Enhanced ozone strongly reduces carbon sink strength of adult beech (Fagus sylvatica)--resume from the free-air fumigation study at Kranzberg Forest.

Ground-level ozone (O(3)) has gained awareness as an agent of climate change. In this respect, key results are comprehended from a unique 8-year free-air O(3)-fumigation experiment, conducted on adult beech (Fagus sylvatica) at Kranzberg Forest (Germany). A novel canopy O(3) exposure methodology was employed that allowed whole-tree assessment in situ under twice-ambient O(3) levels. Elevated O(3) significantly weakened the C sink strength of the tree-soil system as evidenced by lowered photosynthesis and 44% reduction in whole-stem growth, but increased soil respiration. Associated effects in leaves and roots at the gene, cell and organ level varied from year to year, with drought being a crucial determinant of O(3) responsiveness. Regarding adult individuals of a late-successional tree species, empirical proof is provided first time in relation to recent modelling predictions that enhanced ground-level O(3) can substantially mitigate the C sequestration of forests in view of climate change.

The challenge of making ozone risk assessment for forest trees more mechanistic.

Upcoming decades will experience increasing atmospheric CO2 and likely enhanced O3 exposure which represents a risk for the carbon sink strength of forests, so that the need for cause-effect related O3 risk assessment increases. Although assessment will gain in reliability on an O3 uptake basis, risk is co-determined by the effective dose, i.e. the plant's sensitivity per O3 uptake. Recent progress in research on the molecular and metabolic control of the effective O3 dose is reported along with advances in empirically assessing O3 uptake at the whole-tree and stand level. Knowledge on both O3 uptake and effective dose (measures of stress avoidance and tolerance, respectively) needs to be understood mechanistically and linked as a pre-requisite before practical use of process-based O3 risk assessment can be implemented. To this end, perspectives are derived for validating and promoting new O3 flux-based modelling tools.

Synopsis of the CASIROZ case study: carbon sink strength of Fagus sylvatica L. in a changing environment--experimental risk assessment of mitigation by chronic ozone impact.

Databases are needed for the ozone (O(3)) risk assessment on adult forest trees under stand conditions, as mostly juvenile trees have been studied in chamber experiments. A synopsis is presented here from an integrated case study which was conducted on adult FAGUS SYLVATICA trees at a Central-European forest site. Employed was a novel free-air canopy O(3) fumigation methodology which ensured a whole-plant assessment of O(3) sensitivity of the about 30 m tall and 60 years old trees, comparing responses to an experimental 2 x ambient O(3) regime (2 x O(3), max. 150 nl O(3) l (-1)) with those to the unchanged 1 x ambient O(3) regime (1 x O(3)=control) prevailing at the site. Additional experimentation on individual branches and juvenile beech trees exposed within the forest canopy allowed for evaluating the representativeness of young-tree and branch-bag approaches relative to the O(3) sensitivity of the adult trees. The 2 x O(3) regime did not substantially weaken the carbon sink strength of the adult beech trees, given the absence of a statistically significant decline in annual stem growth; a 3 % reduction across five years was demonstrated, however, through modelling upon parameterization with the elaborated database. 2 x O(3) did induce a number of statistically significant tree responses at the cell and leaf level, although the O(3) responsiveness varied between years. Shade leaves displayed an O(3) sensitivity similar to that of sun leaves, while indirect belowground O(3) effects, apparently mediated through hormonal relationships, were reflected by stimulated fine-root and ectomycorrhizal development. Juvenile trees were not reliable surrogates of adult ones in view of O(3) risk assessment. Branch sections enclosed in (climatized) cuvettes, however, turned out to represent the O(3) sensitivity of entire tree crowns. Drought-induced stomatal closure decoupled O(3) intake from O(3) exposure, as in addition, also the "physiologically effective O(3) dose" was subject to change. No evidence emerged for a need to lower the "Critical Level for Ozone" in risk assessment of forest trees, although sensitive tree parameters did not necessarily reflect a linear relationship to O(3) stress. Exposure-based concepts tended to overestimate O(3) risk under drought, which is in support of current efforts to establish flux-related concepts of O(3) intake in risk assessment.

Tree internal signalling and defence reactions under ozone exposure in sun and shade leaves of European beech (Fagus sylvatica L.) trees.

The influence of free-air ozone (O(3)) fumigation on the levels of gene transcripts and compounds of defence and signalling were analysed in leaves of adult beech trees from the "Kranzberg Forest" research site in 2003 and 2004. This includes the precursor of the stress hormone ethylene, ACC (1-aminocyclopropane-1-carboxylic acid), conjugated salicylic acid, lignin content as well as of the expression level of genes connected with oxidative stress and stress signalling. At this site mature beech trees were exposed to an enhanced O(3) regime by a free-air O(3) canopy exposure system. Levels of conjugated ACC and conjugated salicylic acid in leaves were increased under O (3) fumigation whereas lignin content was only slightly enhanced. Quantitative real-time RT-PCR (qRT-PCR) was performed on transcripts of genes connected with lignin, salicylic acid, and ethylene formation, the shikimate pathway, abscisic acid biosynthesis as well as with the antioxidative system. Genes which showed O(3)-dependent increases included FSCOMT (caffeic-acid O-methyltransferase) connected with lignin formation, the stress response genes FSACS2 (ACC synthase) and FSPR1 (PR10 - pathogenesis-related protein), as well as FSNCED1 (9-cis-epoxicarotenoid dioxygenase), the rate-limiting enzyme of the ABA synthesis. For FSNCED1 expression level, a significant O(3) effect was found with an 8-fold (sun) and 7-fold (shade) induction in July 2003 and a 3-fold and 2.5-fold induction in July 2004. While the observed effects were not continuous, elevated O(3) is concluded to have the potential to disrupt the defence and signalling system.

Beech leaf colonization by the endophyte Apiognomonia errabunda dramatically depends on light exposure and climatic conditions.

Ozone and light effects on endophytic colonization by Apiognomonia errabunda of adult beech trees (Fagus sylvatica) and their putative mediation by internal defence compounds were studied at the Kranzberg Forest free-air ozone fumigation site. A. errabunda colonization was quantified by "real-time PCR" (QPCR). A. errabunda-specific primers allowed detection without interference by DNA from European beech and several species of common genera of plant pathogenic fungi, such as Mycosphaerella, Alternaria, Botrytis, and Fusarium. Colonization levels of sun and shade leaves of European beech trees exposed either to ambient or twice ambient ozone regimes were determined. Colonization was significantly higher in shade compared to sun leaves. Ozone exhibited a marginally inhibitory effect on fungal colonization only in young leaves in 2002. The hot and dry summer of 2003 reduced fungal colonization dramatically, being more pronounced than ozone treatment or sun exposure. Levels of soluble and cell wall-bound phenolic compounds were approximately twice as high in sun than in shade leaves. Acylated flavonol 3- O-glycosides with putatively high UV-B shielding effect were very low in shade canopy leaves. Ozone had only a minor influence on secondary metabolites in sun leaves. It slightly increased kaempferol 3- O-glucoside levels exclusively in shade leaves. The frequently prominent hydroxycinnamic acid derivative, chlorogenic acid, was tested for its growth inhibiting activity against Apiognomonia and showed an IC50 of approximately 8 mM. Appearance of Apiognomonia-related necroses strongly correlated with the occurrence of the stress metabolite, 3,3',4,4'-tetramethoxybiphenyl. Infection success of Apiognomonia was highly dependent on light exposure, presumably affected by the endogenous levels of constitutive phenolic compounds. Ozone exerted only minor modulating effects, whereas climatic factors, such as pronounced heat periods and drought, were dramatically overriding.

Transcriptome analysis of ozone-responsive genes in leaves of European beech (Fagus sylvatica L.).

Suppression subtractive hybridization (SSH) was performed to isolate cDNAs representing genes that are differentially expressed in leaves of Fagus sylvatica upon ozone exposure. 1248 expressed sequence tags (ESTs) were obtained from 2 subtractive libraries containing early and late ozone-responsive genes. Sequences of 1139 clones (91 %) matched the EBI/NCBI database entries. For 578 clones, no putative function could be assigned. Most abundant transcripts were O-methyltransferases, representing 7 % of all sequenced clones. ESTs were organized into 12 functional categories according to the MIPS database. Among them, 12 % (early)/15 % (late) were associated with disease and defence, 19/11 % with cell structure, 4/10 % with signal transduction, and 9/6 % with transcription. The expression pattern of selected ESTs (ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit [rbcS], WRKY-type transcription factor, ultraviolet-B-repressible protein, aquaporine, glutathione S-transferase, catalase, caffeic acid O-methyltransferase, and pathogenesis-related protein 1 [PR1]) was analysed by quantitative real-time RT-PCR (qRT-PCR) which confirmed changed transcript levels upon ozone treatment of European beech saplings. The ESTs characterized will contribute to a better understanding of forest tree genomics and also to a comparison of ozone-responsive genes in woody and herbaceous plants.

Characterization of an Aux/IAA cDNA upregulated in Pinus pinaster roots in response to colonization by the ectomycorrhizal fungus Hebeloma cylindrosporum.

• In an attempt to determine whether fungal auxin affects host plant gene expression during mycorrhizal formation, an auxin upregulated cDNA, Pp-iaa88, was isolated by differential screening of a cDNA library made from auxin-treated Pinus pinaster roots. • Pp-iaa88 codes for a polypeptide that shares extensive homology to auxin-inducible Aux/IAA proteins, which are supposed to act as transcription factors. Cycloheximide did not inhibit auxin-induced mRNA accumulation, indicating that Pp-iaa88 upregulation is a primary (direct) auxin response. • The level of Pp-iaa88 transcripts in roots increased following inoculation with either an indoleacetic acid-overproducing mutant or a wild-type strain of the ectomycorrhizal fungus Hebeloma cylindrosporum. With both strains, mRNA accumulation was detectable as soon as fungal hyphae reached the root and it increased during differentiation of symbiotic structures. The kinetics of Pp-iaa88 transcript accumulation was closely connected with the dynamics of symbiosis establishment and was more rapid with the mutant than with the wild-type strain. • As a putative transcription factor expressed at the very early stages of symbiosis establishment, Pp-iaa88 could play a key role in mycorrhizal formation.

The lipid/protein interface as xenobiotic target site: kinetic analysis of the nicotinic acetylcholine receptor.

Membrane proteins are known to be solvated and functionally activated by a fixed number of lipid molecules whose multiple binding can be described by Adair-type binding equations. Lipophilic xenobiotics such as general anesthetics may act by competitive displacement of protein-bound lipids. A kinetic equation is now presented for various binding stoichiometries of lipid and xenobiotic, and microscopic binding constants of anesthetics and organic solvents are derived from two independent assay systems for the enhancement of agonist binding to the nicotinic acetylcholine receptor. These constants lead to the first available free energy estimate (-6.4 kcal/mol) for the binding of membrane lipid to an integral membrane protein.

Ozone-sensitive arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death.

We have isolated a codominant Arabidopsis mutant, radical-induced cell death1 (rcd1), in which ozone (O(3)) and extracellular superoxide (O(2)(*)-), but not hydrogen peroxide, induce cellular O(2)(*)- accumulation and transient spreading lesions. The cellular O(2)(*)- accumulation is ethylene dependent, occurs ahead of the expanding lesions before visible symptoms appear, and is required for lesion propagation. Exogenous ethylene increased O(2)(*)--dependent cell death, whereas impairment of ethylene perception by norbornadiene in rcd1 or ethylene insensitivity in the ethylene-insensitive mutant ein2 and in the rcd1 ein2 double mutant blocked O(2)(*)- accumulation and lesion propagation. Exogenous methyl jasmonate inhibited propagation of cell death in rcd1. Accordingly, the O(3)-exposed jasmonate-insensitive mutant jar1 displayed spreading cell death and a prolonged O(2)(*)- accumulation pattern. These results suggest that ethylene acts as a promoting factor during the propagation phase of developing oxyradical-dependent lesions, whereas jasmonates have a role in lesion containment. Interaction and balance between these pathways may serve to fine-tune propagation and containment processes, resulting in alternate lesion size and formation kinetics.

Gene induction of stilbene biosynthesis in Scots pine in response to ozone treatment, wounding, and fungal infection.

The S-adenosyl-L-methionine:pinosylvin-O-methyltransferase (PMT) gene was sequenced from Scots pine (Pinus sylvestris). The open reading frame is arranged in two exons spaced by one 102-bp intron. Promoter regulatory elements such as two "CAAT" boxes and one "TATA" box were identified. Several cis-regulatory elements were recognized: stress-responsive elements (Myb-responsive elements) as well as G, H, and GC boxes. Moreover, elicitor-responsive elements (W boxes) and a sequence resembling the simian virus 40 enhancer core were found. In phloem and needles of control trees, the transcripts of stilbene synthase (STS) and PMT were hardly detectable. Increased ozone fumigation up to 0.3 microL L(-1) enhanced the transcript level of STS and PMT in needles but not in healthy phloem. Wounding, e.g. mock inoculation, of stem-phloem was characterized by a transient increase in STS and PMT transcripts, which was more pronounced in the case of fungal inoculation. Combination of fungal-challenge or mock treatment with ozone resulted in a positive interaction at 0.3 microL L(-1). Scots pine stilbene formation appeared to be induced via STS and PMT gene expression upon ozone and fungal stress as well as wounding. The broad stress-responsiveness is in agreement with the range of various cis-acting elements detected in the STS and PMT promoters.

Active oxygen species as mediators of plant immunity: three case studies.

A burst of active oxygen species (AOS) is known to be involved in local cell death as part of plant defence against pathogens. It is, however, under dispute to what extent AOS can induce pathogen resistance and immunity throughout the plant. Three experimental strategies that reveal a primary role for AOS and a surprisingly low chemical and spatial specificity are now described for tobacco and Arabidopsis thaliana plants. Ozone is a gaseous AOS that was applied to non-transgenic plants. Hydrogen peroxide or singlet oxygen are AOS that were induced by high-light treatment of transgenic plants that contained antisense constructs inhibiting catalase activity or chlorophyll biosynthetic enzymes. In all cases, activated oxygen species, cellular lesions, ethylene and salicylic acid, and components of major plant defence systems (systemic acquired resistance, hypersensitive response) were induced, as was resistance towards pathogens (tobacco mosaic virus, Pseudomonas syringae or Peronospora parasitica). It is concluded that active oxygen species can act as mediators of plant immunity so that new non-pesticidal plant protection strategies could be developed.

Elevated UV-B radiation reduces genome stability in plants.

Long-term depletion of the stratospheric ozone layer contributes to an increase in terrestrial solar ultraviolet-B radiation. This has deleterious effects on living organisms, such as DNA damage. When exposed to elevated ultraviolet-B radiation (UV-B; 280-315 nm), plants display a wide variety of physiological and morphological responses characterized as acclimation and adaptation. Here we show, using special sun simulators, that elevated solar UV-B doses increase the frequency of somatic homologous DNA rearrangements in Arabidopsis and tobacco plants. Increases in recombination are accompanied by a strong induction of photolyase and Rad51 gene expression. These genes are putatively involved in major DNA repair pathways, photoreactivation and recombination repair. In mutant Arabidopsis plants that are deficient in photoreactivating ultraviolet-induced cyclobutane pyrimidine dimers, recombination under elevated UV-B regimes greatly exceeds wild-type levels. Our results show that homologous recombination repair pathways might be involved in eliminating UV-B-induced DNA lesions in plants. Thus, increases in terrestrial solar UV-B radiation as forecasted for the early 21st century may affect genome stability in plants.

Taxonomic distribution of plant glutathione S-transferases acting on xenobiotics.

Soluble and microsomal glutathione S-transferase activities for five model xenobiotics (nitrobenzene derivatives), two pesticidal xenobiotics (atrazine and fluorodifen), and a natural substrate (cinnamic acid), were determined in 59 different plant species and four plant cell suspension cultures. These enzyme activities were widely distributed over the plant kingdom with certain species showing particularly high activities. Marine macroalgae had a remarkably broad substrate range that included the substrates atrazine and fluorodifen. It is concluded that the evolutionary 'green liver' concept derived for xenobiotic metabolism in higher plant species is also valid for the constitutive soluble and microsomal glutathione S-transferases of lower plant species.

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

Ozone/biotic disease interactions: molecular biomarkers as a new experimental tool.

Current climate change scenarios predict a further increase of tropospheric ozone which is well known to inhibit plant photosynthesis and growth processes. Ozone can also predispose plants to enhanced biotic attack, as proposed in particular for necrotrophic fungi, root-rot fungi and bark beetles. However, at present it does not seem possible to predict whether increased ambient ozone will lead to a higher or lower disease likelihood in particular plant-pathogen systems. It has been stated repeatedly in the literature that periods of high ambient ozone are essentially non-coincident with infection periods of most fungal pathogens. This implies minimal interactive risks. However, it now appears that the various ozone-induced metabolic changes can persist in plants over days or months. Visible ozone symptoms also may be greatly delayed. Certain stress transcripts, proteins and metabolites have been developed as ozone biomarkers in controlled exposure experiments, but these biomarkers remain to be examined on field sites. A simple epidemiological scenario based on 'memory' time spans of ozone effects is proposed as a tool to make ozone-plant disease interactions more predictable.

Fast, sensitive and selective liquid chromatographic-tandem mass spectrometric determination of tumor-promoting diterpene esters.

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to detect tumor-promoting diterpene esters of the tigliane and ingenane types within plant extracts. Fractionation on a C18 high-performance liquid chromatography (HPLC) column was followed by MS-MS-multiple reaction monitoring (MRM) using the precursor-->product ion pairs of m/z 311-->293 and 293-->265 for phorbol esters. The ion pairs m/z 313-->295 and 295-->267 were used for ingenol and deoxyphorbol esters. In a second run, the characteristic ions at m/z 311 and 313 were followed in precursor ion scan mode. These quasi-molecular ions were utilized to obtain full scan spectra of the compounds in product ion scan mode. Due to its selectivity, the present on-line method can be applied for plant cultivar selection and plant product control without time-consuming extraction procedures and complex bioassays.

Tumor promoters in commercial indoor-plant cultivars of the Euphorbiaceae.

Certain decorative indoor-plant cultivars are derived from toxic wild plant species. Native members of the Euphorbiaceae (spurge) contain highly irritating and tumor-promoting diterpene esters. Plant breeders and gardeners are constantly searching for less toxic cultivars of the popular Euphorbiaceae indoor plants. In this investigation, 22 commercial cultivars of Euphorbiaceae indoor plants were examined for tumor promoter contents by high-performance liquid chromatography (HPLC). Cultivars of E. milii (E. lomii hybrids), and in particular E. leuconeura, contained ingenol derivatives, whereas cultivars of E. pulcherrima and Codiaeum variegatum were devoid of these compounds. Tumor-promoting activity was assessed by induction of a luciferase reporter gene, which was placed under the control of an Epstein-Barr virus early antigen promoter. The response was closely correlated with ingenol ester content; the latex of the two E. leuconeura cultivars tested gave the strongest response. The HPLC and bioassay methods used in this study provide a basis for the development of nontoxic indoor-plant cultivars and perhaps for consumer-oriented labeling.

Xenobiotic-metabolizing enzymes in the canine respiratory tract.

Airway epithelial surface is the primary target of airborne pollutants. To estimate the distribution of xenobiotic-metabolizing enzymes in the respiratory tract of dogs, epithelia from different airway sites of four animals were analyzed for metabolism of sulfite (sulfite oxidase) and formaldehyde (formaldehyde dehydrogenase and aldehyde dehydrogenase). In addition, glutathione S-transferases were assayed using several model substrates. Enzyme activities were compared with those found in liver parenchyma. The activity of sulfite oxidase was found to be comparable in nose, trachea, and proximal and medium bronchi, but appeared to be lower in lung parenchyma of most animals. In contrast, hepatic sulfite oxidase activity of these animals was substantially higher compared to that in airway epithelia. The activity of glutathione-dependent formaldehyde dehydrogenase (FDH) appeared to be highest in nose and lowest in distal bronchi, lung, and liver parenchyma. The distribution pattern of the glutathione-independent aldehyde dehydrogenase (AldDH) in the respiratory tract was different from that of FDH. Levels of AldDH were about 5- to 10-fold lower than those of FDH, suggesting that AldDH is of minor importance for pulmonary formaldehyde detoxification. With regard to ethanol detoxification by a class I alcohol dehydrogenase (ADH), no measurable enzyme activity could be detected at most respiratory sites contrary to the high activity found in liver parenchyma. Regarding glutathione S-transferases (GSTs), different distributions of enzyme activities were found in the large and small airways when using three substrates. The 1-chloro-2,4-dinitrobenzene (CDNB)-related activities in the cytosolic fraction of the upper (nose, trachea) and lower airways (proximal, medium and distal bronchi) were higher than those in the microsomal fraction. Interestingly, there was no difference between CDNB-related activities in the cytosolic and microsomal fraction of the liver. Highest cytosolic activities were found in the nose, and were comparable to those detected in the liver parenchyma. The cytosolic 1,2-dichloro-4-nitrobenzene (DCNB)-related activities in the nose, proximal bronchi, and lung parenchyma were appeared to be markedly higher than those in trachea and medium and distal bronchi, while the microsomal activities were not detectable at most respiratory sites. In contrast, distinctly higher activities were measured in both fractions of liver tissue. Cytosolic 1, 2-epoxy-3-(p-nitrophenoxy)-propane (EPNP)-related activities were present in upper and lower airways including lung parenchyma at comparable levels, while in liver tissue the mean activities were distinctly lower. No EPNP-related activities were found in the microsomal fractions. In conclusion, most xenobiotic-metabolizing enzymes investigated in this study could be detected in epithelia of various respiratory sites. The most outstanding result revealed higher levels of FDH activity in the nose and downstream to the medium bronchi in comparison to those found in the small airways, lung, and liver tissue. Similarly, the EPNP-related GST exhibited a distinctly higher activity at all respiratory sites compared to the activity in liver tissue, suggesting a different regulation of this enzyme in lung and liver.