qualitatively the same but quantitatively different. All the strains contain the following phospholipids: cardiolipin, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine; and triglycerides, diglycerides, monoglycerides, ergosterol, and sterol esters as the neutral lipids; mono- and di-galactosyl diglyceride as the major glycolipids along with small amounts of the corresponding mannose analogs. Phosphatidylethanolamine and phosphatidylcholine constitute the bulk of the phospholipids. The mutant (UFA2) contains a higher level of glycerides and lower levels of sterol (both free and esterified form), phospholipids, and glycolipids than the wild type. Aspergillus niger contains C16 to C18 saturated and unsaturated fatty acids. Small amounts of long-chain (C20 to C24) and short-chain (C10 to C14) saturated and unsaturated acids are also present. Linoleic, oleic, and palmitic are the major acids, stearic and linolenic acids being minor ones. UFA2 grows only in the presence of unsaturated fatty acid (C16 or C18) and accumulates a higher concentration of supplemented acid which influences its fatty acid profile.     

Fatty acids of lipids from cultured soybean and rape cells

Lipids from cultured cells, leaves and seeds of two varieties each of soybean (Glycine max) and oil seed rape (Brassica napus) were separated into neutral lipids, glycolipids and phospholipids and their fatty acids were analysed. Usually, the fatty acid composition differed between corresponding fractions from cultured cells, leaves and seeds. Differences were least marked in (i) the phospholipids from cultured cells and leaves of soybean and (ii) the neutral lipids from cultured cells and seeds of rape. In the cultured cells, the fatty acid composition of the phospholipids differed from that of the glycolipids and neutral lipids, and fatty acids of chain length greater than C₁₈ comprised a large proportion of the fatty acids of the glycolipids.     

Changes in respiration, photosynthesis, adenosine 5'-triphosphate, and total adenylate content of ozonated pinto bean foliage as they relate to symptom expression

The effect of 0.25 to 0.30 microliter per liter ozone on photosynthesis and respiration and on the ATP and total adenylate content of the primary leaves of pinto beans (Phaseolus vulgaris L.) was examined. Changes in these parameters over a 72-hour time period were correlated with the development of symptoms of ozone toxicity. Toxicity symptoms normally appeared within 24 hours. The content of ATP and total adenylates increased immediately following a 3-hour exposure to ozone. Photosynthesis was depressed initially, but returned to normal within 24 hours. Respiration was not always altered initially, but it was significantly stimulated within 24 hours. We interpret the results to mean that the changes in adenylate content and photosynthesis are early events in the initiation of ozone damage and that the change in respiration is a consequence rather than a cause of cellular injury.     

The effect of benzylaminopurine on fatty-acid composition of membrane lipids in shoots of Karelian birch in vitro

The effect of the synthetic analogue of natural cytokinin 6-benzylaminopurine (BAP) on the fatty-acid composition of membrane lipids in shoots of Karelian (Curly) birch Betula pendula Roth var. carelica (Mercklin) Hämet-Ahti under in vitro conditions was studied. It was found that the addition of BAP (in a concentration of 0.25, 0.5, 1.0, or 2 mg/L) to the nutrient medium leads to an increase in the proportion of saturated fatty acids in phospholipids, whereas in the control variant unsaturated fatty acids predominated in phospholipids. In glycolipids, on the contrary, BAP caused an increase in the synthesis of unsaturated fatty acids (predominantly, linolenic), but the degree of its effect depended on the concentration of the hormone. In particular, when the maximum amount of the studied concentration (2.0 mg/L) was used, the use of BAP was shown to decrease the proportion of linolenic acid. It is suggested that the revealed changes in the composition of glycolipids are indicators of the stimulating effect of cytokinin (up to a certain concentration) on the structure and function of chloroplasts. Due to the photomixotrophy of shoot cultures, chloroplasts are an important factor determining not only their photosynthetic activity, but also cell proliferation and the direction of in vitro morphogenesis.     

Ethylene, Ethane, Acetaldehyde, and Ethanol Production By Plants under Stress

Red pine (Pinus resinosa Ait.) and paper birch (Betula papyrifera Marsh.) seedlings exposed to sulfur dioxide produced acetaldehyde and ethanol, and exhibited increased production of ethylene and ethane. Gas chromatographic measurement of head space gas from incubation tubes containing leaves or seedlings was a simple method of simultaneously measuring all four compounds. Increased ethylene production had two phases, a moderate increase from the beginning of the stress period and a large increase just prior to appearance of leaf lesions. Ethane production in SO₂-stressed plants did not increase until lesions appeared. Acetaldehyde and ethanol production began within 6 hours at 0.3 microliter per liter SO₂ and 24 hours at 0.1 microliter per liter SO₂ and continued throughout a 6-day fumigation. Production of acetaldehyde and ethanol continued when plants were removed to clean air for up to 2 days. A higher concentration of SO₂ (0.5 microliter per liter) induced acetaldehyde and ethanol production within 2 hours of the start of fumigation of birch and pine seedlings. A number of other stresses, including water deficit, freezing, and ozone exposure induced production of acetaldehyde and ethanol. Production of these compounds was not due to hypoxia, as the O₂ partial pressure in the incubation vessels did not decline. Increasing the O₂ partial pressure to 300 millimeters Hg did not affect production of these compounds. Production of ethylene, acetaldehyde, and ethanol declined when more than 80% of the leaf area became necrotic, while ethane production was linearly related to the percentage of necrosis. A number of woody and herbaceous plant species produced acetaldehyde and ethanol in response to freezing stress, while others did not. Measurement of these four compounds simultaneously in the gas phase may be a valuable method for monitoring plant stress, particularly air pollution stress.     

Changes of lipid composition in non-cultured and cultured porcine embryos

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.     

Chlorophyll fluorescence assay for ozone injury in intact plants

A chlorophyll fluorescence induction (Kautsky effect) assay predicted ozone-induced injury in bean leaves (Phaseolus vulgaris) at least 20 hours before any visible sign of leaf necrosis. The extent of injury, which could be predicted during exposure to ozone, depended on concentration, exposure time, and leaf development stage. Much more injury occurred in light than in darkness and long exposures to lower ozone concentrations were more injurious than brief exposures to higher ones. The first detectable effect was on the photosynthetic water-splitting enzyme systems, followed by inhibition of electron transport between the photosystems. The fluorescence assay provides a simple, rapid, nondestructive method for observing effects of ozone on plants.     

Fatty acids of rice coleoptiles in air and anoxia

The metabolism of lipids, like that of other components, was adversely and strongly affected when rice (Oryza sativa L.) coleoptiles were grown anaerobically. In aerobic coleoptiles, the amounts of total fatty acid, phospholipid, and total lipid per coleoptile increased by 2.5- to 3-fold between days three and seven, whereas under anoxia, the increases were all less than 60%. The total amount of lipid at day seven in anoxia was less than 30% of that in air. In air, the total fatty acid content at day three was 25 nanomoles per coleoptile and this increased to over 71 nanomoles per coleoptile at day seven. All acids except 18:0 showed substantial increases. In anoxia, the corresponding values for total fatty acids were 24 nanomoles and 27 nanomoles. The small increases were confined to the saturated fatty acids; no significant increase occurred in unsaturated fatty acids. A minor fatty acid constituent (16:1) increased from 0.09 to 1.99 nanomoles per coleoptile between days three and seven in air. This component was never observed in any fatty acid preparation from anaerobic coleoptiles. The major phospholipids under all conditions were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid. A small amount of unidentified phosphoester, not present on thin layer chromatography plates from aerobic coleoptiles, was seen in extracts of anaerobic coleoptiles. The fatty acyl substituents of each of the phospholipids were analyzed at days three and seven in coleoptiles grown aerobically and in anoxia. Each phospholipid had its own distinctive fatty acid composition which remained fairly constant under all treatments; 16:0 and 18:2 were the most abundant fatty acids in every phospholipid class. In air, the percentages of total fatty acids that were in the phospholipids were 86% on day three and 87% on day seven. In anoxia, the values at the corresponding ages were 47 and 57%. Since no net synthesis of unsaturated fatty acids occurred in anaerobic conditions, the small increase in total unsaturated acids in the phospholipids between days three and seven must have occurred at the expense of fatty acids preexisting in the neutral lipid. No unusual pathways of biosynthesis or unusual precursors are required to explain the presence of unsaturated fatty acids in the rice coleoptile. The present study and results of experiments where coleoptiles were fed [¹⁴C]acetate (BB Vartapetian et al. 1978 Plant Sci Lett 13:321-328) clearly show that unsaturated fatty acid synthesis in rice coleoptiles requires O₂, as it does in other plants.     

Critical Assessment of Glyco- and Phospholipid Separation by Using Silica Chromatography

Phospholipid-derived fatty acids (PLFAs) are commonly used to characterize microbial communities in situ and the phylogenetic positions of newly isolated microorganisms. PLFAs are obtained through separation of phospholipids from glycolipids and neutral lipids using silica column chromatography. We evaluated the performance of this separation method for the first time using direct detection of intact polar lipids (IPLs) with high-performance liquid chromatography–mass spectrometry (HPLC-MS). We show that under either standard or modified conditions, the phospholipid fraction contains not only phospholipids but also other lipid classes such as glycolipids, betaine lipids, and sulfoquinovosyldiacylglycerols. Thus, commonly reported PLFA compositions likely are not derived purely from phospholipids and perhaps may not be representative of fatty acids present in living microbes.     

Control of plastidic glycolipid synthesis and its relation to chlorophyll formation

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv “Halo”) and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [¹⁴C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [¹⁴C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [¹⁴C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [¹⁴C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [¹⁴C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.     

Lipids and fatty acid composition of developing winged bean seeds

The moisture, lipids and fatty acid composition of developing winged bean (Psophocarpus tetragonolobus) seeds were studied. The moisture content decreased steadily as the seeds matured. The lipid content increased gradually and reached a maximum ca 6 weeks after flowering (WAF). In the early stage (2 WAF) of the developing seeds there were more polar lipids (glycolipids and phospholipids) than neutral lipids but, as the seeds developed, neutral lipids gradually accumulated while the polar lipids decreased until 6 WAF. Thereafter, both the neutral lipid and polar lipid levels remained little changed. The amounts of palmitic and stearic acids decreased, but the level of behenic acid increased as the seeds matured. On the other hand, the oleic acid content increased while that of linolenic acid decreased rapidly as the seeds matured. The concentration of linoleic acid, however, fluctuated during the development of the seeds.     

Changes in Lipids of Bean Seeds (Phaseolus vulgaris) and Corn Caryopses (Zea mays) Aged in Contrasting Environments

Seeds of French bean (Phaseolus vulgaris L. cv. The Prince)and caryopses of sweet corn (maize; Zea mays L.F1 hybrid Firstof All) were stored in environments of 79% relative humidityand 25 °C, 80% r.h. and 40 °C or 100% r.h. and 45 °C,giving ageing (loss of gcrminability and vigour) over periodsof months, weeks or days, respectively. The relationship betweenchanges in lipids and changes in germinability or vigour wasunaffected in general by the speed of ageing. In the corn caryopsesthere was no evidence of hydrolysis of phospholipids or peroxidationof fatty acids during ageing. In the bean seeds phosphatidicacid increased during the ageing period and phosphatyl cholinedeclined. The percentage of fatty acids as hnolenic acid initiallyfell during bean ageing, but in the slower ageing conditionsit rose again as germination reached zero. In bean, the presenceof phosphatidic acid could be a sensitive indictator of lossof vigour, but relative proportions of the different fatty acidswould be a misleading indicator of quality. Rapid artificialageing may be an adequate model, in some species, of ageingat moderate speeds and of ageing under some ambient conditions. French bean (Phaseolus vulgaris L. cv. The Prince), sweet corn (maize, Zea mays L., Fl hybrid First of All), seed ageing, phospholipids, fatty acids     

Phytotoxicity of Air Pollutants: Evidence for the Photodetoxification of SO(2) but Not O(3)

Pisum sativum L. cv Alsweet (garden pea) and Lycopersicon esculentum flacca Mill. (tomato) were used to evaluate the phytotoxicity of SO₂ and O₃ in the light and dark. Plants were grown in controlled environment chambers and exposed to SO₂ or O₃ in the light or dark at the same environmental conditions at which they were grown. The pea plants were treated with fusicoccin to ensure open stomata in the dark; the stomata of the tomato mutant remained open in the dark. Both species exhibited 64% to 80% less foliar necrosis following exposure to SO₂ (0.5 to 1.0 microliter per liter for 2 hours) in the light than in the dark. The decrease in SO₂ injury for light versus dark exposed plants was greater in fully expanded than expanding leaves. Both species exhibited 30% greater foliar necrosis following exposure to O₃ (0.2 microliter per liter for 2 hours) in the light than dark. The increase in O₃ injury in the light versus dark was similar for leaves at all stages of expansion. Leaf conductance to water vapor was 7% to 11% and 23% higher in the light than dark for fusicoccin-treated peas and tomato plants, respectively, indicating greater foliar uptake of both pollutants in the light than dark. Thus, the decreased SO₂ toxicity in the light was not associated with pollutant uptake, but rather the metabolism of SO₂. In contrast, the increased toxicity of O₃ in the light was at least in part associated with increased uptake or could not be separated from it.