New flux based dose-response relationships for ozone for European forest tree species.

To derive O3 dose-response relationships (DRR) for five European forest trees species and broadleaf deciduous and needleleaf tree plant functional types (PFTs), phytotoxic O3 doses (PODy) were related to biomass reductions. PODy was calculated using a stomatal flux model with a range of cut-off thresholds (y) indicative of varying detoxification capacities. Linear regression analysis showed that DRR for PFT and individual tree species differed in their robustness. A simplified parameterisation of the flux model was tested and showed that for most non-Mediterranean tree species, this simplified model led to similarly robust DRR as compared to a species- and climate region-specific parameterisation. Experimentally induced soil water stress was not found to substantially reduce PODy, mainly due to the short duration of soil water stress periods. This study validates the stomatal O3 flux concept and represents a step forward in predicting O3 damage to forests in a spatially and temporally varying climate.

Impacts of increasing ozone on Indian plants.

Increasing anthropogenic and biogenic emissions of precursor compounds have led to high tropospheric ozone concentrations in India particularly in Indo-Gangetic Plains, which is the most fertile and cultivated area of this rapidly developing country. Current ozone risk models, based on European and North American data, provide inaccurate estimations for crop losses in India. During the past decade, several ozone experiments have been conducted with the most important Indian crop species (e.g. wheat, rice, mustard, mung bean). Experimental work started in natural field conditions around Varanasi area in early 2000's, and the use of open top chambers and EDU (ethylene diurea) applications has now facilitated more advanced studies e.g. for intra-species sensitivity screening and mechanisms of tolerance. In this review, we identify and discuss the most important gaps of knowledge and future needs of action, e.g. more systematic nationwide monitoring for precursor and ozone formation over Indian region.

Biochemical and growth acclimation of birch to night temperatures: genotypic similarities and differences.

The responses of plants to environmental factors are connected to the time of day. In this study, silver birch (Betula pendula) was grown in growth chambers at five different night temperatures (6-22 °C), using gradual changes during the evening and morning hours. Despite the increased night respiration and unaffected daytime net photosynthesis (per square metre), the carbon uptake (biomass) of birch did not decrease, probably due to enhanced biochemical processes on warmer nights and the advantage of higher temperatures during the evening and morning hours. The plant stem height, internode length, stem dry weight (DW), stem mass fraction and specific leaf area increased with warmer night temperatures. Changes in growth and metabolite concentrations were partly nonlinear along the temperature gradient. Thus, the temperature effect depends on the temperature window considered. Genotypes had both common and genotype-specific biochemical responses to night temperatures. The common responses among genotypes were related to growth responses, whereas the unique responses may indicate genotype-specific differences in acclimation. The differences in genotypic growth and metabolite levels are valuable for assessing genotype qualities and understanding the connections between the metabolome and growth.

Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridiumperfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate.

Nucleotide-binding cystathionine beta-synthase (CBS) domains serve as regulatory units in numerous proteins distributed in all kingdoms of life. However, the underlying regulatory mechanisms remain to be established. Recently, we described a subfamily of CBS domain-containing pyrophosphatases (PPases) within family II PPases. Here, we express a novel CBS-PPase from Clostridium perfringens (CPE2055) and show that the enzyme is inhibited by AMP and activated by a novel effector, diadenosine 5',5-P1,P4-tetraphosphate (AP(4)A). The structures of the AMP and AP(4)A complexes of the regulatory region of C. perfringens PPase (cpCBS), comprising a pair of CBS domains interlinked by a DRTGG domain, were determined at 2.3 A resolution using X-ray crystallography. The structures obtained are the first structures of a DRTGG domain as part of a larger protein structure. The AMP complex contains two AMP molecules per cpCBS dimer, each bound to a single monomer, whereas in the activator-bound complex, one AP(4)A molecule bridges two monomers. In the nucleotide-bound structures, activator binding induces significant opening of the CBS domain interface, compared with the inhibitor complex. These results provide structural insight into the mechanism of CBS-PPase regulation by nucleotides.

Advances in understanding ozone impact on forest trees: messages from novel phytotron and free-air fumigation studies.

Recent evidence from novel phytotron and free-air ozone (O3) fumigation experiments in Europe and America on forest tree species is highlighted in relation to previous chamber studies. Differences in O3 sensitivity between pioneer and climax species are examined and viewed for trees growing at the harsh alpine timberline ecotone. As O3 apparently counteracts positive effects of elevated CO2 and mitigates productivity increases, response is governed by genotype, competitors, and ontogeny rather than species per se. Complexity in O3 responsiveness increased under the influence of pathogens and herbivores. The new evidence does not conflict in principle with previous findings that, however, pointed to a low ecological significance. This new knowledge on trees' O3 responsiveness beyond the juvenile stage in plantations and forests nevertheless implies limited predictability due to complexity in biotic and abiotic interactions. Unravelling underlying mechanisms is mandatory for assessing O3 risks as an important component of climate change scenarios.

Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states in Aspergillus flavus urate oxidase.

Urate oxidase (Uox) catalyses the oxidation of urate to allantoin and is used to reduce toxic urate accumulation during chemotherapy. X-ray structures of Uox with various inhibitors have been determined and yet the detailed catalytic mechanism remains unclear. Neutron crystallography can provide complementary information to that from X-ray studies and allows direct determination of the protonation states of the active-site residues and substrate analogues, provided that large, well-ordered deuterated crystals can be grown. Here, we describe a method and apparatus used to grow large crystals of Uox (Aspergillus flavus) with its substrate analogues 8-azaxanthine and 9-methyl urate, and with the natural substrate urate, in the presence and absence of cyanide. High-resolution X-ray (1.05-1.20 A) and neutron diffraction data (1.9-2.5 A) have been collected for the Uox complexes at the European Synchrotron Radiation Facility and the Institut Laue-Langevin, respectively. In addition, room temperature X-ray data were also collected in preparation for joint X-ray and neutron refinement. Preliminary results indicate no major structural differences between crystals grown in H(2)O and D(2)O even though the crystallization process is affected. Moreover, initial nuclear scattering density maps reveal the proton positions clearly, eventually providing important information towards unravelling the mechanism of catalysis.

Performance and secondary chemistry of two hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) clones in long-term elevated ozone exposure.

The effects of moderately elevated ozone (ca. 35 ppb) on the growth and secondary chemistry of the leaves of two soil-grown Finnish hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) clones with different ozone sensitivities were studied at an open-air exposure field in Kuopio, Finland. Stomatal conductance, photosynthetic rate, and chlorophyll fluorescence were measured during the third growing season. Foliar phenolic concentrations, ergosterol concentration of fine roots, and final dry mass of the trees were determined at the end of the third growing season. Elevated ozone increased the ectomycorrhizal status of the fine roots but had no effect on gas exchange or on the final biomass of either of the clones, indicating equal sensitivity to ozone and no effect of elevated ozone on the intraspecific competitive ability of the clones after three growing seasons. However, in agreement with the data from potted plants of the same clones after two growing seasons, significant differences between the clones were found in all parameters measured. A negative correlation between growth and high concentrations of foliar phenolics indicated that allocation to secondary chemistry also was costly in terms of growth under high resource availability.

Free-air exposure systems to scale up ozone research to mature trees.

Because seedlings and mature trees do not necessarily respond similarly to O(3) stress, it is critically important that exposure systems be developed that allow exposure of seedlings through to mature trees. Here we describe three different O(3) Free-Air Exposure Systems that have been used successfully for exposure at all growth stages. These systems of spatially uniform O(3) release have been shown to provide reliable O(3) exposure with minimal, if any, impact on the microclimate. This methodology offers a welcome alternative to chamber studies which had severe space constraints precluding stand or community-level studies and substantial chamber effects on the microclimate and, hence physiological tree performance.

Risk assessments for forest trees: the performance of the ozone flux versus the AOT concepts.

Published ozone exposure-response relationships from experimental studies with young trees performed at different sites across Europe were re-analysed in order to test the performance of ozone exposure indices based on AOTX (Accumulated exposure Over a Threshold of X nmol mol(-1)) and AF(st)Y (Accumulated Stomatal Flux above a threshold of Y nmol m(-2) s(-1)). AF(st)1.6 was superior, as compared to AOT40, for explaining biomass reductions, when ozone sensitive species with differing leaf morphology were included in the analysis, while this was not the case for less sensitive species. A re-analysis of data with young black cherry trees, subject to different irrigation regimes, indicated that leaf visible injuries were more strongly related to the estimated stomatal ozone uptake, as compared to the ozone concentration in the air. Experimental data with different clones of silver birch indicated that leaf thickness was also an important factor influencing the development of ozone induced leaf visible injury.

Northern environment predisposes birches to ozone damage.

Ozone sensitivity of silver birch ( BETULA PENDULA Roth) has been thoroughly investigated since early 1990's in Finland. In our long-term open-field experiments the annual percentage reduction in basal diameter and stem volume increment were the best non-destructive growth indicators for ozone impact when plotted against AOTX. Remarkable differences in defence strategies, stomatal conductance, and defence compounds (phenolics), clearly indicate that external exposure indices are ineffective for accurate risk assessment for birch. For flux-based approaches, site-specific values for G(max) and G(dark) are necessary, and determinants for detoxification capacity, ageing of leaves, and cumulative ozone impact would be needed for further model development. Increasing CO(2) seems to counteract negative ozone responses in birch, whereas exposure to spring time frost may seriously exacerbate ozone damage in northern conditions. Therefore, we need to proceed towards incorporating the most important climate change factors in any attempts for ozone risk assessment.

Comparison of different stomatal conductance algorithms for ozone flux modelling.

A multiplicative and a semi-mechanistic, BWB-type [Ball, J.T., Woodrow, I.E., Berry, J.A., 1987. A model predicting stomatal conductance and its contribution to the control of photosynthesis under different environmental conditions. In: Biggens, J. (Ed.), Progress in Photosynthesis Research, vol. IV. Martinus Nijhoff, Dordrecht, pp. 221-224.] algorithm for calculating stomatal conductance (g(s)) at the leaf level have been parameterised for two crop and two tree species to test their use in regional scale ozone deposition modelling. The algorithms were tested against measured, site-specific data for durum wheat, grapevine, beech and birch of different European provenances. A direct comparison of both algorithms showed a similar performance in predicting hourly means and daily time-courses of g(s), whereas the multiplicative algorithm outperformed the BWB-type algorithm in modelling seasonal time-courses due to the inclusion of a phenology function. The re-parameterisation of the algorithms for local conditions in order to validate ozone deposition modelling on a European scale reveals the higher input requirements of the BWB-type algorithm as compared to the multiplicative algorithm because of the need of the former to model net photosynthesis (A(n)).

Components of apoplastic ascorbate use in Betula pendula leaves exposed to CO2 and O3 enrichment.

Here, the aim was to estimate loads imposed on the apoplastic ascorbate (ASC) pool by enzymatic and nonenzymatic reactions in Betula pendula exposed to doubled CO2 and O3 concentrations in open-top chambers. Leaf apoplastic extracts were analysed for peroxidase and oxidase activities in vitro, using different substrates. Partial loads in vivo were deduced using measured kinetic constants and substituted-enzyme catalysis approaches. Ascorbate use in O3 scavenging was calculated using measured stomatal conductances and ASC concentrations. Under elevated O3, stomatal conductance and O3 uptake were higher. O3 fluxes to the plasmalemma were levelled off by higher apoplastic ASC concentrations. The effect of CO2 enrichment on ASC concentrations under elevated O3 was minor. Under ambient O3, the ascending hierarchy of ASC users was: peroxidases, O3 scavenging, oxidases, coniferyl alcohol re-reduction. Under elevated O3, ASC use in O3 scavenging was higher than by oxidases. The redox state of ASC was not depressed by O3; there was no leaf injury. The cell wall/plasmalemma/cytosol system in birch had sufficient capacity to maintain ASC redox status in the apoplast, without necessity to restrict O3 uptake by stomatal closure.

Ozone-induced H2 O2 accumulation in field-grown aspen and birch is linked to foliar ultrastructure and peroxisomal activity.

• Saplings of three aspen (Populus tremuloides) genotypes and seedlings of paper birch (Betula papyrifera) were exposed to elevated ozone (1.5× ambient) and 560 p.p.m. CO2 , singly and in combination, from 1998 at the Aspen-FACE (free-air CO2 enrichment) site (Rhinelander, USA). • The plants were studied for H2 O2 accumulation within the leaf mesophyll, number of peroxisomes, level of gene expression for catalase (Cat), and changes in ultrastructure. • In tolerant clones, ozone-elicited excess H2 O2 production was restricted to the apoplast, without any ultrastructural injuries. This was associated with ozone-induced proliferation of peroxisomes and increased transcript levels of Cat. In sensitive plants, ozone-induced H2 O2 accumulation continued from the cell wall to the plasma membrane, cytosol and chloroplasts, particularly in older leaves. However, chloroplastic precipitation was absent in the presence of elevated CO2 . In the most sensitive aspen clone, H2 O2 accumulation was found in conjunction with chloroplast injuries, low number of peroxisomes and low cell wall volume, whereas in birch a simultaneous increase in cell wall thickness indicated defence activation. • Our results indicate that oxidative stress manifests as H2 O2 effects on leaf ultrastructure in sensitive trees exposed to elevated ozone. However, CO2 enrichment appears to alleviate chloroplastic oxidative stress.

Responses of selected birch (Betula pendula Roth) clones to ozone change over time.

A long-term free air ozone fumigation experiment was conducted to study changes in physiological ozone responses during tree ontogeny and exposure time in ozone sensitive and tolerant clones of European white birch (Betula pendula Roth), originated from south and central Finland. The trees were grown in soil in natural microclimatic conditions under ambient ozone (control) and 1.4-1.7 x ambient (elevated) ozone from May 1996 to October 2001, and were measured for stem and foliage growth, net photosynthesis, stomatal conductance, stomatal density, visible injuries, foliar starch content and bud formation. After 6 years of exposure, the magnitude of ozone-induced growth reductions in the sensitive clone was 12-48% (significant difference), levels similar or greater than those reported earlier for 2- and 3-year-old saplings undergoing shorter exposures. In the tolerant clone, growth of these larger trees was reduced by 1-38% (significant difference in stem volume), although the saplings had previously been unaffected. In both clones, ozone stress led to significantly reduced leaf-level net photosynthesis but significantly increased stomatal conductance rates during the late summer, resulting in a lower carbon gain for bud formation and the onset of visible foliar injuries. Increasing ozone sensitivity with duration of exposure was explained by a change in growth form (relatively reduced foliage mass), a lower photosynthesis to stomatal conductance ratio during the late summer, and deleterious carry-over effects arising from the reduced number of over-wintering buds.

Genotypic variation in growth and physiological responses of Finnish hybrid aspen (Populus tremuloides x P. tremula) to elevated tropospheric ozone concentration.

Saplings of six Finnish hybrid aspen (Populus tremuloides Michx. x P. tremula L.) clones were exposed to 0, 50, 100 and 150 ppb ozone (O3) for 32 days in a chamber experiment to determine differences in O3 sensitivity among genotypes. Based on the chamber experiment, three clones with intermediate sensitivity to O3 were selected for a free-air O3 enrichment experiment in which plants were exposed for 2 months to either ambient air (control) or air containing 1.3 x the ambient O3 concentration. We measured stem height and radial growth, number of leaves, dry mass and relative growth rate of leaves, stem and roots, visible leaf injuries, net photosynthesis and stomatal conductance of the clones. There was high clonal variation in susceptibility to O3 in the chamber experiment, indicated by foliar injuries and differential reductions in growth and net photosynthesis. In the free-air O3 enrichment experiment, ozone caused a shift in resource allocation toward stem height growth, thereby altering the shoot to root balance. In both experiments, low O3 concentrations tended to stimulate growth of most clones, whereas 100 and 150 ppb O3 in the chamber experiment impaired growth of most clones. However, growth of the most O3-tolerant clone was not significantly affected by any O3 treatment.

Effects of long-term open-field ozone exposure on leaf phenolics of European silver birch (Betula pendula Roth).

The response of phenolic compounds as a result of long-term low open-field ozone exposure was studied in ozone-sensitive and ozone-tolerant clones of European silver birch (Betula pendula Roth). The saplings were exposed to 1.5-1.6 times the ambient (elevated) ozone and ambient air (as control) over three growing seasons from May 1996 until August 1998. Quantification by modified Folin-Ciocalteau assay showed a 16.2% increase in total phenolics in elevated ozone plants as compared to that in controls and a corresponding 9.9% increase of 10 phenolic compounds quantified by HPLC. Five nonflavonoids and five flavonoids showed 8.4% and 11.4% increases, respectively. The phenolic results indicated slightly higher ozone sensitivity of clone 5 as compared to clone 2. The most ozone-responsive phenolic compounds in clone 2 and clone 5 were (+)-catechin (CT), chlorogenic acid (CGA), 5-p-coumaroylquinic acid (5CQA), 3-p-coumaroylquinic acid (3CQA), myricetin galactopyranoside (MG), quercetin-3-O-glucuronopyranoside (QGR), and quercetin-3-O-arabinofuranoside (QA). Increased phenolic content in ozone-exposed plants was related to impaired growth and accelerated leaf senescence, indicated by enhanced autumn leaf yellowing and lower chlorophyll and Mg content. The change in carbon allocation towards defensive phenolics at the expense of growth was greater in the ozone-sensitive clone as compared to tolerant clone.

Impacts of elevated CO2 and/or O3 on leaf ultrastructure of aspen (Populus tremuloides) and birch (Betula papyrifera) in the aspen FACE experiment.

Impacts of elevated atmospheric O3 and/or CO2 on three clones of aspen (Populus tremuloides Michx.) and birch (Betula papyrifera Marsh.) were studied to determine, whether or not elevated CO2 ameliorates O3-induced damage to leaf cells. The plants were exposed for 3 years at the Aspen FACE exposure site in Wisconsin (USA) prior to sampling for ultrastructural investigations on 19 June 1999. In the aspen clones, elevated CO2 increased chloroplast cover index, leaf and spongy mesophyll layer thickness, intercellular air space volume in mesophyll, amount of starch in chloroplasts and cytoplasmic lipids but decreased the number of plastoglobuli in chloroplasts. In contrast, elevated O3 decreased chloroplast cover index, starch content, and the proportion of cytoplasm and intercellular space in mesophyll, and increased the proportion of vacuoles, the amount of condensed vacuolar tannins and the number of plastoglobuli. Ozone also caused structural thylakoid injuries (dilation, distortion) and stromal condensation in chloroplasts, which was ameliorated by elevated CO2 by 5-66% in aspen clones and by 2-10% in birch. Birch ultrastructure was less affected by elevated CO2 or O3 stress compared to aspen. In the most O3-sensitive aspen clone, thinner leaves and cell walls, lower proportion of cell wall volume, and higher volume for vacuoles was found compared to more-tolerant clones.

Effects of elevated CO2 and O3 on aspen clones varying in O3 sensitivity: can CO2 ameliorate the harmful effects of O3?

To determine whether elevated CO2 reduces or exacerbates the detrimental effects of O3 on aspen (Populus tremuloides Michx.). aspen clones 216 and 271 (O3 tolerant), and 259 (O3 sensitive) were exposed to ambient levels of CO2 and O3 or elevated levels of CO2, O3, or CO2 + O3 in the FACTS II (Aspen FACE) experiment, and physiological and molecular responses were measured and compared. Clone 259. the most O3-sensitive clone, showed the greatest amount of visible foliar symptoms as well as significant decreases in chlorophyll, carotenoid, starch, and ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) concentrations and transcription levels for the Rubisco small subunit. Generally, the constitutive (basic) transcript levels for phenylalanine ammonialyase (PAL) and chalcone synthase (CHS) and the average antioxidant activities were lower for the ozone sensitive clone 259 as compared to the more tolerant 216 and 271 clones. A significant decrease in chlorophyll a, b and total (a + b) concentrations in CO2, O3, and CO2 + O3 plants was observed for all clones. Carotenoid concentrations were also significantly lower in all clones; however. CHS transcript levels were not significantly affected, suggesting a possible degradation of carotenoid pigments in O3-stressed plants. Antioxidant activities and PAL and 1-aminocyclopropane-l-carboxylic acid (ACC)-oxidase transcript levels showed a general increase in all O3 treated clones, while remaining low in CO2 and CO2 + O3 plants (although not all differences were significant). Our results suggest that the ascorbate-glutathione and phenylpropanoid pathways were activated under ozone stress and suppressed during exposure to elevated CO2. Although CO2 + O2 treatment resulted in a slight reduction of O3-induced leaf injury, it did not appear to ameliorate all of the harmful affects of O3 and, in fact. may have contributed to an increase in chloroplast damage in all three aspen clones.

Leukocytes as a coronary risk factor in a dyslipidemic male population.

The role of an elevated serum leukocyte count (WBC) as a coronary risk factor was investigated using a nested case-control design in dyslipidemic middle-aged men (n = 420) participating in the Helsinki Heart Study, a coronary primary prevention trial. Baseline WBC was significantly higher, 6.93 (2.11) x 10(9)/L in subjects with cardiac events, than in controls, 6.26 (1.88) x 10(9)/L; p less than 0.002. This association was time-dependent, however, since the difference was not significant for events occurring during the second half of the 5-year study. Using nonsmokers in the lowest WBC tertile as the reference sample, the relative risks in the highest WBC tertile were 1.86 (95% confidence intervals [CI] 0.81 to 4.28) for nonsmokers and 3.07 (95% CI 2.23 to 8.19) for smokers. Logistic regression analysis including smoking in the model disclosed an independent contribution of elevated WBC to coronary heart disease. We conclude that elevated leukocyte count was a coronary risk factor even in this dyslipidemic population.

Alternative approaches to the measurement of consumption and price of alcoholic beverages, Canada, 1957-1983.

Alternative accepted methods of aggregating prices and quantities of beer, spirits and wine could yield different conclusions for intertemporal comparisons. In this study Fisher's index is used to measure changes in per capita consumption and price of alcoholic beverages in Canada, 1957-83. The results are typically very close to those obtained from measures based on pure alcohol content.