Ozone-induced changes in the chloroplast structure of conifer needles, and their use in ozone diagnostics.

Ozone induces characteristic symptoms in the chloroplasts of the needles of several coniferous species. Chloroplasts are (1) reduced in size and (2) the stroma is electron dense. Moreover, (3) these chloroplast alterations are more pronounced in the outer mesophyll cell layers and in the upper side of the needle compared to the inner layers and lower side. The syndrome, including the three symptoms (1)-(3), is found in the green needles of Scots pine and Norway spruce not only in the experimental fumigations, but also in mature trees in the field, and has potential for diagnosis of ozone stress. For sound ozone diagnostics all three symptoms must be present in the samples studied. The symptoms in relation to needle anatomy and physiology is discussed, and recommendations for sampling and analysis are given.

A cumulative ozone uptake-response relationship for the growth of Norway spruce saplings.

Norway spruce saplings [Picea abies (L.) Karst.] were exposed during four growing seasons to different ozone treatments in open-top chambers: charcoal filtered air (CF), non-filtered air (NF) and non-filtered air with extra ozone (NF+, 1.4xambient concentrations). The CF and NF+ ozone treatments were combined with phosphorous deficiency and drought stress treatments. The total biomass of the trees was harvested at different intervals during the experimental period. The ozone uptake to current-year needles of the Norway spruce saplings was estimated using a multiplicative stomatal conductance simulation model. There was a highly significant correlation between the reduction of total biomass and the estimated cumulative ozone uptake, which did not vary when different thresholds were applied for the rate of ozone uptake. The reduction of the total biomass was estimated to 1% per 10 mmol m(-2) cumulated ozone uptake, on a projected needle area basis.

The effects of tropospheric ozone and elevated carbon dioxide on potato (Solanum tuberosum L. cv. Bintje) growth and yield.

An open-top chamber experiment with field grown potato (Solanum tuberosum L. cv. Bintje) was performed in the south-west of Sweden in 1998. The experiment was aimed to investigate individual and interactive impacts of elevated ozone (O(3)) and carbon dioxide (CO(2)) on potato growth and yield. Treatments were ambient and elevated CO(2) with or without the addition of 20 nmol mol(-1) O(3). In addition, plants were grown in ambient air plots. Plants grown in elevated CO(2) and O(3) had a reduced tuber yield, compared to control, by 2% and 8%, respectively. These effects were, however, not statistically significant. Elevated CO(2) caused a significantly larger number of tubers of lower average size, while elevated O(3) induced significantly lower tuber dry matter content. The number of tuber initials (tubers <15 mm) found at harvest was significantly lower in elevated O(3). Visible O(3) leaf injury appeared earlier and the number of yellow leaves 89 days after emergence was significantly larger in elevated O(3). In elevated CO(2), haulm dry weight was significantly (15%) lower at harvest and the haulm/tuber ratio was significantly reduced. There were significant interactive effects of O(3) and CO(2) on the final number of leaves and on stem height. Plants in elevated CO(2) reached final stem height earlier than in the other treatments. In summary, it was concluded: (a) that anticipated future O(3) levels are likely to have a negative impact on potato growth, although tuber yield effects may be hard to demonstrate experimentally in this plant due to large variation; (b) that whether any possible beneficial effect of elevated CO(2) on the magnitude of tuber yield will be expressed, depend on the climatic conditions prevailing; and (c) that potato growth responses to CO(2) and O(3) are not simply additive.

Impact of ozone on the growth of birch (Betula pendula) saplings.

Saplings of one half-sib family of birch, Betula pendula, were exposed to three levels of ozone in open-top chambers (OTCs) during two growing seasons 1997-1998. The ozone treatments were non-filtered air (NF, accumulated daylight AOT40 over the two growing seasons of 3.0 l l-1 h), non-filtered air with extra ozone (NF+, accumulated daylight AOT40 of 27.3 l l-1 h) and non-filtered air with additional extra ozone (NF++, accumulated daylight AOT40 of 120 l l-1 h). The birch saplings, including the roots, were harvested after the first and second growing seasons. After the first growing season, the NF++ treatment reduced the total wood biomass by 22%, relative to the NF treatment. There was no further reduction of the total wood biomass in the NF++ treatment after the second growing season. The root biomass was reduced by 30% after the first growing season. The shoot/root ratio, as well as the proportional biomass of leaves, were increased by ozone during both years. The ozone impact on the relative growth rate was estimated to -2% per 10 l l-1 h daylight AOT40 per growing season.

Impact of ozone and reduced water supply on the biomass accumulation of Norway spruce saplings.

Norway spruce saplings [Picea abies (L.) Karst.] were exposed during four growing seasons to two different ozone treatments in open-top chambers: charcoal filtered air (CF), and non-filtered air with extra ozone (NF+, 1.4xambient concentrations). Within each ozone treatment the saplings were either kept well watered or treated with a 7-8 week period with reduced water supply each growing season. The total biomass of the trees was measured in April and September during each of the last three growing seasons. NF+ significantly reduced the total biomass accumulation of Norway spruce saplings during the fourth growing season. No interaction between ozone and reduced water supply could be detected. The magnitude of the ozone impact after 4 years of exposure was an 8% reduction of the total plant biomass and a 1.5% reduction of the RGR. The reduced water supply reduced the total biomass 29% and the RGR 12%.

An ozone flux-response relationship for wheat.

Six open-top chamber experiments with field-grown wheat Triticum aestivum L. (five with spring wheat and one with winter wheat) were combined to test which of the two ozone exposure indices, AOT40 and CFO(3), that provided the most consistent relationship between relative yield loss and ozone exposure. AOT40 is the accumulated exposure over a threshold ozone concentration of 40 nl l(-1), while CFO(3) is the cumulative flux of ozone (uptake) to the flag leaves. The ozone uptake of the flag leaves was estimated using a stomatal conductance model, sensitive to phenology, light, vapour pressure deficit (VPD) and temperature in combination with measurements of the boundary layer conductance in the open-top chambers. Both indices were calculated for the grain-filling period, defined as the time from anthesis until 2 weeks before harvest. The duration of the grain-filling period was shown to be closely related to the rate of accumulation of thermal time above a base temperature of 0 degrees C. The CFO(3) index provided a much more consistent pattern in terms of ozone effects compared to the AOT40 index. This was especially the case for spring wheat, for which a linear regression between relative yield and CFO(3) using all five data sets is presented. According to the stomatal conductance model, VPD limited daytime stomatal conductance in warm and dry years, while temperature was the most important limiting factor during daytime in cool and humid years. The effect of light was mainly to delimit the time period of the day during which substantial uptake of ozone took place. It is concluded that, compared to the AOT40 index, the more mechanistically relevant flux-based index CFO(3) will estimate larger yield loss in the relatively humid parts of western and northern Europe, while smaller yield loss will be estimated for the dry summer climates in south and central Europe. The use of an ozone flux threshold, similar to the cut-off concentration 40 nl l(-1) in AOT40, did not improve the performance of the CFO(3) index.

Tropospheric ozone decreases biomass production in radish plants (Raphanus sativus) grown in rural south-west Sweden.

Potted plants of radish (Raphanus sativus L., cv. Cherry Belle) were grown in the ambient air for 5 weeks, with or without the application of a soil drench of the anti-ozonant ethylenediurea (EDU). The 24-h mean ozone concentration during the experimental period was 31 nl l(-1). Towards the end of the experiment two ozone episodes, with maximum concentrations around 70 and 115 nl l(-1), occurred. No visible injury that could be attributed to ozone was observed on any of the plants. Shoot and hypocotyl biomass were significantly lower in the non-EDU-treated plants than in the EDU-treated plants. The non-EDU-treated plants had a 32% lower hypocotyl biomass and a 22% lower shoot biomass. The shoot:hypocotyl ratio of the non-EDU-treated plants was higher than that of the EDU-treated plants, although the difference was not statistically significant. EDU treatment increased the leaf area and decreased the chlorophyll content of the leaves. These differences were, however, not statistically significant. It is suggested that the ambient rural ozone climate in southern Sweden has the potential to decrease biomass production in Cherry Belle radishes in the absence of visible injury.

Clover as an indicator plant for phytotoxic ozone concentrations: visible injury in relation to species, leaf age and exposure dynamics.

Visible ozone injury on leaves of three clover species was investigated in relation to species, leaf age and exposure dynamics. It was shown that ozone episodes in south-west Sweden cause visible injury to subterranean clover (Trifolium subterraneum L.), white clover (Trifolium repens L.) and red clover (Trifolium pratense L.). Trifolium subterranean was most sensitive to ozone, whilst T. pratense was least sensitive. Application of the anti-ozonant, ethylenediurea (EDU), reduced the extent of visible ozone injury, but did not give complete protection with the concentrations used. Similarly, filtration of the air reduced the extent of visible injury in T. subterraneum enclosed in open-top chambers. EDU-treated plants of T. subterranean accumulated more biomass than the non-EDU treated plants after a period with rather high ozone concentrations, while the opposite occurred for T. pratense. Experiments with T. subterraneum in open-top chambers also showed that older leaves were more sensitive to ozone than younger leaves and that a shorter period with higher ozone concentrations produced more ozone injury than a longer period with lower ozone concentrations, although the two periods had the same number of ppb-hours.

Exposure of oats, Avena sativa L., to filtered and unfiltered air in open-top chambers: effects on grain yield and quality.

Field grown oats, Avena sativa L. cv Vital, were exposed to filtered and unfiltered air from anthesis until harvest in open-top chambers at a site in south-west Sweden. Ambient plots were used to study the influence of the chamber itself. With the exception of the number of grains per panicle, which was significantly higher in the charcoal-filtered treatment, no significant filtration effects were obtained for any of the plant growth parameters studied), i.e. grain yield, number of panicles per square metre, 1000-grain weight, straw yield and harvest index.) The chamber had a significant negative effect on grain yield, 1000-grain weight and straw yield. None of the yield quality parameters that were measured, such as crude protein content, crude fibre content, fat content, volume weight of the grain and water content of the grain at harvest, were significantly influenced by either air filtration, or the presence of the chamber. The chlorophyll content of the flag leaves was higher in the charcoal-filtered treatment than in the non-filtered and ambient air treatments towards the end of the experiment, indicating that filtering of the air delayed senescence. The decline of the shoot area after the onset of plant senescence proceeded faster in both chamber treatments. The faster development in the chamber was explained by the faster accumulation of thermal time in the chamber.

Long-term exposure of Norway spruce, Picea abies (L.) Karst., to ozone in opentop chambers: III. Effects on the light response of net photosynthesis in shoots of different ages.

Rates of photosynthesis at different photosynthetic photon flux densities (PPFD; 0-1530 µmol m-2 s-1 ) were measured on three shoot age classes of one clone of Norway spruce, Picea abies (L.) Karst. exposed to three different concentrations of ozone for three seasons (1985-7) in open-top chambers. The treatments were charcoal-filtered air (CF), non-filtered air (NF) and non-filtered air + ozone (NF +). The rate of net photosynthesis was determined using an infrared gas analyzer (IRGA) connected to a small cuvette, equipped with an artificial light source. In 1- and 2-yr-old shoots the apparent quantum yield decreased with increasing shoot age and ozone concentration, while no effect was found in the current-year shoots. The decrease could probably partly be attributed to a lower efficiency of light capture due to a lower content of chlorophyll. No effect was observed on the Kok-effect or on the convexity of the light response curve in any of the treatments or age classes. The significantly higher light compensation point found in current year shoots from the NF+ treatment compared to that of shoots from the CF and NF treatments was suggested to be attributed to an increase in dark respiration. The effect of ozone was larger on photosynthesis at light saturation than on the apparent quantum yield. It is therefore suggested that ozone has a greater influence on the photosynthetic efficiency at high light conditions than at low light conditions.

Long-term exposure of Norway spruce, Picea abies (L.) Karst., to ozone in open-top chambers: IV. Effects on the stomatal and non-stomatal limitation of photosynthesis and on the carboxylation efficiency.

Rates of photosynthesis at different concentrations of CO2 (0-1850 µmol mol-1 ) were measured using one clone of Norway spruce, Picea abies (L.) Karst. exposed to three different concentrations of ozone for four seasons (1985-8) in open-top chambers. The treatments were non-filtered air+ozone (NF+), non-filtered air (NF) and charcoal-filtered air (CF). The measurements were made on shoots of four different age classes. No significant effects were found on photosynthesis or on leaf conductance to CO2 in current year shoots. In 1- to 3-yr-old shoots, leaf conductance to CO2 and rates of net photosynthesis at both 330 µmol mol-1 CO2 (Pn 330), and saturating concentrations of CO2 (Pn max), decreased with increasing shoot age and ozone concentration, although this was only significant for Pn 330 in 3-yr-old shoots. In current year shoots the intercellular concentration of CO2 was significantly higher in NF+-treated shoots compared with CF- and NF-treated shoots, while in the 2- and 3-yr-old shoots it was significantly higher in NF- and NF +-treated shoots compared with CF-treated shoots. The carboxylation efficiency significantly decreased in 2- and 3-yr-old shoots from the NF and NF+ treatments compared with shoots from the CF treatment. The gas phase limitation of photosynthesis decreased with increasing shoot age and ozone concentration.

Stromal low temperature compartment derived from the inner membrane of the chloroplast envelope.

Leaf discs of four dicotyledonous species, when incubated at temperatures of 4 to 18 degrees C (optimum at 12 degrees C) for 30 or 60 minutes, responded by accumulations of membranes in the chloroplast stroma in the space between the inner membrane of the envelope and the thylakoids. The accumulated membranes, here referred to as the low temperature compartment, were frequently continuous with the envelope membrane and exhibited kinetics of formation consistent with a derivation from the envelope. Results were similar for expanding leaves of garden pea (Pisum sativum), soybean (Glycine max), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum). We suggest that the stromal low temperature compartment may be analogous to the compartment induced to form between the transitional endoplasmic reticulum and the Golgi apparatus at low temperatures. The findings provide evidence for the possibility of a vesicular transfer of membrane constituents between the inner membrane of the chloroplast envelope and the thylakoids of mature chloroplasts in expanding leaves.

Yield and grain quality of spring wheat (Triticum aestivum L., cv. Drabant) exposed to different concentrations of ozone in open-top chambers.

Spring wheat (Triticum aestivum L., cv. Drabant) was exposed to different concentrations of ozone in open-top chambers for two growing seasons, 1987 and 1988, at a site located in south-west Sweden. The chambers were placed in a field of commercially grown spring wheat. The treatments were charcoal-filtered air (CF), non-filtered air (NF) and non-filtered air plus extra ozone (NF(+)). In 1988, one additional ozone concentration (NF(++)) was used. Grain yield was affected by the ozone concentration of the air. Air filtration resulted in an increase in grain yield of about 7% in both years, compared to NF. The addition of ozone (NF(+), NF(++)) reduced grain yield and increased the content of crude protein of the grain in both years. Filtration of the air had no significant effect on the content of crude protein, compared to NF. The results showed a strong positive chamber effect on grain yield in the cold and wet summer of 1987. In 1988, there was no net chamber effect on grain yield. The relative differences between the CF, NF and NF(+) treatments with respect to grain yield were of the same magnitude in the two years, despite the very different weather conditions.

Long-term exposure of Norway spruce, Picea abies (L.) Karst., to ozone in open top chambers: II. Effects on the ultrastructure of needles.

One clone of spruce, Picea abies (L.) Karst., was exposed to different concentrations of ozone in open-top chambers for three seasons. The treatments were filtered air (CF), non-filtered air (NF) and non-filtered air + ozone (NF+). Changes in ultrastructure occurring in the 1985 flush of needles were followed for three years. Ultrastructural changes due to ozone first occurred in the chloroplasts. A decrease in length of the chloroplasts, an increase of the density of the stroma, an accumulation of ribosome-like granules and a decrease in the size of starch grains, were observed. The second organelle to be affected was the microbody and the last the mitochondria. At this later stage the cytoplasm had also disintegrated. Structural changes always appeared first in the outer cell layer facing the sky. The changes then gradually advanced in the tissue towards the lower part of the needle. In the NF+ treatment changes were observed at the first sampling event in November, 1 month after the end of the first exposure period. At this time the needles were about 6 months old. In the NF treatment the same changes appeared when the needles were about 14 months old. No changes were observed in the CF treatment. Changes of the same type as those observed in the mesophyll tissue were also seen in stomatal and endodermal cells. In albuminous cells a general breakdown of the organelles was observed.

Long-term exposure of Norway spruce, Picea abies (L.) Karst., to ozone in open-top chambers: I. Effects on the capacity of net photosynthesis, dark respiration and leaf conductance of shoots of different ages.

Rates of net photosynthesis were measured on Norway spruce, Picea abies (L.) Karst. exposed to three different concentrations of ozone for three seasons (1985-7) in open-top chambers. The treatments were non-filtered air + ozone (NF +), non-filtered air (NF) and charcoal-filtered air (CF). Ozone was added daily between 11.00-18.00 local time. The study was performed during September and October, 1987. The rate of net photosynthesis was determined using an infrared gas analyser (IRGA) connected to a small cuvette, which was equipped with an artificial light source for unilateral illumination. The measurements were made on shoots of three different age classes: current year, 1 year old and 2 years old. Measurements took place at saturating light intensity (1150 µmol m1 s1 ), at a temperature of 16.6 ±0.7°C, and at a relative humidity of 67.5 ± 5.3%. In current-year shoots the rate of net photosynthesis expressed on a needle-area basis was lowest in the CF treatment and highest in the NF treatment. In 1- and 2-year-old shoots, a decreased rate of the net photosynthetic assimilation rate, expressed on a needle area basis, was observed in trees from the NF and NF+ treatment compared with that of trees from the CF treatment. The effect increased with increasing ozone concentration and shoot age. A significant increase in the rate of respiration, expressed on a dry-weight basis, was observed in current year shoots from trees exposed to NF+. A significant decrease of the leaf conductance to carbon dioxide was obtained in 2-year-old shoots exposed to NF and NF+. A decrease in the content of chlorophyll was observed in 1- and 2-year-old needles from trees exposed to NF+.

Localization of DNA in Mature and Young Wheat Chloroplasts Using the Fluorescent Probe 4'-6-Diamidino-2-phenylindole.

The spatial organization of chloroplast DNA in developing and dividing wheat chloroplasts was studied in the light microscope using the fluorescent probe 4'-6-diamidino-2-phenylindole, which binds specifically to DNA.The DNA of wheat chloroplasts was localized at the periphery of the plastid, frequently in a discrete band. No relocalization of the DNA was observed during plastid replication. This peripheral location of the DNA was shown to differ from the central random location of DNA in tobacco and spinach chloroplasts.

Comparative analyses of the effect of triacontanol on photosynthesis, photorespiration and growth of tomato (C3-plant) and maize (C 4-plant).

Tomato (C3-plants) and maize (C4-plants) were grown in a nutrient solution to which triacontanol was added twice a week. After about 4 weeks the triacontanol treatment caused a significant increase in the dry weight of the tomato plants. Leaf area and dry weight measurements of tomato leaves at different stages of development showed that the largest increase in growth was obtained when triacontanol treatment was initiated before bud formation. In maize, no effect of the triacontanol treatment on dry wieght was observed. Photosynthesis was inhibited by 27% in young leaves from triacontanol-treated tomato plants and 39% in the controls, when the oxygen concentration was raised from 2% to 21%. In maize no change in photosynthesis could be observed, neither after altered oxygen concentration nor after triacontanol treatment. The difference in the response of C3- and C4-plants to triacontanol indicates that it regulates processes related to photosynthesis.

Influence of Cell Age on Chlorophyll Formation in Light-grown and Etiolated Wheat Seedlings.

A method is described for relating the age of a cereal leaf cell to its distance from the leaf base. The rates of chlorophyll synthesis per plastid in the first leaf of light-grown and of greening etiolated seedlings of wheat (Triticum aestivum, var. Maris Dove) increase with cell age. Normally developing plastids of light-grown wheat take over 24 hours to reach the chlorophyll a/b ratio characteristic of mature wheat chloroplasts (4.5), but mature etioplasts need only 8 hours light to achieve this a/b ratio. Plastid greening potential depends only on cell age, whereas the chlorophyll a/b ratio is influenced both by cell age and by light.

Chloroplast Division and DNA Synthesis in Light-grown Wheat Leaves.

Light-grown 7-day-old wheat seedlings (Triticum aestivum, var. Maris Dove) showed an increase of 200% in plastids per cell between 1.7 and 4.5 centimeters from the leaf base. This increase was the result of divisions of young chloroplasts at various stages of development, and was well separated in distance, and therefore in time from the region of cell division in the basal meristem. [(3)H]Thymidine was incorporated into plastid DNA throughout the zone of plastid division, but not above it.

Appearance and development of P700 oxidation and photosystem I activity in etio-chloroplasts prepared from greening barley leaves.

The development of photosystem I activity of plastids isolated from greening barley (Hordeum distichum, L.) leaves was studied. The electron transport activity in photosystem I was measured as anthraquinone-mediated oxygen uptake and as light induced absorbance changes of the reaction centre molecule P700. P700 oxidation was observed after one hour of greening though an electron transport leading to oxygen uptake was observed after 30 minutes. Phenazine methosulphate had no effect on the oxidation of P700 until after four hours of greening. The ratio chlorophyll/P700 decreased from about 2300/l at one hour to 640/l at sixteen hours of greening. The light intensity dependence of the electron transport of photosystem I showed that the photosynthetic units gradually increased in size as the greening proceeded. The increase of the rate of the oxygen uptake, calculated on plastid basis, decreased after eight hours while the P700 content, calculated on plastid basis, increased continuously between three and sixteen hours. Chromatographic separations and fluorimetric analyses of the chlorophyll pigments showed that the reaction centre molecule could not be protochlorophyllide or chlorophyllide.