Manipulation of glutathione metabolism in transgenic plants.

There is clear potential for the genetic manipulation of key enzymes involved in stress metabolism in transgenic plants. However, the data emerging so far from such experiments are equivocal. The detailed analysis of stress responses in progeny of primary transgenics, coupled with comparisons with control transgenic plants that do not contain the GR transgene, allows us to take into account the possible variation in response to stress associated with regeneration of plants from tissue culture. The picture that is now beginning to emerge with respect to the role of GR in stress protection is that, although there are clearly benefits to be had from overexpression of the enzymes, there is no direct correlation between enzyme levels and stress tolerance. It may be that overexpression of the cytosolic isoform (gor2) will prove to be of greater benefit. Furthermore, the types of stresses to which transgenic plants have been exposed in order to assess the consequences of oxidative stress tolerance cannot reproduce those that will experienced in field conditions. Only when plants with higher GR levels and increased glutathione synthesis capacity are grown in field trials will it be possible to make a full assessment of the benefits of engineering plants with altered glutathione metabolism.

Chlorophyll fluorescence decay profiles of O3 -exposed spruce needles as measured by time-correlated single photon counting.

Chlorophyll fluorescence decay profiles have been measured in the wavelength range 680-720 nm for needles from Norway spruce [Picea abies (L.) Karst] trees which have been exposed to ozone. All profiles required three exponential components of lifetime 100-150 ps, 400-600 ps and 3.5-5.0 ns to fit the experimental data. Compared to control samples, the ozone-treated needles exhibited a greater amount of the longest-lived chlorophyll fluorescence and a redistribution in intensity for both the other components from 720 + to 690 nm. These observations are interpreted in terms of disruption of energy transfer and break-up of light-harvesting complexes on exposure to ozone. The potential for use of the technique in monitoring forest decline is discussed.

Relationship between foliar injury and changes in antioxidant levels in red and Norway spruce exposed to acidic mists.

Elevated levels of total glutathione and enhanced activities of glutathione reductase and ascorbate peroxidase were found in needles of red spruce which had been exposed to acidic mists. Reduced levels of ascorbate were also detected. Such observations suggest that oxidative stress is involved in processes which resist foliar injury caused by acidic misting. Different ionic compositions of the acidic mists applied had pronounced effects on the levels of these antioxidants and the activities of related enzymes. Sulphate was found to be most effective in causing increases in glutathione, while additions of ammonium and/or nitrate mitigated the effects of sulphuric acid. Moreover, it is the composition of ions in the applied mists, rather than the levels of acidity, that determines the extent of the overall response of red spruce. By contrast, although acidic mists caused similar increases in glutathione content of Norway spruce needles, no statistically significant changes of ascorbate or related enzymic activities were found.

Effects of summer ozone on membrane lipid composition during subsequent frost hardening in Norway spruce [Picea abies (L.) Karst].

Norway spruce trees [Picea abies (L.) Karst] were exposed to ozone during the summers of three consecutive years and the fatty acid composition of the chloroplast membrane lipid, monogalactosyl diglyceride (MCDG) was analysed over the last 14 months. Significant variations in the degree of unsaturation and the percentage of linolenic acid (18:3) were not found, either on a seasonal basis or in relation to ozone treatment. The proportion of octadecatetraenoic acid (18:4) and other Δ5 fatty acids increased during autumn in trees grown in filtered air, but this seasonal effect was less pronounced in ozone-treated trees. The ratio of two forms of linoleate, Δ5,9 18:2 and Δ9,12 18:2 also increased significantly during the frost hardening period but not in trees previously exposed to ozone. This implies that two separate biosynthetic pathways exist, one for the Δ12 and one for the Δ12 desaturated series, which operate at different relative rates, depending on environmental conditions. It is suggested that ozone may interfere with the biosynthesis of 18:4 during frost hardening by inhibiting the Δ5 desaturation of oleate.

Enhanced ethylene emissions from red and norway spruce exposed to acidic mists.

Acidic cloudwater is believed to cause needle injury and to decrease winter hardiness in conifers. During simulations of these adverse conditions, rates of ethylene emissions from and levels of 1-aminocyclopropane-1-carboxylic acid (ACC) in both red and Norway spruce needles increased as a result of treatment with acidic mists but amounts of 1-malonyl(amino)cyclopropane-1-carboxylic acid remained unchanged. However, release of significant quantities of ethylene by another mechanism independent of ACC was also detected from brown needles. Application of exogenous plant growth regulators such as auxin, kinetin, abscisic acid and gibberellic acid (each 0.1 millimolar) had no obvious effects on the rates of basal or stress ethylene production from Norway spruce needles. The kinetics of ethylene formation by acidic mist-stressed needles suggest that there is no active inhibitive mechanism in spruce to prevent stress ethylene being released once ACC has been formed.

Induction of ascorbate peroxidase and glutathione reductase activities by interactions of mixtures of air pollutants.

The response of ascorbate peroxidase and glutathione reductase activities in peas (Pisum sativum var. Waverex) was investigated after three weeks of exposure to mixed fumigations with SO2, NO2 and O3 (0.050 parts per million each) and increasing concentrations of O3 (0-0.150 parts per million). The results show that plants respond similarly to a high concentration (0.150 parts per million) of a single air pollutant (ozone) and to mixtures of air pollutants (SO2, NO2 and O3) when individual concentrations are low (0.050 parts per million each). In both cases, levels of ascorbate peroxidase and glutathione reductase activities were approximately twice those to be found in plants grown in charcoal-filtered air (p less than 0.01).

FOLIAR ENTRY AND INCORPORATION OF ATMOSPHERIC NITROGEN DIOXIDE INTO BARLEY PLANTS OF DIFFERENT NITROGEN STATUS.

The uptake of atmospheric NO2 through leaf surfaces and the influence on uptake of combined nitrogen by the roots were investigated in hydroponically grown barley plants exposed to 0.3 µI 1-1 nitrogen dioxide (NO2 ). The response to NO2 was dependent on the nitrogen status of the plant at the time of exposure. Only with low nitrate supplies to the roots did exposure of leaves to atmospheric NO2 cause significant increases in the nitrogen (N) content of barley. Levels of both nitrate and reduced forms of N increased in plants in response to the pollutant and were directly due to uptake of NO2 by leaves, since the root environment was completely sealed from atmospheric sources of NO2 . Moreover, these increases were not due to the stimulation of nitrate uptake by the roots from the nutrient solution, although levels of activity of both nitrate and nitrite reductases were affected by exposure to NO2 . Atmosphere NO2 did, however, affect the ability of a plant to respond to a change in the amount of nitrate supplied to the roots, and the use of both 18 N-labelled NO2 and nitrate indicated that the cycling of nitrogen within the plant was also influenced by exposure to atmospheric NO2 .

CELLULAR READJUSTMENT OF BARLEY SEEDLINGS TO SIMULATED ACID RAIN.

No changes in buffer capacity at any range of pH were detected in either the shoots or roots of 9 d old hydroponically-grown barley seedlings which had been grown in dilute acid (pH 3 or 4) for the last 4 days of growth. Similar lack of change in buffer capacity was observed in barley which had been intermittently sprayed with acidic mists (pH 3 or 4) as compared to those sprayed with pH 56 media for the last 2 or 4 d of growth. Acidic treatments, especially growth in acidic media or 2 d of occasional spraying, caused significant increases in the levels of plastidic sulphate, as measured by high performance ion chromatography but no changes in the plastidic concentrations of other anions occurred. High field 31 P-NMR spectroscopy of barley shoot tissue showed that the vacuolar pH levels (as revealed by the pH-dependent orthophosphate signals) of those tissues which had been sprayed with acidic solutions (pH 3 or 4) for 2 d were significantly lower than in those similarly sprayed with pH 5.6 solutions. This may mean that ATP-dependent H+ -pumps in the tonoplast are involved in cellular readjustments to the imposed acidity. No growth reductions or visible injury were detected over these short term treatments but these changes of vacuolar pH and plastidic sulphate content may indicate that homeostatic adjustments have an energetic cost which may ultimately account for reductions in plant growth attributed elsewhere to acidic precipitation.

Oxides of nitrogen and their impact upon vegetation.

Annual and daily levels of the two major oxides of nitrogen (NOX), nitric oxide (NO) and nitrogen dioxide (NO2) are still rising in the atmosphere of the urban environment and in close proximity to automobile and airport traffic. Although oxides of nitrogen give rise to other phytotoxic pollutants such as ozone they are also in their own right damaging to plant health. This injury is not normally visible but is reflected in poorer growth and loss of productivity in terms of value and amenity. Few if any reviews have considered the effects of atmospheric concentrations of NOX as separate phytotoxic agents especially at the metabolic level. This paper is an attempt to remedy this deficiency and to promote an understanding of the problem.

Chloroplast development in low light-grown barley seedlings.

Segments of 7-d low light-grown barley laminae cut at 0.5 cm intervals up from the intercalary meristem were examined ultrastructurally and biochemically. The different regions upwards showed the succession of plastid development in light-grown tissues of eoplasts, amyloplasts, amoeboid, immature and mature plastids as described by Whatley (1977). Semi-crystalline bodies were detected in all of them. The eoplast-amyloplast regions are characterised by a greater proportion of mitochondria and high levels of ATP and 3-phosphoglyceric acid, together with low levels of inorganic phosphate conducive to the activation of ADP glucose pyrophosphorylase. The amoeboid and immature plastid regions have higher levels of inhibitory phosphate and starch breakdown may be responsible for the release of metabolites and energy for development. Segments containing amoeboid and immature plastids also have reduced levels of ATP (and 3-phosphoglyceric acid) as photosynthetic components are synthesised. Using ultrastructural assessments of areas of thylakoids, first ß-carotene and violaxanthin, followed by chlorophyll a and lutein and, lastly, chlorophyll b are concentrated in the developing lamellar systems of the immature and mature chloroplasts. The formation of additional membraneous material which spreads these pigment systems over a greater thylakoid area within the plastids is the final stage of plastid morphogenesis in low light-grown seedlings.

Appearance of photochemical function in prothylakoids during plastid development.

1. A method to separate the vesicles of prothylakoids from prolamellar body preparations obtained from etiolated and rapidly greening Avena laminae (0.25--4 h illumination ) is described. The prothylakoid preparations were found to be free from contaminating prolamellar bodies but enriched prolamellar body preparations (enriched prolamellar body preparations) still contained some adhering prothylakoid material. 2. Only existing beta-carotene appears to be transferred from the prolamellar bodies to the prothylakoids during early development and this ceases when freshly synthesized beta-carotene becomes available. 3. Prolamellar body structures proper show no positive association of existing or developing photochemical activities; these are only to be found in the developing prothylakoids. 4. Using methylviologen-linked electron transport-dependent oxygen consumption, Photosystem I activities may be detected with added diaminodurene within 15 min of illumination and within 30 min and 1 h with added tetramethylphenylenediamine and dichlorophenolindophenol, respectively. 5. During the 2nd, and 3rd. h of greening, proton-pumping capability and later ATP formation increased in prothylakoids in the presence of diaminodurene. 6. The first indications of Photosystem II activity using diphenylcarbazide as electron donor are shown at a similar time (2 h) with prothylakoids. The last photochemical activity to appear is the capacity to split water (3 h) and consequently the diphenylcarbazide activity diminished to zero before 8 h of illumination have passed. 7. The lack of effect of uncouplers such as NH4+ prior to 2 h suggests that in spite of some proton-pumping ability there is the possibility of proton-leaky areas existing within prothylakoids. This lack of a persistent proton gradient before 2 h of illumination may explain the different starting times of phenazine methosulfate- and diaminodurene-dependent photophosphorylation (0.25 and 2 h, respectively).

Conjoined mitochondria and plastids in the barley mutant 'albostrians'.

The intercalary meristem and surrounding tissues of the gene induced plastome mutant 'albostrians' of Hordeum vulgare L. were examined in the electron microscope for ultrastructural evidence of membrane continuities between plastids and mitochondria. In well developed tissues the ribosome-deficient plastids were usually in close proximity or appressed to mitochondria of normal appearance. In some sections through the meristemmatic region however the relationship between the two organelles was observed to be of a fused nature. These conjoinings are thought to be similar to those reported in normal living cells using cinephotomicrography but never before observed by transmission electron microscopy.

Control of mitochondrial activities by phytochrome during greening.

Mitochondria isolated from 7-day old darkgrown Avena sativa L. (var. Arnold) laminae given 5 min illumination of red light, followed by varying lengths of darkness up to 3 h, showed at least a twofold increase in the rates of both NADH-dependent oxygen consumption and respiratory chain phosphorylation over those of mitochondria isolated from unilluminated tissue. Similar organelles, isolated from tissue given either far-red or red followed by far-red pretreatment, exhibited rates of both functions of between 25% and 75% below those of the mitochondria from unilluminated tissue. The induction-reversion criteria for phytochrome control of respiration and oxidative phosphorylation were satisfied under all experimental conditions during the greening process.Treatment with continuous far-red light, acting presumably through the 'high irradiance' reaction of phytochrome, served to disengage phytochrome activity from photosynthesis. The stimulation of oxidative phosphorylation still occurred under these conditions, slightly slower but much more prolonged in the absence of ATP from photophosphorylation.

Examination of ribosome-like particles in isolated prolamellar bodies.

Potential methods for the preparation of fractions enriched in prolamellar bodies (PLBs) were examined in detail. Sucrose density gradient centrifugation methods gave fractions consisting almost exclusively of PLBs whilst those methods employing differential centrifugation were quite successful but contained greater quantities of lamellar membranes. Greater difficulty was experienced in obtaining detached PLBs which retained their "ribosome-like" lattice particles. No modification to density gradient procedures was found which retained these particles but the omission of ethylene diaminetetraacetic acid (EDTA) from all media including that of lysis gave a hint that this was possible with differential centrifugal methods. This was developed to produce a successful method for the preparation of PLBs which retain the "ribosome-like" particles of the lattice. Such fractions from Avena sativa L. and Hordeum vulgare L. were treated with ribonuclease which completely removed these particles from the lattice structures implying that they may be "ribosomal" in nature. EDTA apparently has a critical effect on PLB structure at concentration lower than those that effect the chloroplast coupling factor particles but it is not known if it is a direct effort of PLB membranes, on the lattice particles or both.

Distribution of chloroplast coupling factor (CF1) particles on plastid membranes during development.

Samples of internal membrane systems separated from lysates of intact plastids from dark grown Avena sativa L. (vars, Cooba and Mostyn) and Hordeum vulgare L. (vars, Himalaya and Deba Abed) given different periods of illumination before isolation were assayed for trypsin-activated Ca(2+)-dependent ATPase activities and also examined in the electron microscope after treatment in the manner described by Oleszko and Moudinanakis (1974) which assists the visualization of the chloroplast coupling factor (CF1) particles. Concentrations of membrane-attached CF1 particles were not observed on the membrane surfaces of the prolamellar bodies (PLBs) proper but only on the attached extruded lamellar membranes. Increasing lengths of illumination followed by plastid isolation and subsequent membrane separation had the effect of progressively increasing the mean distance between these individual lamellar-attached CF1 particles. Measurements of trypsin-activated Ca(2+)-dependent ATPase activities during similar developmental regimes indicated that functions associated with CE1 particles are relative constant and largely independent of the period of illumination if the values were expressed on a per plastid basis indicating that assembly of CF1 particles may take place in either etioplasts, etiochloroplasts or mature chloroplasts.

Uptake of mevalonate and acetate during plastid development.

1. The envelopes of etioplasts and 1-2h etiochloroplasts are permeable to mevalonate, but plastids from etiolated tissue illuminated for longer than 4h show progressive impermeability towards mevalonate. 2. Acetate permeates the envelopes of 1-4h etiochloroplasts but does not significantly cross the envelopes of etioplasts or 8-24h etiochloroplasts. 3. A translocator system exists within the plastid envelopes for mevalonate which relies on malate as a counter-exchange anion.

Movement of labelled metabolites from mitochondria to plastids during development.

The exchange of metabolites from isolated mitochondria of Avena sativa L. to isolated etioplasts or 1-24 h etio-chloroplasts, from the same laminae, has been investigated. The results confirm the synchronised changes in the permeabilities of the inner membranes of each during plastid development. They also indicated the possibility of directed transport of certain metabolites from mitochondria to plastids especially during the early stages of chloroplast maturation. Over 80% of labelled succinate and oxaloacetate previously associated with mitochondria was found to be associated with 1 and 2 h etio-chloroplasts after in vitro incubation, with their respective mitochondria, for only 1 min at 0°C. Other metabolites showed variable but lower rates of transfer and that of aminolevulinic acid was at a much reduced level throughout development.