Next-generation sequencing as a screening tool for foodborne pathogens in fresh produce.

Next generation sequencing (NGS) approaches are increasingly applied to tracing microbial contaminants entering the food chain due to NGS' untargeted nature and ability to investigate non-culturable (and/or difficult to culture) organisms while yielding genomic information about the microbiota. So far, a plethora of microbes has been shown to be associated with fresh produce, but few studies have utilised NGS to identify contamination with human pathogens. This study aims to establish the limit of detection (LoD) for Salmonella and phage MS2 (a Norovirus surrogate) contamination of fresh produce employing NGS approaches on the Illumina MiSeq: 16S amplicon-sequencing, and RNA-seq, using ScriptSeq (Illumina) and NEBNext (New England BioLabs) kits. ScriptSeq proved the most sensitive approach; delivering an LoD of 104 CFU reaction-1 (Colony Forming Units) for Salmonella and 105 PFU reaction-1 (Plaque Forming Units) for phage MS2. Use of the NEBNext kit resulted in detection of Salmonella at 106 CFU reaction-1 and phage MS2 at 107 PFU reaction-1. 16S amplicon-sequencing yielded a similar LoD of 105 CFU reaction-1 for Salmonella but could not detect MS2. The tested NGS methodologies, in combination with bioinformatics approaches applied, proved less sensitive than conventional microbial detection approaches.

Inequality in physical activity, sedentary behaviour, sleep duration and risk of obesity in children: a 12-country study.

OBJECTIVE: Studies examining associations between movement behaviours (i.e. physical activity, sedentary behaviour and sleep duration) and obesity focus on average values of these movement behaviours, despite important within-country and between-country variability. A better understanding of movement behaviour inequalities is important for developing public health policies and behaviour-change interventions. The objective of this ecologic analysis at the country level was to determine if inequality in movement behaviours is a better correlate of obesity than average movement behaviour volume in children from all inhabited continents of the world. METHODS: This multinational, cross-sectional study included 6,128 children 9-11 years of age. Moderate-to-vigorous physical activity (MVPA), total sedentary time (SED) and sleep period time were monitored over 7 consecutive days using waist-worn accelerometry. Screen time was self-reported. Inequality in movement behaviours was determined using Gini coefficients (ranging from 0 [complete equality] to 1 [complete inequality]). RESULTS: The largest inequality in movement behaviours was observed for screen time (Gini of 0.32; medium inequality), followed by MVPA (Gini of 0.21; low inequality), SED (Gini of 0.07; low inequality) and sleep period time (Gini of 0.05; low inequality). Average MVPA (h d-1) was a better correlate of obesity than MVPA inequality (r = -0.77 vs. r = 0.00, p = 0.03). Average SED (h d-1) was also a better correlate of obesity than SED inequality (r = 0.52 vs. r = -0.32, p = 0.05). Differences in associations for screen time and sleep period time were not statistically significant. MVPA in girls was found to be disproportionally lower in countries with more MVPA inequality. CONCLUSIONS: Findings from this study show that average MVPA and SED should continue to be used in population health studies of children as they are better correlates of obesity than inequality in these behaviours. Moreover, the findings suggest that MVPA inequality could be greatly reduced through increases in girls' MVPA alone.

Thresholds of physical activity associated with obesity by level of sedentary behaviour in children.

BACKGROUND: It is unknown whether moderate-to-vigorous physical activity (MVPA) thresholds for obesity should be adapted depending on level of sedentary behaviour in children. OBJECTIVE: The objective of the study is to determine the MVPA thresholds that best discriminate between obese and non-obese children, by level of screen time and total sedentary time in 12 countries. METHODS: This multinational, cross-sectional study included 6522 children 9-11 years of age. MVPA and sedentary time were assessed using waist-worn accelerometry, while screen time was self-reported. Obesity was defined according to the World Health Organization reference data. RESULTS: Receiver operating characteristic curve analyses showed that the best thresholds of MVPA to predict obesity ranged from 53.8 to 73.9 min d-1 in boys and from 41.7 to 58.7 min d-1 in girls, depending on the level of screen time. The MVPA cut-offs to predict obesity ranged from 37.9 to 75.9 min d-1 in boys and from 32.5 to 62.7 min d-1 in girls, depending on the level of sedentary behaviour. The areas under the curve ranged from 0.57 to 0.73 ('fail' to 'fair' accuracy), and most sensitivity and specificity values were below 85%, similar to MVPA alone. Country-specific analyses provided similar findings. CONCLUSIONS: The addition of sedentary behaviour levels to MVPA did not result in a better predictive ability to classify children as obese/non-obese compared with MVPA alone.

Mid-upper arm circumference as a screening tool for identifying children with obesity: a 12-country study.

BACKGROUND: No studies have examined if mid-upper arm circumference (MUAC) can be an alternative screening tool for obesity in an international sample of children differing widely in levels of human development. OBJECTIVE: Our aim is to determine whether MUAC could be used to identify obesity in children from 12 countries in five major geographic regions of the world. METHODS: This observational, multinational cross-sectional study included 7337 children aged 9-11 years. Anthropometric measurements were objectively assessed, and obesity was defined according to the World Health Organization reference data. RESULTS: In the total sample, MUAC was strongly correlated with adiposity indicators in both boys and girls (r > 0.86, p < 0.001). The accuracy level of MUAC for identifying obesity was high in both sexes and across study sites (overall area under the curve of 0.97, sensitivity of 95% and specificity of 90%). The MUAC cut-off value to identify obesity was ~25 cm for both boys and girls. In country-specific analyses, the cut-off value to identify obesity ranged from 23.2 cm (boys in South Africa) to 26.2 cm (girls in the UK). CONCLUSIONS: Results from this 12-country study suggest that MUAC is a simple and accurate measurement that may be used to identify obesity in children aged 9-11 years. MUAC may be a promising screening tool for obesity in resource-limited settings.

The chlorine isotope composition of the moon and implications for an anhydrous mantle.

Arguably, the most striking geochemical distinction between Earth and the Moon has been the virtual lack of water (hydrogen) in the latter. This conclusion was recently challenged on the basis of geochemical data from lunar materials that suggest that the Moon's water content might be far higher than previously believed. We measured the chlorine isotope composition of Apollo basalts and glasses and found that the range of isotopic values [from -1 to +24 per mil (per thousand) versus standard mean ocean chloride] is 25 times the range for Earth. The huge isotopic spread is explained by volatilization of metal halides during basalt eruption--a process that could only occur if the Moon had hydrogen concentrations lower than those of Earth by a factor of approximately 10(4) to 10(5), implying that the lunar interior is essentially anhydrous.

Chlorine isotope homogeneity of the mantle, crust and carbonaceous chondrites.

Chlorine in the Earth is highly depleted relative to carbonaceous chondrites and solar abundances. Knowledge of the Cl concentrations and distribution on Earth is essential for understanding the origin of these depletions. Large differences in the stable chlorine isotope ratios of meteoritic, mantle and crustal materials have been used as evidence for distinct reservoirs in the solar nebula and to calculate the relative proportions of Cl in the mantle and crust. Here we report that large isotopic differences do not exist, and that carbonaceous chondrites, mantle and crust all have the same 37Cl/35Cl ratios. We have further analysed crustal sediments from the early Archaean era to the Recent epoch and find no systematic isotopic variations with age, demonstrating that the mantle and crust have always had the same delta37Cl value. The similarity of mantle, crust and carbonaceous chondrites establishes that there were no nebular reservoirs with distinct isotopic compositions, no isotopic fractionation during differentiation of the Earth and no late (post-core formation) Cl-bearing volatile additions to the crustal veneer with a unique isotopic composition.

Dissecting a wheat QTL for yield present in a range of environments: from the QTL to candidate genes.

Previous studies with 95 bread wheat doubled haploid lines (DHLs) from the cross Chinese Spring (CS)xSQ1 trialled over 24 yearxtreatmentxlocations identified major yield quantitative trait loci (QTLs) in homoeologous locations on 7AL and 7BL, expressed mainly under stressed and non-stressed conditions, respectively. SQ1 and CS contributed alleles increasing yield on 7AL and 7BL, respectively. The yield component most strongly associated with these QTLs was grains per ear. Additional results which focus on the 7AL yield QTL are presented here. Trials monitoring agronomic, morphological, physiological, and anatomical traits revealed that the 7AL yield QTL was not associated with differences in flowering time or plant height, but with significant differences in biomass at maturity and anthesis, biomass per tiller, and biomass during tillering. In some trials, flag leaf chlorophyll content and leaf width at tillering were also associated with the QTL. Thus, it is likely that the yield gene(s) on 7AL affects plant productivity. Near-isogenic lines (NILs) for the 7AL yield QTL with CS or SQ1 alleles in an SQ1 background showed the SQ1 allele to be associated with >20% higher yield per ear, significantly higher flag leaf chlorophyll content, and wider flag leaves. Epidermal cell width and distance between leaf vascular bundles did not differ significantly between NILs, so the yield-associated gene may influence the number of cell files across the leaf through effects on cell division. Interestingly, comparative mapping with rice identified AINTEGUMENTA and G-protein subunit genes affecting lateral cell division at locations homologous to the wheat 7AL yield QTL.

The role of the retinoblastoma protein (Rb) in the nuclear localization of BAG-1: implications for colorectal tumour cell survival.

Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide variety of human cancers, the retinoblastoma protein (Rb) has been shown to be overexpressed in colon cancers, which is linked to the anti-apoptotic function of the protein. However, the mechanisms by which Rb regulates apoptosis are yet to be fully elucidated. We have established that Rb interacts with the anti-apoptotic BAG-1 (Bcl-2 associated athanogene-1) protein, and that a decrease in nuclear localization of BAG-1 is detectable when the interaction between Rb and BAG-1 is disrupted by expression of the E7 viral oncoprotein. Interestingly, although reported as deregulated in colorectal cancers, we have found that BAG-1 expression is also altered in small adenomas, where its localization was found to be predominantly nuclear. In addition, we have established that maintenance of high nuclear BAG-1 in vitro increases the resistance of adenoma-derived cells to gamma-radiation-induced apoptosis. Our work suggests a novel function for Rb, involving modulation of the subcellular localization of BAG-1. We have found predominant nuclear BAG-1 localization in small adenomas, and suggest that BAG-1 may promote colorectal tumour cell survival by making colonic epithelial cells less sensitive to DNA damage.

Nuclear BAG-1 expression inhibits apoptosis in colorectal adenoma-derived epithelial cells.

BAG-1 is an anti-apoptotic protein that is frequently deregulated in a variety of malignancies including colorectal cancer. There are three isoforms: BAG-1L is located in the nucleus, BAG-1M and BAG-1S are located both in the nucleus and the cytoplasm. In colon cancer, the expression of nuclear BAG-1 is associated with poorer prognosis and is potentially a useful predictive factor for distant metastasis. However, the function of BAG-1 in colonic epithelial cells has not been studied. Having previously shown a predominant nuclear localisation of BAG-1 in adenoma-derived cell lines, we wanted to determine the function of nuclear BAG-1 in these non-tumourigenic cells, to identify whether nuclear BAG-1 was implicated in tumour progression in the colon. In the current report we established that nuclear BAG-1 inhibits apoptosis in a colorectal adenoma-derived cell line. We demonstrate that apoptosis induced by gamma-radiation or the vitamin D analogue EB1089 in the non-tumourigenic human colorectal adenoma-derived S/RG/C2 cell line, was preceded by a decrease in nuclear and an increase in cytoplasmic BAG-1 expression. This change in subcellular localisation of BAG-1 was due to the redistribution of the BAG-1M isoform. In addition, we have shown that the maintenance of high nuclear BAG-1 through enforced expression of the nuclear localised BAG-1L isoform enhanced cellular survival after gamma-radiation or exposure to EB1089. Furthermore the expression of cytoplasmic BAG-1S isoform fused with a nuclear localisation signal protected against gamma-radiation induced apoptosis. This demonstrates that nuclear localisation of the BAG-1 protein confers a survival advantage in colorectal adenoma-derived cells and that nuclear BAG-1 could potentially be an important survival factor in colorectal carcinogenesis.

Limitations to CO2 assimilation in ozone-exposed leaves of Plantago major.

• The potential limitations on net leaf carbon assimilation imposed by stomatal conductance, carboxylation velocity, capacity for ribulose 1,5-bisphosphate regeneration and triose phosphate ultilization rate were derived from steady-state gas exchange measurements made over the life-span of two leaves on plants of an 'O3 -sensitive' population of Plantago major grown at contrasting atmospheric O3 concentrations. • Parallel measurements of chlorophyll fluorescence were used to monitor changes in the quantum efficiency of PSII photochemistry, and in vitro measurements of Rubisco activity were made to corroborate modelled gas exchange data. • Data indicated that a loss of Rubisco was predominantly responsible for the decline in CO2 assimilation observed in O3 -treated leaves. The quantum efficiency of PSII was unchanged by O3 exposure. • Stomatal aperture declined in parallel with CO2 assimilation in O3 -treated plants, but this did not account for the observed decline in photosynthesis. Findings suggested that O3 -induced shifts in stomatal conductance result from 'direct' effects on the stomatal complex as well as 'indirect effects' mediated through changes in intercellular CO2 concentration. Leaves on the same plant exposed to equivalent levels of O3 showed striking differences in their response to the pollutant.

Phenological weighting of ozone exposures in the calculation of critical levels for wheat, bean and plantain.

This paper presents phenological weighting factors to be applied to AOT40 (accumulated ozone exposure above a threshold of 40 nl l(-1)) ozone exposure-response relationships for crops at different growth stages. The quantification of such factors represents a step-forward in the derivation of Level II critical levels for ozone. The weighting factors presented are derived from published literature on the sensitivity of wheat (Triticum aestivum), bean (Phaseolus vulgaris) and plantain (Plantago major) to ozone at different growth stages. Weighting functions were calculated using either multiple linear regression or the reciprocal residual mean square (RMS(-1)). The resulting weights were transformed into multiplication factors to be applied to the monthly AOT40 during the 3-month assessment period of critical level exceedance. Interspecific differences were too large to allow for the development of a unified weighting function for the three species considered. For wheat grain yield, the derived multiplication factors varied by almost four-fold (0.40, 1.06, 1.54), while those for bean pod yield varied by only about 25% (0.85, 1.01, 1.14). The available data for plantain were restricted to short-term studies conducted under controlled conditions. These data were not suitable for the derivation of weighting factors comparable to those derived for bean and wheat. Based on known differences in wheat development and phenology across Europe, the need for a geographic differentiation of the time period for the calculation of the critical level exceedances is also discussed and examples provided of the adoption of the derived weightings in the mapping of critical level exceedances. Differences between critical level exceedance maps using weighted and unweighted AOT40 calculations are discussed.

Long-term responses of the green-algal lichen Parmelia caperata to natural CO2 enrichment.

Acclimation to elevated CO2 was investigated in Parmelia caperata originating from the vicinity of a natural CO2 spring, where the average daytime CO2 concentration was 729 ± 39 µmol mol-1 dry air. Thalli showed no evidence of a down-regulation in photosynthetic capacity following long-term exposure to CO2 enrichment in the field; carboxylation efficiency, total Ribulose bisphosphate carboxylase/oxygenase (Rubisco) content, apparent quantum yield of CO2 assimilation, and the light-saturated rate of CO2 assimilation (measured under ambient and saturating CO2 concentrations) were similar in thalli from the naturally CO2 enriched site and an adjacent control site where the average long-term CO2 concentration was about 355 µmol mol-1. Thalli from both CO2 environments exhibited low CO2 compensation points and early saturation of CO2 uptake kinetics in response to increasing external CO2 concentrations, suggesting the presence of an active carbon-concentrating mechanism. Consistent with the lack of significant effects on photosynthetic metabolism, no changes were found in the nitrogen content of thalli following prolonged exposure to elevated CO2. Detailed intrathalline analysis revealed a decreased investment of nitrogen in Rubisco in the pyrenoid of algae located in the elongation zone of thalli originating from elevated CO2, an effect associated with a reduction in the percentage of the cell volume occupied by lipid bodies and starch grains. Although these differences did not affect the photosynthetic capacity of thalli, there was evidence of enhanced limitations to CO2 assimilation in lichens originating from the CO2-enriched site. The light-saturated rate of CO2 assimilation measured at the average growth CO2 concentration was found to be significantly lower in thalli originating from a CO2-enriched atmosphere compared with that of thalli originating and measured at ambient CO2. At lower photosynthetic photon flux densities, the light compensation point of net CO2 assimilation was significantly higher in thalli originating from elevated CO2, and this effect was associated with higher usnic acid content.

Ozone levels in Chongqing: a potential threat to crop plants commonly grown in the region?

Chongqing, a city with a population of >2.5 million, constitutes the biggest industrial and commercial centre in southwest China. Recent industrialization has led to an increasing air pollutant problem which is exacerbated by the topography and prevailing climate of the region. To date, interest has remained firmly focused on the levels of traditional air pollutants (sulphur dioxide [SO2], oxides of nitrogen [NOx], smoke and suspended particulate matter [SPM]), with little attention paid to photochemical oxidants such as ozone (O3). This paper reports the first assessment of ambient O3 levels in southwest China. Measurements were made in and around Chongqing using a combination of UV-absorption (at a site located in the northern sector of the city) and passive samplers (at 20 sites located in and around the city) between 1993 and 1996. The 7-h daily mean O3 concentrations ranged between 2 and 16 ppb (x10(9)) during the winter months, increasing to 18-41 ppb during the summer (June-August), when peak hourly mean O3 concentrations of 93 ppb were attained. Ozone exposures across the region commonly approached (or exceeded) UN-ECE and WHO short-term guidelines for the protection of crops. In addition, controlled chamber studies, in which 11 cultivars of Chinese crops commonly grown in the Chongqing region were screened for relative O3 sensitivity, indicated the potential for subtle effects on the growth of a number of crop plants, if ambient O3 levels continue to rise in the region. Employing ozone exposures somewhat higher than those experienced in the field, several cultivars of commonly grown Chinese cereal, vegetable and salad crops were found to be sensitive to O3 in terms of growth, but this was not always associated with the appearance of visible symptoms of injury and, in contrast to what was generally expected, only three species showed significant O3-induced reductions in root:shoot partitioning. There is a clear and urgent need for a comprehensive study of ambient air quality and its impact on crops and natural vegetation in this, as in other, rapidly developing regions of China.

The impact of elevated CO2 on growth and photosynthesis in Agrostis canina L. ssp. monteluccii adapted to contrasting atmospheric CO2 concentrations.

The aim of this study was to characterise growth and photosynthetic capacity in plants adapted to long-term contrasting atmospheric CO2 concentrations (C a). Seeds of Agrostis canina L. ssp. monteluccii were collected from a natural CO2 transect in central-western Italy and plants grown in controlled environment chambers at both ambient and elevated CO2 (350 and 700 µmol mol-1) in nutrient-rich soil. Seasonal mean C a at the source of the plant material ranged from 610 to 451 µmol CO2 mol-1, derived from C4 leaf stable carbon isotope discrimination (δ13C). Under chamber conditions, CO2 enrichment stimulated the growth of all populations. However, plants originating from elevated C a exhibited higher initial relative growth rates (RGRs) irrespective of chamber CO2 concentrations and a positive relationship was found between RGR and C a at the seed source. Seed weight was positively correlated with C a, but differences in seed weight were found to explain no more than 34% of the variation in RGRs at elevated CO2. Longer-term experiments (over 98 days) on two populations originating from the extremes of the transect (451 and 610 µmol CO2 mol-1) indicated that differences in growth between populations were maintained when plants were grown at both 350 and 700 µmol CO2 mol-1. Analysis of leaf material revealed an increase in the cell wall fraction (CWF) in plants grown at elevated CO2, with plants originating from high C a exhibiting constitutively lower levels but a variable response in terms of the degree of lignification. In vivo gas exchange measurements revealed no significant differences in light and CO2 saturated rates of photosynthesis and carboxylation efficiency between populations or with CO2 treatment. Moreover, SDS-PAGE/ LISA quantification of leaf ribulose bisphosphate carboxylase/oxygenase (Rubisco) showed no difference in Rubisco content between populations or CO2 treatments. These findings suggest that long-term adaptation to growth at elevated CO2 may be associated with a potential for increased growth, but this does not appear to be linked with differences in the intrinsic capacity for photosynthesis.

Embryonic expression of Lim-1, the mouse homolog of Xenopus Xlim-1, suggests a role in lateral mesoderm differentiation and neurogenesis.

cDNAs encoded by the mouse homolog (Lim-1) of the Xenopus LIM-class homeobox gene Xlim-1 have been isolated from an 8.5-day mouse embryo cDNA library. Nucleotide and deduced amino acid sequences show a high degree of identity with Xlim-1 in the LIM and homeodomains, and 85% identity over the whole protein. An interspecific back-cross has been used to show close linkage of Lim-1 to the endogenous proviral marker Mpmv-4 on mouse chromosome 11. Whole mount in situ hybridization studies have been carried out on mouse embryos between 6.5 and 10.5 days. In mid- to late-streak stage embryos, Lim-1 is expressed in a restricted region of mesoderm in the primitive streak, with the highest level of signal at the anterior. At 7.5 days, transcripts can be seen in a horseshoe-shaped pattern in the periphery of the node, as well as along both sides of the immediately adjacent notochord. In addition, transcripts are present in presumptive lateral and intermediate mesoderm. Later, expression becomes progressively restricted to intermediate mesoderm, the nephrogenic cords, and eventually mesonephric ducts and tubules. By 10.5 days Lim-1 transcripts also appear in restricted regions of the central nervous system (CNS) that are associated with sensory function. The lateral diencephalon, hindbrain, and presumed commissural neurons in the dorsal spinal cord all show Lim-1 expression. In the adult, Lim-1 is expressed in the cerebellum/medulla and kidney, and at very low levels in the cerebrum. These data suggest that in the mouse embryo Lim-1 plays a role in early mesoderm formation and later specification of a differentiated phenotype in subsets of cells of the mesonephros and sensory neurons of the CNS.

Physiological effects of ozone on cultivars of watermelon (Citrullus lanatus) and muskmelon (Cucumis melo) widely grown in Spain.

Two cultivars of watermelon (Citrullus lanatus) and muskmelon (Cucumis melo), which are widely grown in Spain, were exposed to ozone (70 nl litre(-1), 6 h d(-1)) for 21 days. Ozone sensitivity was assessed by recording the extent of visible injury, changes in fast-fluorescence kinetics, the relative-growth rate (R) of root (RR) and shoot (RS), and effects on the number of flowers produced per plant. Leaf gas exchange was measured in order to provide some indication of the factors underlying differential response to ozone. After 9-10 days of fumigation, all the cultivars developed typical visible symptoms of zone injury on the older leaves. However, significant (P < 0.05) changes in fast-fluorescence kinetics were detected prior to the development of visible foliar injury, indicating that detectable effects of ozone on primary photochemical processes supersede the appearance of visible symptoms. In both muskmelon and watermelon, there was a marked reduction in the rate of CO(2) assimilation as a result of exposure to ozone, and this was accompanied by a parallel decrease in stomatal conductance. Mean plant-relative-growth rate (R) was markedly (P < 0.01) suppressed by ozone in the two cultivars of watermelon, but there were no significant effects on R in muskmelon. Ozone reduced root growth relative to the shoot in three out of four cultivars-an effect that may be of considerable ecological significance. Moreover, exposure to ozone reduced flower production in both muskmelon and watermelon, which indicated effects on yield. There was no correlation between a variety of methods used to assess ozone sensitivity and visible injury, and reasons for this are discussed. This observation draws clear attention to the dangers in ranking plants for ozone sensitivity purely on the basis of visible symptoms. It is concluded from this study that ozone-insensitive genotypes should be identified and considered for planting in the major areas of melon production concentrated on the Mediterranean coast of Spain.

On the ecophysiology of the Clusiaceae in Trinidad: expression of CAM in Clusia minor L. during the transition from wet to dry season and characterization of three endemic species.

A study was made of photosynthesis and expression of crassulacean acid metabolism (CAM) in naturally exposed and shaded populations of Clusia minor L. during the transition from wet to dry season in Trinidad (mid-February to mid-April, 1990). At the start of the dry season, plants from exposed and shaded habitats showed a capacity for CAM either through the fixation of external or internal (respiratory) CO2 . Exposed plants showed continuous uptake of CO2 over 24 h although dark fixation accounted for only a small proportion of CO2 fixed over the day. The expression of CAM was considerably enhanced as the dry season progressed with substantial increases in the overnight accumulation of titratable acidity, particularly in leaves of exposed plants. This was accompanied by a reduction in day-time photosynthesis and an increase in dark fixation, with shaded plants showing only night-time fixation of CO2 . The magnitude of CAM in C. minor was substantial with a maximum ΔH+ of 1410 mol m-3 measured in leaves from exposed branches. Both malic and citric acids were accumulated overnight. The highest citric:malic acid ratios were found in young leaves from exposed plants with 250 mol m-3 malic and 125 mol m-3 citric acid accumulated near the time of maximum CAM activity. Photosynthetic efficiency, measured as light responses of O2 evolution, also varied on a daily basis dependent on the incident photosynthetic photon flux density (PPFD). Apparent quantum yield and photosynthetic capacity showed marked reductions depending on the degree of exposure, suggesting that photoinhibitory responses are important under natural conditions. An analysis of three members of the Clusiaceae endemic to Trinidad showed that each had the capacity to induce CAM activity, despite being found in a narrow range of habitats which have higher rainfall than those of C. minor. However, despite the variable expression of CAM activity, carbon isotope composition suggested that when integrated throughout the year, carbon accumulation is predominantly mediated via the C3 pathway in all the species studied.

The influence of CO2 and O3 , singly and in combination, on gas exchange, growth and nutrient status of radish (Raphanus sativus L.).

Five days after emergence radish (Raphanus sativus L. ev. Cherry Belle) plants were transferred to a phytotron at the GSF München, where they were exposed in four large controlled climate chambers to two atmospheric concentrations of CO2 , ('ambient', daily means of ∼ 385 µmol-1 ; elevated, daily means of ∼ 765 µmol mol-1 ) and two O3 regimes ('non-polluted' air, 24 h mean of 20 nmol mol-1 ; polluted air, 24 h mean of 73 nmol mol-1 ). Leaf gas-exchange measurements were made at intervals, and visible O3 damage, effects on growth, dry matter partitioning and mineral composition were assessed at a final whole-plant harvest after 27 d. In 'non-polluted air' CO2 enrichment resulted in a progressive stimulation in Asat , whilst there was a decline in g which decreased E (i.e. improved WUEi ). The extra carbon fixed in elevated CO2 stimulated growth of the root (+ hypocotyl) by 43 %, but there was no significant effect on shoot growth or leaf area. Moreover, a decline in SLA and LAR in CO2 -enriched plants suggested that less dry matter was invested in leaf area expansion. Tissue concentrations of N, S, P, Mg and Ca were lower (particularly in the root + hypocotyl) in elevated CO2 , indicating that total uptake of these nutrients was not affected by CO2 , and there was an increase in the C:N ratio in root (+ hypocotyl) tissue. In contrast, O3 depressed Asat , (∼ 26%) and induced slight stomatal closure, with the result that WUE, declined. All plants exposed to 'polluted' air developed typical visible symptoms of O3 injury, and effects on carbon assimilation were reflected in reduced growth, with shoot growth maintained at the expense of the root. In addition, O3 increased the P and K concentration in shoot and root (+ hypocotyl) tissue, indicating enhanced uptake of these nutrients from the growth medium. However, there was no affect of O3 on tissue concentrations of N, S, Mg and Ca. Interactions between the gases were complex, and often subtle. In general, elevated CO2 counteracted (at least in part) the detrimental effects of phytotoxic concentrations of O3 , whilst conversely, O3 reduced the impact of elevated CO2 . Moreover, there were indications that cumulative changes in source: sink relations in O3 -exposed plants may limit plant response to CO2 -enrichment to an even greater extent in the long-term. The future ecological significance of interactions between CO2 and O3 are discussed.

Persistent stimulation of CO2 assimilation and stomatal conductance by summer ozone fumigation in Norway spruce.

CO(2) assimilation rate, stomatal conductance and chlorophyll content of current and previous years' needles of Norway spruce were measured in May 1988, 205 days after the cessation of ozone fumigation during the summer of 1987. Rates of assimilation were consistently higher for both needle year age classes for ozone fumigated trees in comparison to control trees, although only statistically significant for part of the day for current year's needles. A 26% and 48% stimulation, overall, in mean daily rates of assimilation for current and previous years' needles of ozone fumigated trees was observed. This was due to an enhanced apparent quantum yield and light saturated rate of assimilation of ozone fumigated trees. The temperature response regression of assimilation versus temperature was also greater, such that at any given temperature, assimilation was higher for ozone treated trees than control trees. Stomatal conductance was greater for ozone fumigated trees than the controls, but this was only marginally statistically significant. Moreover, there was a consistent increase in chlorophyll content in both year classes in ozone-treated trees. These results are discussed in relation to a possible long term effect of ozone fumigation upon the processes of conifer winter hardening and spring de-hardening.

Persistent effects of ozone on needle water loss and wettability in Norway spruce.

Four-year-old, seed-grown trees of Norway spruce (Picea abies (L.) Karst.) were exposed in open-top chambers to charcoal-filtered air (8 h daily mean 54 microg O(3) m(-3)) over three consecutive summers (1986-1988). In mid-May 1988, before the third season of fumigation and more than 7 months after exposure to ozone the previous summer had terminated, daily rates of transpiration from intact shoots and water loss from excised needles were measured together with the amount of wax on the needle surface. In mid-July, 92 days after the beginning of the third year of exposure, the wettability of needles was assessed by measuring the contact angle of water droplets on the surface of needles. Exposure to 156 microg O(3) m(-3) resulted in a 16% increase in daily transpiration in current year's needles and a 28% increase in 1-year old needles. These effects were associated with slower stomatal closure in response to increasing water deficit in the needles previously exposed to 156 microg m(-3) ozone. The long-lasting nature of such ozone-induced effects could predispose trees to drought and winter desiccation. No significant effects of ozone were found on the amount of wax covering the needle surface, but a marked increase in the wettability of needles exposed to ozone was observed. The far reaching physiological consequences of these effects in the field and the possibility that similar disturbances may contribute to the decline of high-altitude forests of Norway spruce in Europe are discussed.