Ozone uptake at night is more damaging to plants than equivalent day-time flux.

MAIN CONCLUSION: Plants exposed to equivalent ozone fluxes administered during day-time versus night-time exhibited greater losses in biomass at night and this finding is attributed to night-time depletion of cell wall-localised ascorbate. The present study employed Lactuca sativa and its closest wild relative, L. serriola, to explore the relative sensitivity of plants to ozone-induced oxidative stress during day-time versus night-time. By controlling atmospheric ozone concentration and measuring stomatal conductance, equivalent ozone uptake into leaves was engineered during day and night, and consequences on productivity and net CO2 assimilation rate were determined. Biomass losses attributable to ozone were significantly greater when an equivalent dose of ozone was taken-up by foliage at night compared to the day. Linkages between ozone impacts and ascorbic acid (AA) content, redox status and cellular compartmentation were probed in both species. Leaf AA pools were depleted by exposure of plants to darkness, and then AA levels in the apoplast and symplast were monitored on subsequent transfer of plants to the light. Apoplast AA appeared to be more affected by light-dark transition than the symplast pool. Moreover, equivalent ozone fluxes administered to leaves with contrasting AA levels resulted in contrasting effects on the light-saturated rate of CO2 assimilation (Asat) in both species. Once apoplast AA content recovered to pre-treatment levels, the same ozone flux resulted in no impacts on Asat. The results of the present investigation reveal that plants are significantly more sensitive to equivalent ozone fluxes taken-up at night compared with those during the day and were consistent with diel shifts in apoplast AA content and/or redox status. Furthermore, findings suggest that some thought should be given to weighing regional models of ozone impacts for extraordinary night-time ozone impacts.

A method for analysing small samples of floral pollen for free and protein-bound amino acids.

Pollen provides floral visitors with essential nutrients including proteins, lipids, vitamins and minerals. As an important nutrient resource for pollinators, including honeybees and bumblebees, pollen quality is of growing interest in assessing available nutrition to foraging bees. To date, quantifying the protein-bound amino acids in pollen has been difficult and methods rely on large amounts of pollen, typically more than 1 g. More usual is to estimate a crude protein value based on the nitrogen content of pollen, however, such methods provide no information on the distribution of essential and non-essential amino acids constituting the proteins.Here, we describe a method of microwave-assisted acid hydrolysis using low amounts of pollen that allows exploration of amino acid composition, quantified using ultra high performance liquid chromatography (UHPLC), and a back calculation to estimate the crude protein content of pollen.Reliable analysis of protein-bound and free amino acids as well as an estimation of crude protein concentration was obtained from pollen samples as low as 1 mg. Greater variation in both protein-bound and free amino acids was found in pollen sample sizes <1 mg. Due to the variability in recovery of amino acids in smaller sample sizes, we suggest a correction factor to apply to specific sample sizes of pollen in order to estimate total crude protein content.The method described in this paper will allow researchers to explore the composition of amino acids in pollen and will aid research assessing the available nutrition to pollinating animals. This method will be particularly useful in assaying the pollen of wild plants, from which it is difficult to obtain large sample weights.

A CAM- and starch-deficient mutant of the facultative CAM species Mesembryanthemum crystallinum reconciles sink demands by repartitioning carbon during acclimation to salinity.

In the halophytic species Mesembryanthemum crystallinum, the induction of crassulacean acid metabolism (CAM) by salinity requires a substantial investment of resources in storage carbohydrates to provide substrate for nocturnal CO(2) uptake. Acclimation to salinity also requires the synthesis and accumulation of cyclitols as compatible solutes, maintenance of root respiration, and nitrate assimilation. This study assessed the hierarchy and coordination of sinks for carbohydrate in leaves and roots during acclimation to salinity in M. crystallinum. By comparing wild type and a CAM-/starch-deficient mutant of this species, it was sought to determine if other metabolic sinks could compensate for a curtailment in CAM and enable acclimation to salinity. Under salinity, CAM deficiency reduced 24 h photosynthetic carbon gain by >50%. Cyclitols were accumulated to comparable levels in leaves and roots of both the wild type and mutant, but represented only 5% of 24 h carbon balance. Dark respiration of leaves and roots was a stronger sink for carbohydrate in the mutant compared with the wild type and implied higher maintenance costs for the metabolic processes underpinning acclimation to salinity when CAM was curtailed. CAM required the nocturnal mobilization of >70% of primary carbohydrate in the wild type and >85% of carbohydrate in the mutant. The substantial allocation of carbohydrate to CAM limited the export of sugars to roots, and the root:shoot ratio declined under salinity. The data suggest a key role for the vacuole in regulating the supply and demand for carbohydrate over the day/night cycle in the starch-/CAM-deficient mutant.

Enhanced nitrogen deposition exacerbates the negative effect of increasing background ozone in Dactylis glomerata, but not Ranunculus acris.

The combined impacts of simulated increased nitrogen (N) deposition (75 kg Nha(-1)yr (-1)) and increasing background ozone (O(3)) were studied using two mesotrophic grassland species (Dactylis glomerata and Ranunculus acris) in solardomes, by means of eight O(3) treatments ranging from 15.5 ppb to 92.7 ppb (24h average mean). A-C(i) curves were constructed for each species to gauge effects on photosynthetic efficiency and capacity, and effects on biomass partitioning were determined after 14 weeks. Increasing the background concentration of O(3) reduced the healthy above ground and root biomass of both species, and increased senesced biomass. N fertilisation increased biomass production in D. glomerata, and a significantly greater than additive effect of O(3) and N on root biomass was evident. In contrast, R. acris biomass was not affected by high N. The study shows the combined effects of these pollutants have differential implications for carbon allocation patterns in common grassland species.

A new stable isotope approach identifies the fate of ozone in plant-soil systems.

* We show that the stable isotope (18)O can be used to trace ozone into different components of the plant-soil system at environmentally relevant concentrations. * We exposed plants and soils to (18)O-labelled ozone and used isotopic enrichment in plant dry matter, leaf water and leaf apoplast, as well as in soil dry matter and soil water, to identify sites of ozone-derived (18)O accumulation. * It was shown that isotopic accumulation rates in plants can be used to infer the location of primary ozone-reaction sites, and that those in bare soils are dependent on water content. However, the isotopic accumulation rates measured in leaf tissue were much lower than the modelled stomatal flux of ozone. * Our new approach has considerable potential to elucidate the fate and reactions of ozone within both plants and soils, at scales ranging from plant communities to cellular defence mechanisms.

A new method for using 18O to trace ozone deposition.

Isotopically labelled ozone ((18)O(3)) is an ideal tool to study the deposition of O(3) to plants and soil, but no studies have made use of it due to the technical difficulties in producing isotopically enriched ozone. For (18)O(3) to be used in fumigation experiments, it has to be purified and stored safely prior to fumigations, to ensure that the label is present predominantly in the form of O(3), and to make efficient use of isotopically highly enriched oxygen. We present a simple apparatus that allows for the safe generation, purification, storage, and release of (18)O(3). Following the purification and release of O(3), about half (by volume) of the (18)O is present in the form of O(3). This means that for a given release of (18)O(3) into the fumigation system, a roughly identical volume of (18)O(2) is released. However, the small volume of this concurrent (18)O(2) release (100 nmol mol(-1) in our experiment) results in only a minor shift of the much larger atmospheric oxygen pool, with no detectable consequence for the isotopic enrichment of either soil or plant materials. We demonstrate here the feasibility of using (18)O as an isotopic tracer in O(3) fumigations by exposing dry soil to 100 nmol mol(-1) (18)O(3) for periods ranging from 1 to 11 h. The (18)O tracer accumulation in soil samples is measured using gas chromatography/isotope ratio mass spectrometry (GC/IRMS), and the results show a linear increase in (18)O/(16)O isotope ratio over time, with significant differences detectable after 1 h of exposure. The apparatus is adapted for use with fumigation chambers sustaining flow rates of 1 m(3) min(-1) for up to 12 h, but simple modifications now allow larger quantities of O(3) to be stored and continuously released (e.g. for use with open-top chambers or FACE facilities).

The use of ozone in the remediation of polycyclic aromatic hydrocarbon contaminated soil.

The potential of using ozone for the removal of phenanthrene from several different soils, both alone and in combination with biodegradation using a microbial inoculant (Pseudomonas alcaligenes PA-10), was examined. The greater the water content of the soil the less effective the ozone treatment, with air-dried soils showing the greatest removal of phenanthrene; while soils with higher levels of clay also reduced the effectiveness of the ozone treatments. However, at least a 50% reduction in phenanthrene levels was achieved in air-dried soil after an ozone treatment of 6 h at 20 ppm, with up to 85% removal of phenanthrene achieved in sandy soils. The biodegradation results indicate that P. alcaligenes PA-10 may be useful as an inoculant for the removal of PAHs from contaminated soils. Under the conditions used in our experiments, however, pre-ozonation did not enhance subsequent biodegradation of phenanthrene in the soils. Similar levels of phenanthrene removal occurred in both non-ozonated and ozonated Cruden Bay soil inoculated with P. alcaligenes PA-10. However, the biodegradation of phenanthrene in ozonated Boyndie soil was much slower. This may be due to the release of toxic products in this soil during ozonation.

The function of ascorbate oxidase in tobacco.

The function of the apoplastic enzyme ascorbate oxidase (AO) was investigated in tobacco (Nicotiana tabacum). The abundance of AO mRNA was up-regulated by light. Cytosolic ascorbate peroxidase (APX1) transcripts were also highest in the light. In contrast, L-galactono-gamma-lactone dehydrogenase, stromal APX, and thylakoid APX transcripts remained constant over the day/night cycle. Salicylic acid inhibited growth, increased expression of the pathogenesis-related protein (PR) 1a, and decreased AO transcript abundance. In contrast, the application of auxin enhanced growth and increased AO and PR 1a gene expression. Therefore, AO transcript abundance varied in a manner similar to hormone-mediated changes in plant growth. To study the effects of modified AO expression on growth, transformed tobacco plants expressing AO in the sense and antisense orientations were generated. The resultant large changes in apoplastic AO activity in the transformed tobacco plants had little effect on whole leaf ascorbate (AA) content, but they had dramatic effects on apoplastic AA levels. Enhanced AO activity oxidized the apoplastic AA pool, whereas decreased AO activity increased the amount of AA compared with dehydroascorbate. A relationship was observed between AO activity and plant height and biomass. Native AO transcript levels were no longer subject to light/dark regulation in AO sense and antisense plants. Taken together, these data show that there is an interaction between hormone, redox, and light signals at the level of the apoplast via modulation of ion of AA content.

Impacts of ozone on Plantago major: apoplastic and symplastic antioxidant status.

The aim of this work was to examine the correspondence between apoplastic/symplastic antioxidant status and previously reported plant age-related shifts in the ozone (O3 ) resistance of Plantago major L. Seed-grown plants were fumigated in duplicate controlled environment chambers with charcoal/Purafil?-filtered air (CFA) or CFA plus 70 nmol mol-1 O3 for 7 h d-1 over a 42 d period. Measurements of stomatal conductance and antioxidants were made after 14, 28 and 42 d fumigation, on leaves at an equivalent stage of development (youngest fully expanded leaf, measured c. 9 d after emergence). Ozone exposure resulted in a similar decline in stomatal conductance across plant ages, indicating that increases in O3 resistance with plant age were mediated through changes in the tolerance of leaf tissue rather than enhanced pollutant exclusion. Leaf apoplastic washing fluid was found to contain 'unspecific' peroxidase, ascorbate peroxidase, superoxide dismutase and ascorbate, but not glutathione and the enzymes required to facilitate the regeneration of ascorbate from its oxidized forms. A weak induction in the activity of certain symplastic antioxidants was found after 14 d O3 fumigation, despite a lack of visible symptoms of injury, but shifts in symplastic antioxidant enzyme activity were not consistent with previously observed increases in resistance to O3 with plant age. By contrast, changes in 'unspecific' peroxidase activity and in the small pool of ascorbate in the leaf apoplast were found to accompany age-related shifts in O3 resistance. It is concluded that constituents of the leaf apoplast may constitute a potentially important front line defence against O3 .

Relationships between ozone resistance and climate in European populations of Plantago major.

The relative ozone resistance of 20 European and two American populations of Plant ago major was examined, and relationships with climatic factors at the source of the plant material were explored using data provided by participants in the ICP-Crops initiative (International Co-operative Programme to Investigate the Effects of Air Pollutants and Other Stresses on Agricultural and Semi-Natural Vegetation). Plants grown from seed were exposed to either charcoal/Purafil® filtered air (CF < 5 nmol mol-1 O3 ) or CF + ozone (70 nmol mol-1 O3 7 h d-1 ) over a 2-wk period in controlled environment chambers, and effects on mean plant relative growth rate (R) and allometric root/shoot growth (K) determined. Ozone resistance (R%) was calculated from (R03 /RCF ) × 100. Populations exhibited contrasting sensitivities to ozone, without the development of typical visible symptoms of injury. A positive relationship was found between relative ozone resistance and descriptors of the ozone-climate at the site of seed collection for the year of, and the 2 yr before, seed collection. The best predictors of inherent ozone resistance were shown to be cumulative ozone exposure indices calculated according to current United Nations Economic Commission for Europe (UN-ECE) critical level guidelines for the pollutant (i.e. the accumulated hourly average ozone exposure over a threshold level of 40 nmol mol-1 (AOT40) or 30 nmol mol-1 (AOT30) calculated during daylight hours for the consecutive 3-month period of the year experiencing the highest ozone concentrations). No relationships were found between ozone resistance and climatic factors (temperature, precipitation, sunshine hours, humidity) or the concentrations of other air pollutants (SO2 , NO2 , NO). These findings support the view that current ambient levels of ozone in many regions of Europe are high enough to promote evolution of resistance to the pollutant in native plant populations. The significance of these findings to the debate over the establishment of separate critical levels for the protection of natural and semi-natural vegetation is discussed.

Potential effects of rising tropospheric concentrations of CO2 and O3 on green-algal lichens.

Pormelia sulcata Taylor was used as a model to examine the effects of elevated CO2 and/or O3 on green algal lichens. Thalli were exposed for 30 d in duplicate controlled-environment chambers to two atmospheric concentrations of CO2 ('ambient' [350µmol mol-1 ] and 'elevated' [700µmol mol-1 ] 24 h d-1 ) and two O3 regimes ('non-polluted' air [CF, < 5 nmol mol-1 ] and 'polluted' air [15 nmol mol-1 overnight rising to a midday maximum of 75 nmol mol-1 ]), in a factorial design. Elevated CO2 , or elevated O3 depressed the light saturated rate of CO2 , assimilation Asat ) measured at ambient CO2 , by 30% and 18%, respectively. However, despite this effect ultrastructure) studies revealed increased lipid storage in cells of the photobiont in response to CO2 -enrichment. Simultaneous exposure to elevated O3 reduced CO2 -induced lipid accumulation and reduced Asat in an additive manner. Gold-antibody labelling revealed that the decline in photosynthetic capacity induced by elevated CO2 and/or O3 was accompanied by a parallel decrease in the concentration of Rubiscoa in the algal pyrenoid (r= 0.93). Interestingly, differences in the amount of Rubisco protein were not correlated with changes in pyrenoid volume. Measurements of in vivo chlorophyll-fluorescence induction kinetics showed that the decline in Asat induced by elevated CO2 , and/or O2 , was not associated with significant changes in the photochemical efficiency of photosystem (PS) II. Although the experimental conditions inevitably imposed some stress on the thalli, revealed as a significant decline in the efficiency of PS II photochemistry, and enhanced starch accumulation in the photobiont over the fornication period, the study shows that the green-algal lichen symbiosis might be influenced by future changes in atmospheric composition. Photosynthetic capacity, measured at ambient CO2 , was found to be reduced after a controlled 30 d exposure to elevated CO2 , and/or O3 and this effect was associated with a parallel decline in the amount of Rubisco in the pyrenoid of algal chloroplasts.