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Nitric oxide (NO) is an important signalling molecule that is involved in many different physiological processes in plants. Here, we report about a NO-fixing mechanism in Arabidopsis, which allows the fixation of atmospheric NO into nitrogen metabolism. We fumigated Arabidopsis plants cultivated in soil or as hydroponic cultures during the whole growing period with up to 3 ppmv of NO gas. Transcriptomic, proteomic and metabolomic analyses were used to identify non-symbiotic haemoglobin proteins as key components of the NO-fixing process. Overexpressing non-symbiotic haemoglobin 1 or 2 genes resulted in fourfold higher nitrate levels in these plants compared with NO-treated wild-type. Correspondingly, rosettes size and weight, vegetative shoot thickness and seed yield were 25, 40, 30, and 50% higher, respectively, than in wild-type plants. Fumigation with 250 ppbv 15NO confirmed the importance of non-symbiotic haemoglobin 1 and 2 for the NO-fixation pathway, and we calculated a daily uptake for non-symbiotic haemoglobin 2 overexpressing plants of 250 mg N/kg dry weight. This mechanism is probably important under conditions with limited N supply via the soil. Moreover, the plant-based NO uptake lowers the concentration of insanitary atmospheric NOx, and in this context, NO-fixation can be beneficial to air quality.

Plants have to cope with various abiotic stresses including UV-B radiation (280-315 nm). UV-B radiation is perceived by a photoreceptor, triggers morphological responses and primes plant defence mechanisms such as antioxidant levels, photoreapir or accumulation of UV-B screening pigments. As poplar is an important model system for trees, we elucidated the influence of UV-B on overall metabolite patterns in poplar leaves grown under high UV-B radiation. Combining non-targeted metabolomics with gas exchange analysis and confocal microscopy, we aimed understanding how UV-B radiation triggers metabolome-wide changes, affects isoprene emission, photosynthetic performance, epidermal light attenuation and finally how isoprene-free poplars adjust their metabolome under UV-B radiation. Exposure to UV-B radiation caused a comprehensive rearrangement of the leaf metabolome. Several hundreds of metabolites were up- and down-regulated over various pathways. Our analysis, revealed the up-regulation of flavonoids, anthocyanins and polyphenols and the down-regulation of phenolic precursors in the first 36 h of UV-B treatment. We also observed a down-regulation of steroids after 12 h. The accumulation of phenolic compounds leads to a reduced light transmission in UV-B-exposed plants. However, the accumulation of phenolic compounds was reduced in non-isoprene-emitting plants suggesting a metabolic- or signalling-based interaction between isoprenoid and phenolic pathways.

Sprouts contaminated with human pathogens are able to cause food-borne diseases due to the favorable growth conditions for bacteria during germination and because of minimal processing steps prior to consumption. We have investigated the potential of hot humid air, i.e., aerated steam, to treat alfalfa and mung bean seeds which have been artificially contaminated with Escherichia coli O157:H7, Salmonella enterica subsp. enterica serovar Weltevreden, and Listeria monocytogenes Scott A. In addition, a recently collected E. coli O178:H12 isolate, characterized by a reduced heat sensitivity, was exposed to the treatment described. Populations of E. coli O157:H7 and S. enterica on alfalfa and mung bean seeds could be completely eliminated by a 300-s treatment with steam at 70 ± 1°C as revealed by enrichment studies. L. monocytogenes and E. coli O178:H12 could not be completely eliminated from artificially inoculated seeds. However, bacterial populations were reduced by more than 5 log CFU/g on alfalfa and by more than 4 log CFU/g on mung bean seeds. The germination rate of mung beans was not affected by the 300-s treatment compared to the germination rate of untreated seeds whereas that of alfalfa seeds was significantly lower by 11.9%. This chemical-free method is an effective alternative to the 20,000-ppm hypochlorite treatment presently recommended by the U.S. Food and Drug Administration (FDA).

Quorum sensing auto-inducers of the N-acyl homoserine lactone (AHL) type produced by Gram-negative bacteria have different effects on plants including stimulation on root growth and/or priming or acquirement of systemic resistance in plants. In this communication the influence of AHL production of the plant growth promoting endophytic rhizosphere bacterium Acidovorax radicis N35 on barley seedlings was investigated. A. radicis N35 produces 3-hydroxy-C10-homoserine lactone (3-OH-C10-HSL) as the major AHL compound. To study the influence of this QS autoinducer on the interaction with barley, the araI-biosynthesis gene was deleted. The comparison of inoculation effects of the A. radicis N35 wild type and the araI mutant resulted in remarkable differences. While the N35 wild type colonized plant roots effectively in microcolonies, the araI mutant occurred at the root surface as single cells. Furthermore, in a mixed inoculum the wild type was much more prevalent in colonization than the araI mutant documenting that the araI mutation affected root colonization. Nevertheless, a significant plant growth promoting effect could be shown after inoculation of barley with the wild type and the araI mutant in soil after two months cultivation. While A. radicis N35 wild type showed only a very weak induction of early defense responses in plant RNA expression analysis, the araI mutant caused increased expression of flavonoid biosynthesis genes. This was corroborated by the accumulation of several flavonoid compounds such as saponarin and lutonarin in leaves of root inoculated barley seedlings. Thus, although the exact role of the flavonoids in this plant response is not clear yet, it can be concluded, that the synthesis of AHLs by A. radicis has implications on the perception by the host plant barley and thereby contributes to the establishment and function of the bacteria-plant interaction.

Marine macroalgae represent an important part of coastal ecosystems in temperate and polar areas. Increasing surface UV radiation (UVR) levels due to stratospheric ozone thinning may lead to deleterious effects on the physiology of marine macroalgae. Only few studies concentrate on the response of macroalgal gene expression towards elevated UVR. Analyzing gene expression in marine algae is challenging regarding RNA extraction, enrichment of differentially expressed transcripts and identification of resulting sequences. Thus, we focused on the optimization of the appropriate techniques using the widely distributed green marine macroalga Acrosiphonia arcta. We successfully used a combination of suppression subtractive hybridization (SSH) with microarray-based screening of size-selected cDNAs libraries and were able to substantially improve the outcome compared to standard techniques.

The indirect defences of plants are comprised of herbivore-induced plant volatiles (HIPVs) that among other things attract the natural enemies of insects. However, the actual extent of the benefits of HIPV emissions in complex co-evolved plant-herbivore systems is only poorly understood. The observation that a few Quercus robur L. trees constantly tolerated (T-oaks) infestation by a major pest of oaks (Tortrix viridana L.), compared with heavily defoliated trees (susceptible: S-oaks), lead us to a combined biochemical and behavioural study. We used these evidently different phenotypes to analyse whether the resistance of T-oaks to the herbivore was dependent on the amount and scent of HIPVs and/or differences in non-volatile polyphenolic leaf constituents (as quercetin-, kaempferol- and flavonol glycosides). In addition to non-volatile metabolic differences, typically defensive HIPV emissions differed between S-oaks and T-oaks. Female moths were attracted by the blend of HIPVs from S-oaks, showing significantly higher amounts of (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E)-β-ocimene and avoid T-oaks with relative high fraction of the sesquiterpenes α-farnesene and germacrene D. Hence, the strategy of T-oaks exhibiting directly herbivore-repellent HIPV emissions instead of high emissions of predator-attracting HIPVs of the S-oaks appears to be the better mechanism for avoiding defoliation.

Flavonols, phenylalanine-derived secondary metabolites, have protective and regulatory functions in plants. In Arabidopsis thaliana, they are consecutively glycosylated at their 3-OH and 7-OH groups. UGT78D1 and UGT78D2 are the major flavonol 3-O-glycosyltransferases in Arabidopsis leaves. The ugt78d1 ugt78d2 double mutant, which was strongly compromised in the initial 3-O-glycosylation, showed a severe and specific repression of flavonol biosynthesis, retaining only one-third of the wild-type level. This metabolic phenotype was associated with a repressed transcription of several flavonol biosynthetic genes including the committed step chalcone synthase [(CHS) or TRANSPARENT TESTA 4 (TT4)]. Furthermore, the committed step of the upstream, general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL), was down-regulated in its enzyme activity and in the transcription of the flavonol-related PAL1 and PAL2. However, a complete blocking of flavonoid biosynthesis at CHS released PAL inhibition in a tt4 ugt78d1 ugt78d2 line. PAL activity was even enhanced in the flavonol synthase 1 mutant, which compromises the final formation of flavonol aglycones. The dependence of the PAL feedback inhibition on flavonols was confirmed by chemical complementation of tt4 ugt78d1 ugt78d2 using naringenin, a downstream flavonoid intermediate, which restored the PAL repression. Although aglycones were not analytically detectable, this study provides genetic evidence for a novel, flavonol-dependent feedback inhibition of the flavonol biosynthetic pathway and PAL. It was conditioned by the compromised flavonol-3-O-conjugation and a decrease in flavonol content, yet dependent on a residual, flavonol synthase 1 (FLS1)-related capacity to form flavonol aglycones. Thus, this regulation would not react to a reduced metabolic flux into flavonol biosynthesis, but it might prevent the accumulation of non-glycosylated, toxic flavonols.

HIV replication assays are important tools for HIV drug discovery efforts. Here, we present a full HIV replication system (EASY-HIT) for the identification and analysis of HIV inhibitors. This technology is based on adherently growing HIV-susceptible cells, with a stable fluorescent reporter gene activated by HIV Tat and Rev. A fluorescence-based assay was designed that measures HIV infection by two parameters relating to the early and the late phases of HIV replication, respectively. Validation of the assay with a panel of nine reference inhibitors yielded effective inhibitory concentrations consistent with published data and allowed discrimination between inhibitors of early and late phases of HIV replication. Finer resolution of the effects of reference drugs on different steps of HIV replication was achieved in secondary time-of-addition assays. The EASY-HIT assay yielded high Z ′ scores (>0.9) and signal stabilities, confirming its robustness. Screening of the LOPAC 1280 library identified 10 compounds (0.8%), of which eight were known to inhibit HIV, validating the suitability of this assay for screening applications. Studies evaluating anti-HIV activities of natural products with the EASY-HIT technology led to the identification of three novel inhibitory compounds that apparently act at different steps of HIV-1 replication. Furthermore, we demonstrate successful evaluation of plant extracts for HIV-inhibitory activities, suggesting application of this technology for the surveillance of biological extracts with anti-HIV activities. We conclude that the EASY-HIT technology is a versatile tool for the discovery and characterization of HIV inhibitors.

Tropospheric ozone causes severe oxidative stress in plants. To investigate the transcriptional responsiveness of adult trees to ozone, fully-expanded sun and shade leaves of mature beech trees were harvested at four time points over the entire vegetation period in 2005 and 2006. Microarray analyses were conducted on leaves from trees grown in the field under ambient and twice-ambient ozone concentrations at Kranzberger Forst (Bavaria). Beech trees changed their transcript levels in response to ozone. In the years 2005 and 2006 different transcription patterns were observed; this may have been a result of different weather conditions and ozone uptake. Furthermore, we obtained differences in mRNA expression patterns between shade and sun leaves. In the ozone-treated sun leaves of 2005, slightly up- and down-regulated transcript levels were detected, particularly in the spring and autumn, whereas shade leaves clearly exhibited reduced mRNA levels, particularly at the end of the vegetation period. In 2006, this pattern could not be confirmed, and in the autumn, four other transcripts were slightly up-regulated in ozone-treated shade leaves. In addition, two other transcripts were found to be influenced in sun leaves in the spring/summer. While we detected changes in the levels of only a few transcripts, the observed effects were not identical in both years. In conclusion, elevated ozone exhibited very small influence on the transcription levels of genes of mature beech trees.

In 2006, a controlled infection study was performed in the 'Kranzberger Forst' to address the following questions: (1) Will massive artificial inoculation with Apiognomonia errabunda override the previously observed inhibitory effect of chronic ozone? (2) Can biochemical or molecular markers be detected to account for the action of ozone? To this end six adult beech trees were chosen, three ozone fumigated (2x ozone) and three control trees (ambient = 1x ozone). Spore-sprayed branches of sun and shade crown positions of each of the trees, and uninoculated control branches, were enclosed in 100-L plastic bags for one night to facilitate infection initiation. Samples were taken within a five-week period after inoculation. A. errabunda infestation levels quantified by real-time PCR increased in leaves that were not fumigated with additional ozone. Cell wall components and ACC (ethylene precursor 1-amino cyclopropane-1-carboxylic acid) increased upon ozone fumigation and may in part lead to the repression of fungal infection.

The shikimate pathway plays a central role in the formation of aromatic intermediates in the production of stilbenes, flavonoids and lignins. Ozone effects on the levels of transcripts in this pathway were studied in saplings of European beech. Complementary DNA (cDNA) clones of all genes of this pathway were isolated, and quantitative real-time RT-PCR (qRT-PCR) using RNA isolated from leaves of ozone-treated saplings showed a strong induction of 3-deoxy-d -arabino-heptulosonate-7-phosphate synthase 1 (DAHPS1), DAHPS3, 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQD/SD), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and chorismate mutase (CM) transcripts. In contrast, DAHPS2, 3-dehydroquinate synthase (DHQS), shikimate kinase (SK), and chorismate synthase (CS) transcripts were only weakly induced. Earliest induction could be observed after 2 days of ozone treatment for DAHPS1, SK, EPSPS and CM. The coordinated regulation was evident for 3–5 weeks after the onset of ozone fumigation, and increased transcript levels were still detectable after another 7 weeks. Western blot analyses of DAHPS3 and DHQD/SD showed an increased protein level in agreement with the increased transcription levels. Ozone-dependent leaf lesions appeared 7 weeks after onset of ozone exposure. Strongly elevated were levels of conjugates of salicylic (SA) and gentisic acids (GA), either derived directly from chorismate, the key product of the shikimate pathway, or via phenylalanine, cinnamic, and benzoic acids. Concentrations of cell wall-bound phenolic compounds increased in both control and ozone-treated saplings with the latter showing slightly higher levels. Interestingly, however, this increase of cell wall-bound phenolics was accompanied by a decrease of soluble phenolics, which may indicate their deposition into the cell wall.

To prevent carbon (C) loss to consumers, trees need to defend their primary production. The tree-internal conflict in resource allocation between growth and defence demands has been the subject of various hypotheses but still requires quantification. A conceptual approach to approximating the C amount dispensable in favour of primary production at the expense of defence is demonstrated which is based on nine defence-related metabolite groups. Quantification is exemplified at the level of sun and shade leaves of adult Fagus sylvatica and Picea abies trees, two species contrasting in foliage type, under oxidative stress as induced by ozone exposure. The difference between maximum and minimum metabolite levels sampled several times throughout four consecutive growing seasons were conceived as dispensable between growth and defence-related metabolism and expressed in proportion of the mean annual gross primary production (GPP) of the foliage. In both species, this proportion amounted to between 2 and 5% of GPP (on a molar C basis). Remaining uncertainties are discussed as concerning functional overlap of substances between growth and defence-related metabolism, estimated classification of metabolite turnover rates and “third-party” trade-offs across C demands. Given the complexity of plant defence, simplification is needed for modelling allocation trade-offs in plants. The presented conceptual approach meets this need in approximating C transfer capacities between competing physiological demands and in stimulating empirical assessments towards mechanistic understanding.

The shikimate pathway plays a pivotal role in the formation of aromatic secondary compounds in plants. It finally leads to the formation of, for example, stilbenes, flavonoids, and lignins. Ozone effects on transcript levels of the shikimate pathway were studied in leaves of European beech saplings in the greenhouse (300 nl l−1, 8 h/day, 30 days), and in leaves of adult beech trees at the Kranzberg Forest free-air ozone fumigation site (ambient and twice ambient ozone levels) between June and October 2004. Quantitative real-time RT-PCR (qRT-PCR) with RNA isolated from beech saplings showed a strong induction of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase3 (DAHPS3), 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQD/SD), 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), and chorismate mutase (CM) starting at day seven from the onset of ozone treatment. In contrast, sun leaves of adult European beech showed only a weak elevation of shikimate pathway transcripts throughout the vegetation period studied. In addition, we examined lignin content and monomeric structure in leaves of European beech saplings from the greenhouse experiment. Leaves exhibiting highest amount of ozone-dependent lesions had the highest lignin content as determined according to Klason. Moreover, with increasing leaf damage, the syringyl (S) monomer content decreased, whereas the content of guaiacyl (G) and p-hydroxyphenyl (H) units increased. These results overall suggest that the composition of lignin is affected by ozone and is already regulated at early steps of the shikimate pathway.

BACKGROUND AND AIMS Patterns and variations in concentration of carbon-based secondary compounds in plant tissues have been explained by means of different complementary and, in some cases, contradictory plant defence hypotheses for more than 20 years. These hypotheses are conceptual models which consider environmental impacts on plant internal demands. In the present study, a mathematical model is presented, which converts and integrates the concepts of the 'Growth-Differentiation Balance' hypothesis and the 'Protein Competition' model into a dynamic plant growth model, that was tested with concentration data of polyphenols in leaves of juvenile apple, beech and spruce trees. The modelling approach is part of the plant growth model PLATHO that considers simultaneously different environmental impacts on the most important physiological processes of plants. METHODS The modelling approach for plant internal resource allocation is based on a priority scheme assuming that growth processes have priority over allocation to secondary compounds and that growth-related metabolism is more strongly affected by nitrogen deficiency than defence-related secondary metabolism. KEY RESULTS It is shown that the model can reproduce the effect of nitrogen fertilization on allocation patterns in apple trees and the effects of elevated CO(2) and competition in juvenile beech and spruce trees. The analysis of model behaviour reveals that large fluctuations in plant internal availability of carbon and nitrogen are possible within a single vegetation period. Furthermore, the model displays a non-linear allocation behaviour to carbon-based secondary compounds. CONCLUSIONS The simulation results corroborate the underlying assumptions of the presented modelling approach for resource partitioning between growth-related primary metabolism and defence-related secondary metabolism. Thus, the dynamical modelling approach, which considers variable source and sink strengths of plant internal resources within different phenological growth stages, presents a successful translation of existing concepts into a dynamic mathematical model.

The major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles are flavonol 3-o-glycosides (F3Gs) esterified with hydroxycinnamic acids at positions 3" and 6". Acylation is the last step in biosynthesis and is catalysed by position-specific hydroxycinnamoyl transferases (3" and 6"HCT). The UV-B dependence of these enzyme activities was studied in primary needles of Scots pine seedlings grown under different UV-B conditions in environmentally controlled sun simulators. 6"HCT activity was induced upon UV-B irradiation while 3"HCT activity was not induced but showed high constitutive values. To investigate the biosynthesis of diacylated F3Gs during needle development under natural conditions, the HCT activities and metabolite contents were analysed in needles of field-grown mature pine trees. Accumulation of diacylated compounds as well as of 6"HCT activity occurred transiently in the first year of needle development only. In contrast, 3"HCT activity exhibited broad maxima in two consecutive years during needle growth. The data suggest that acylated F3Gs are first formed as soluble compounds which are then translocated into the cell wall to be bound by their hydroxycinnamoyl residues.

Flavonol 3-O-glucosides esterified with ferulic or p-coumaric acid at positions 3'' and 6'' are the major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles. The last steps in the biosynthesis of these compounds are catalyzed by enzymes that transfer the acyl part of hydroxycinnamic acid CoA esters to flavonol 3-O-glucosides. A newly developed enzyme assay revealed three flavonol 3-O-glucoside hydroxycinnamoyltransferases (HCTs) in Scots pine needles with specificities for positions 3'', 4'' or 6''. The positions of the acyl groups were identified by cochromatography with reference compounds and by NMR spectroscopy. The enzymes were characterized by molecular mass, isoelectric point, and also pH and temperature optima. Substrate specificities for flavonol glycosides and hydroxycinnamic acid CoA esters as well as kinetic properties of 3''- and 6''HCT suggested that acylation preferably occurs with glucosides and p-coumaroyl-CoA. In addition, acylation takes place in a well-defined order, beginning at position 6'' followed by acylation at position 3''. These results give the first detailed characterization of flavonol 3-O-glycoside HCTs involved in the protection of plant tissues against UV-B (280-315 nm) radiation.

Only a few studies have investigated use of the Dental Anxiety Scale in dental fear-induced neuroendocrine changes. The present study examined 19 female patients, each at two timepoints across an educational and a treatment session within a periodontitis therapy. Subjective measures included a visual analogue scale, the STAI State scale, and the Dental Anxiety Scale. Salivary cortisol was measured in parallel across all four timepoints. Although patients were significantly more aroused and anxious prior to the treatment session, salivary cortisol remained unchanged. However, patients with high Dental Anxiety were significantly more aroused and anxious and showed significantly higher salivary cortisol during the educational session than those with low scores on Dental Anxiety. In conclusion, the Dental Anxiety Scale differentiated mean neuroendocrine change between patients scoring low and high for Dental Anxiety and its use as an accurate tool to identify patients with high dental anxiety should be further studied predictively.

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.

Concentrations of soluble phenolics and lignin in the phloem of ponderosa pines inoculated with the pathogen Heterobasidion annosum were assessed over a period of 2 years in a 35-year-old plantation in northern California, USA. The major effect of the pathogen on phloem-soluble phenolics consisted of a significant accumulation of ferulicacid glucoside: 503 ± 27 μg/g fresh weight (FW), compared with 366 ± 26 μg/g FW for mock-treated and 386 ± 27 μg/g FW for control trees. Lignin content was negatively correlated with ferulic acid glucoside concentration, and there was an indication of lignin reduction in the cell walls of inoculated trees. Lignin had a negative effect on the in vitro growth of two common bark beetle fungal associates, Ceratocystiopsis brevicomi and Ophiostoma minus. For this reason it, is hypothesized that lower lignification may facilitate the growth of beetle-associated fungi, resulting in greater susceptibility of the presymptomatic host to bark beetle colonization.

Mesembryanthemum crystallinum L. (Aizoaceae) is a drought- and salt-tolerant halophyte that is able to endure harsh environmental conditions. Upon irradiation with high light irradiance (1200‐1500 µ mol m − 2 s − 1 ) it displays a rapid cell-specific accumulation of plant secondary metabolites in the upper leaf epidermis; a phenomenon that is not detectable with salt or drought treatment. The accumulation of these compounds, the betacyanins and acylated flavonol glycosides, increases if the plants are exposed to polychromatic radiation with a progressively decreasing short-wave cut-off in the ultraviolet range. The response is localized in the epidermal bladder cells on the tips of young leaves and epidermal layers of fully expanded leaves. It is demonstrated that the accumulation of flavonols and betacyanins can be described by a weakly sigmoid dose function in combination with an exponential decrease of the response function of the plant with increasing wavelength.

Polar auxin transport (PAT) plays key roles in the regulation of plant growth and development. Flavonoids have been implicated in the inhibition of PAT. However, the active flavonoid derivative(s) involved in this process in vivo has not yet been identified. Here, we provide evidence that a specific flavonol bis-glycoside is correlated with shorter plant stature and reduced PAT. Specific flavonoid-biosynthetic or flavonoid-glycosylating steps were genetically blocked in Arabidopsis thaliana. The differential flavonol patterns established were analyzed by high-performance liquid chromatography (HPLC) and related to altered plant stature. PAT was monitored in stem segments using a radioactive [(3)H]-indole-3-acetic acid tracer. The flavonoid 3-O-glucosyltransferase mutant ugt78d2 exhibited a dwarf stature in addition to its altered flavonol glycoside pattern. This was accompanied by reduced PAT in ugt78d2 shoots. The ugt78d2-dependent growth defects were flavonoid dependent, as they were rescued by genetic blocking of flavonoid biosynthesis. Phenotypic and metabolic analyses of a series of mutants defective at various steps of flavonoid formation narrowed down the potentially active moiety to kaempferol 3-O-rhamnoside-7-O-rhamnoside. Moreover, the level of this compound was negatively correlated with basipetal auxin transport. These results indicate that kaempferol 3-O-rhamnoside-7-O-rhamnoside acts as an endogenous PAT inhibitor in Arabidopsis shoots.

BACKGROUND: Pollen of common ragweed (Ambrosia artemisiifolia) is a main cause of allergic diseases in Northern America. The weed has recently become spreading as a neophyte in Europe, while climate change may also affect the growth of the plant and additionally may also influence pollen allergenicity. To gain better insight in the molecular mechanisms in the development of ragweed pollen and its allergenic proteins under global change scenarios, we generated SuperSAGE libraries to identify differentially expressed transcripts. RESULTS: Ragweed plants were grown in a greenhouse under 380ppm CO2 and under elevated level of CO2 (700ppm). In addition, drought experiments under both CO2 concentrations were performed. The pollen viability was not altered under elevated CO2, whereas drought stress decreased its viability. Increased levels of individual flavonoid metabolites were found under elevated CO2 and/or drought. Total RNA was isolated from ragweed pollen, exposed to the four mentioned scenarios and four SuperSAGE libraries were constructed. The library dataset included 236,942 unique sequences, showing overlapping as well as clear differently expressed sequence tags (ESTs). The analysis targeted ESTs known in Ambrosia, as well as in pollen of other plants. Among the identified ESTs, those encoding allergenic ragweed proteins (Amb a) increased under elevated CO2 and drought stress. In addition, ESTs encoding allergenic proteins in other plants were also identified. CONCLUSIONS: The analysis of changes in the transcriptome of ragweed pollen upon CO2 and drought stress using SuperSAGE indicates that under global change scenarios the pollen transcriptome was altered, and impacts the allergenic potential of ragweed pollen.

Changes in the patterns of gene expression were analyzed in ultraviolet-B (UV-B; 280–320 nm) -treated seedlings of European beech by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) of mRNA. Utilizing 12 different downstream primers and 27 degenerated 10-mer upstream primers 50 induced and 29 repressed cDNA bands were detected. Nineteen bands were subcloned and sequenced. Two clones revealed significant homologies to known database sequences: putative nascent polypeptide-associated complex alpha and 60S ribosomal protein L37. However, only the latter was confirmed to be UV-B-induced by northern blot analysis.

Increased levels of both ozone and ultraviolet-B radiation (UV-B) are typical for high-altitude sites. Here we report on interactive effects of both stresses on Scots pine (Pinus sylvestris L.). Scots pine seedlings were exposed in a phytotron to a simulated outdoor climate. To assess effects of ozone and of UV-B radiation on the plants, ozone was added at ambient or at twice-ambient levels, and UV-B was eliminated or added at a low ambient level. Visible needle damage became apparent at enhanced ozone concentrations after 10 d of exposure and increased continuously. The accumulated exposure over a threshold value of 40 nL L−1 ozone (AOT40) revealed that an ozone exposure dose of 10 μL L−1 h was necessary for the development of needle injury, regardless of the UV-B conditions. Cinnamyl alcohol dehydrogenase (CAD) and stilbene synthase (STS) have been reported to be induced by ozone. The mRNAs of these stress-related enzymes were induced only under twice-ambient ozone concentrations. Ozone-induced transient STS transcript levels reached their maximal values between day 1 and day 5, and were more pronounced in the presence of UV-B. Similarly, pinosylvin, as well as pinosylvin methyl ether contents, showed moderate transient increases under these conditions. In contrast, CAD mRNA content and CAD enzyme activity increased continuously under enhanced ozone concentrations. Additional UV-B resulted in suppressed transcript level and enzymatic activity of CAD. The data indicate that in conifer needles, at the level of gene expression, there is an interaction between responses to UV-B and ozone.

Controlled inoculation of spruce seedling needle crowns and of shoots of 4-year-old spruce trees by Sirococcus conigenus led to disease symptoms (discoloration and necrosis) and to the induction of phenolic metabolites. Even upon complete infection, as proved by re-isolation of the pathogen from inoculated seedlings, only 40% of the plants developed visible disease symptoms after 38 days. A Sirococcus-specific polymerase chain reaction (PCR) primer pair, SIRO1 and SIRO6, was designed based on sequences of a RAPD fragment. The primer pair permitted the detection of 1 pg fungal DNA (10–40 genomes) in 1 mg fresh weight spruce tissues (needles, bark, wood), regardless of visible disease symptoms. The amounts of the major phenolic compound of spruce needles, catechin, increased significantly in all of the five spruce provenances as a response to inoculation with Sirococcus. The second major phenolic compound, picein, increased in three of the provenances, whereas the remaining two had high concentrations to begin with and showed no reaction. Minor phenolic compounds increased in response to infection regardless of provenance. In a preliminary field study, Sirococcus infection of spruce was detectable by PCR even in the presence of massive infection by other fungi, such as Rhizospaera spp. and Lophodermium spp.

Long-term depletion of the stratospheric ozone layer contributes to an increase in terrestrial solar ultraviolet-B radiation$^{1,2,3}$. This has deleterious effects on living organisms, such as DNA damage$^{4,5}$. 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$^{6}$. 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$^{7,8}$. 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.

A protocol is described for rapid RNA isolation from various plant species and tissues rich in polyphenolics and polysaccharides. The method is based on the Nucleon PhytoPure™ system without the use of phenol. The procedure can be completed within 1 h and many samples can be processed at the same time. The yield ranged from 240 μg up to 3 mg per gram of tissue with an average purity measured as A260/280 of 1.85. The RNA was of sufficient quality for use in RT-PCR reactions. Quantitation of single-stranded cDNA was carried out with the RiboGreen™ reagent and of PCR products with the PicoGreen™ reagent.

Elevated levels of both ozone and UV-B radiation are typical for high-altitude sites. Few studies have investigated their possible interaction on plants. This study reports interactive effects of O3 and UV-B radiation in four-year-old Norway spruce and Scots pine trees. The trees were cultivated in controlled environmental facilities under simulated climatic conditions recorded on Mt Wank, an Alpine mountain in Bavaria, and were exposed for one growing season to simulated ambient or twice-ambient ozone regimes at either near ambient or near zero UV-B radiation levels. Chlorotic mottling and yellowing of current year needles became obvious under twice-ambient O3 in both species at the onset of a high ozone episode in July. Development of chlorotic mottling in relation to accumulated ozone concentrations over a threshold of 40 nL L–1 was more pronounced with near zero rather than ambient UV-B radiation levels. In Norway spruce, photosynthetic parameters at ambient CO2 concentration, measured at the end of the experiment, were reduced in trees cultivated under twice-ambient O3, irrespective of the UV-B treatment. Effects on photosynthetic capacity and carboxylation efficiency were restricted to trees exposed to near zero levels of UV-B radiation, and twice-ambient O3. The data indicate that UV-B radiation, applied together with O3, ameliorates the detrimental effects of O3. The data also demonstrate that foliar symptoms develop more rapidly in Scots pine than in Norway spruce at higher accumulated ozone concentrations. Symbols and abbreviations: LSD, least significant difference; PAS300, UV-B irradiance weighted according to the plant action spectrum of Green et al. (1974) normalized at 300 (nm); AOT40, (AOT = accumulated over threshold) reflects the sum of hourly ozone concentrations above 40 nL L–1 during daylight hours (> 50 Wm–2) ( Karenlampi & Skarby 1996); A350, net photosynthesis at ambient CO2; G350, stomatal conductance for water vapour at ambient CO2; A2500, net photosynthesis at saturating CO2 (maximal potential photosynthetic activity); CE, carboxylation efficiency; ROS, reactive oxygen species; RuBP, ribulose 1,5-bisphosphate; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; GLM, general linear model.

Conifer needles are highly effective in screening ultraviolet- B radiation (280-320 nm). This ability is mainly attributed to the presence of flavonoids and hydroxycinnamic acids in the epidermal tissue. In two field cabinet experiments with two different clones of Norway spruce we assessed the seasonal accumulation of UV-B screening pigments under near-ambient, and close-to-zero UV-B irradiation. At the beginning of needle development, i. e. in June, kaempferol 3-O-glucoside was the dominant UV-B screening pigment. It was replaced during needle differentiation by the more effective diacylated flavonol glucosides, particulary kaempferol 3-O-(3", 6"-O-di-p-coumaroyl)- glucoside, which reached highest concentrations in July. In addition to the soluble pool of diacylated flavonol glucoside derivatives, a cell wall-bound UV-B screen in the epidermal cell walls was formed during needle differentiation, consisting mainly of p-coumaric acid and kaempferol 3-O- glucoside. An effect of UV-B radiation on the accumulation of diacylated flavonol glucosides was only observed in 1996 with clone 2, when the concentrations of kaempferol 3-O-( 3", 6"-O-di-p-coumaroyl) glucoside were significantly higher in July and August under field, and near-ambient than under close-to-zero UV-B irradiance. For wall-bound p-coumaric acid and kaempferol 3-O- glucoside UV-B radiation enhanced the concentrations of these compounds by approximately 20% in relation to the concentrations in close-to-zero UV-B-treated plants in both field cabinet experiments.

The new sesquiterpenoid 8-deoxy-15-(3′-hydroxy-2′-methyl-propanoyl)-lactucin 3′-sulfate (1) was isolated from the methanolic extract of roots of Reichardia gaditana L. The compound was isolated by silica gel column chromatography (CC) and repeated Sephadex LH-20 CC. Structure elucidation was accomplished by high-resolution mass spectrometry and by 1D- and 2D-NMR spectroscopy. The chemosystematic significance of the new compound is discussed in the context of sesquiterpenoids from other members of the Lactuceae tribe of the Asteraceae family.

Phanerochaete chrysosporium ATCC 34541 has been reported to be unable to mineralize 3,4-dichloroaniline (DCA). However, high mineralization is now shown to occur when a fermentation temperature of 37° and gassing with oxygen are used. Mineralization did not correlate with lignin peroxidase activity. The latter was high under C limitation and low under N limitation, whereas the reverse was true for mineralization. The kinetics of DCA metabolism was studied in low-N and low-C and C- and N-rich culture media by metabolite analysis and 14 CO 2 determination. In all cases, DCA disappeared within 2 days, and a novel highly polar conjugate termed DCAX accumulated in the growth medium. This metabolite was a dead-end product under C and N enrichment. In oxygenated low-C medium and in much higher yield in oxygenated low-N medium, DCAX was converted to DCA-succinimide and then mineralized. DCAX was purified by high-performance liquid chromatography and identified as N -(3,4-dichlorophenyl)-α-ketoglutaryl-δ-amide by high-performance liquid chromatography and mass spectroscopy, gas chromatography and mass spectroscopy, and nuclear magnetic resonance spectroscopy. The formation of conjugate intermediates is proposed to facilitate mineralization because the sensitive amino group of DCA needs protection so that ring cleavage rather than oligomerization can occur.

The air pollutant ozone has recently been found to resemble fungal elicitors — it can induce plant signal molecules such as ethylene and salicylic acid, as well as certain genes and biosynthetic pathways associated with pathogen and oxidative defence. The action of ambient ozone on the plant defence system may predispose the plant to enhanced attack by pathogens, but may also lead to induced resistance. The results mean that ozone can be regarded as a new experimental tool for analyzing stress responses.

Two enzyme assays using either radiolabelled or non-labelled substrates were applied to detect dihydroflavonol 4-reductase (DFR) activity in lignocellulosic tissues. The successful detection of DFR in these tissues rich in polyphenols relied on the addition of sufficient amounts of polyethylene glycol. DFR activity is measured in sapwood and inner bark of different conifer species. © 1997 Elsevier Science Ltd. All rights reserved

The indirect defences of plants are comprised of herbivore-induced plant volatiles (HIPVs) that among other things attract the natural enemies of insects. However, the actual extent of the benefits of HIPV emissions in complex co-evolved plant-herbivore systems is only poorly understood. The observation that a few Quercus robur L. trees constantly tolerated (T-oaks) infestation by a major pest of oaks (Tortrix viridana L.), compared with heavily defoliated trees (susceptible: S-oaks), lead us to a combined biochemical and behavioural study. We used these evidently different phenotypes to analyse whether the resistance of T-oaks to the herbivore was dependent on the amount and scent of HIPVs and/or differences in non-volatile polyphenolic leaf constituents (as quercetin-, kaempferol- and flavonol glycosides). In addition to non-volatile metabolic differences, typically defensive HIPV emissions differed between S-oaks and T-oaks. Female moths were attracted by the blend of HIPVs from S-oaks, showing significantly higher amounts of (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E)-β-ocimene and avoid T-oaks with relative high fraction of the sesquiterpenes α-farnesene and germacrene D. Hence, the strategy of T-oaks exhibiting directly herbivore-repellent HIPV emissions instead of high emissions of predator-attracting HIPVs of the S-oaks appears to be the better mechanism for avoiding defoliation.

Treatment of Pinus sylvestris L. cell-suspension cultures with an elicitor preparation from the pine needle pathogen Lophodermium seditiosum, resulted in a severalhundredto thousandfold accumulation of the stilbenes pinosylvin and pinosylvin 3-O-methyl ether in methanolic cell extracts. There was a simultaneous induction of the biosynthetic enzymes phenylalanine ammonia-lyase (E.C. 4.3.1.5.) and stilbene synthase (pinosylvin-forming, E.C. 2.3.1.146). For the first time, an incorporation of stilbenes into the cell wall fraction as well as stilbene excretion into the extracellular space was demonstrated in addition to intracellular accumulation.

The major flavone glycoside in parsley leaves has been identified as 6″- O -malonylapiin. This compound as well as apiin were induced approximately

SUMMARY Interactions between ozone and biotic stress caused by a pathogen, Heterobasidion annosum (Fr.) Bref., in mycorrhizal [Hegeloma crustuliniforme (Bull, ex St. Am.) Quel.] and non-mycorrhizal Scots pine seedlings were investigated using a semi-axenic model system. Ozone exposure (200 nl 1−1, 8 h d−1 for 28 d) increased disease incidence significantly, but mycorrhizal infection completely prevented this negative effect. The presence of the pathogen on the root systems was necessary for the induction of changes in the soluble and wall-bound secondary compounds of roots and needles; ozone alone did not induce such changes. Mycorrhizal infection appeared to have a dampening effect on the induction of these compounds. H. annosum induced a significant accumulation of the two pine stilbenes both locally and systemically in the more susceptible seedlings. In these seedlings ozone had a significant positive effect on the accumulation of both stilbenes in the roots, but it reduced pinosylvin and had no effect on pinosylvin 3-methyl ether in the needles. The catechin content of the roots decreased in the same infected seedlings, but to a larger degree upon ozone treatment. One compound of as yet unknown structure accumulated gradually in the infected roots over the experimental period, and could thus be associated with resistance. Its accumulation was little affected by ozone treatment. Among the root cell wall-bound phenolics analyzed, only lignin-like material showed significant changes. The presence of the pathogen was again necessary for induction, but ozone had an inhibitory effect on this response. Pure pinosylvin applied through the hypocotyls of excised seedlings was shown to be phytotoxic, with the needles displaying discoloration and wilting as observed after pathogenic inoculation, and being characterized by a lower chlorophyll content and increased transpiration. Accumulation of pinosylvin in the needles was detected at amounts comparable to those found in the main experiment.

Spruce needle cinnamyl alcohol dehydrogenase (CAD) was induced in seedlings and four-year-old trees two-to-four-fold by ozone concentrations that did not lead to visible damage. CAD thus acted as an early biochemical ozone marker. A highly purified CAD preparation consisting of two ∼42 000 M r subunits was prepared from spruce cell cultures. A polyclonal antibody was strongly inhibitory. The enzyme had high specificity for coniferyl alcohol and NADP + , and was also detected in spruce seedlings and spruce cambial sap.

Plants respond to low levels of UV-B radiation with a coordinated photomorphogenic response that allows acclimation to this environmental stress factor. The key players in this UV-B response are COP1 (an E3 ubiquitin ligase), UVR8 (a β-propeller protein), and HY5 (a bZIP transcription factor). We have shown previously that an elevated UV-B–specific response is associated with dwarf growth, indicating the importance of balancing UV-B–specific signaling. Negative regulators of this pathway are not known, however. Here, we describe two highly related WD40-repeat proteins, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2, that interact directly with UVR8 as potent repressors of UV-B signaling. Both genes were transcriptionally activated by UV-B in a COP1-, UVR8-, and HY5-dependent manner. rup1 rup2 double mutants showed an enhanced response to UV-B and elevated UV-B tolerance after acclimation. Overexpression of RUP2 resulted in reduced UV-B–induced photomorphogenesis and impaired acclimation, leading to hypersensitivity to UV-B stress. These results are consistent with an important regulatory role for RUP1 and RUP2, which act downstream of UVR8–COP1 in a negative feedback loop impinging on UVR8 function, balancing UV-B defense measures and plant growth.

Long-term effects of ultraviolet (UV) radiation on flavonoid biosynthesis were investigated in Arabidopsis thaliana using the sun simulators of the Helmholtz Zentrum München. The plants, which are widely used as a model system, were grown (1) at high photosynthetically active radiation (PAR; 1,310 micromol m(-2) s(-1)) and high biologically effective UV irradiation (UV-B(BE) 180 mW m(-2)) during a whole vegetative growth period. Under this irradiation regime, the levels of quercetin products were distinctively elevated with increasing UV-B irradiance. (2) Cultivation at high PAR (1,270 micromol m(-2) s(-1)) and low UV-B (UV-B(BE) 25 mW m(-2)) resulted in somewhat lower levels of quercetin products compared to the high-UV-B(BE) conditions, and only a slight increase with increasing UV-B irradiance was observed. On the other hand, when the plants were grown (3) at low PAR (540 micromol m(-2) s(-1)) and high UV-B (UV-B(BE) 180 mW m(-2)), the accumulation of quercetin products strongly increased from very low levels with increasing amounts of UV-B but the accumulation of kaempferol derivatives and sinapoyl glucose was less pronounced. We conclude (4) that the accumulation of quercetin products triggered by PAR leads to a basic UV protection that is further increased by UV-B radiation. Based on our data, (5) a combined effect of PAR and different spectral sections of UV radiation is satisfactorily described by a biological weighting function, which again emphasizes the additional role of UV-A (315-400 nm) in UV action on A. thaliana.

Polyamine metabolism was examined in tobacco (Nicotiana tabacum L.) exposed to a single ozone treatment (5 or 7 hours) and then postcultivated in pollutant-free air. The levels of free and conjugated putrescine were rapidly increased in the ozone-tolerant cultivar Bel B and remained high for 3 days. This accumulation was preceded by a transient rise of l-arginine decar-boxylase (ADC, EC 4.1.1.19) activity. The ozone-sensitive cultivar Bel W3 showed a rapid production of ethylene and high levels of 1-aminocyclopropane-1-carboxylic acid after 1 to 2 hours of exposure. Induction of putrescine levels and ADC activity was weak in this cultivar and was observed when necrotic lesions developed. Leaf injury occurred in both lines when the molar ratio of putrescine to 1-aminocyclopropane-1-carboxylic acid or ethylene fell short of a certain threshold value. Monocaffeoyl-putrescine, an effective scavenger for oxyradicals, was detected in the apo-plastic fluid of the leaves of cv Bel B and increased upon exposure to ozone. This extracellular localization could allow scavenging of ozone-derived oxyradicals at the first site of their generation. Induction of either polyamine or ethylene pathways may represent a control mechanism for inhibition or promotion of lesion formation and thereby contribute to the disposition of plants for ozone tolerance.

Plants in alpine habitats are exposed to many environmental stresses, in particular temperature and radiation extremes. Recent field experiments on Arnica montana L. cv. ARBO indicated pronounced altitudinal variation in plant phenolics. Ortho-diphenolics increased with altitude compared to other phenolic compounds, resulting in an increase in antioxidative capacity of the tissues involved. Factors causing these variations were investigated by climate chamber (CC) experiments focusing on temperature and ultraviolet (UV)-B radiation. Plants of A. montana L. cv. ARBO were grown in CCs under realistic climatic and radiation regimes. Key factors temperature and UV-B radiation were altered between different groups of plants. Subsequently, flowering heads were analyzed by HPLC for their contents of flavonoids and caffeic acid derivatives. Surprisingly, increased UV-B radiation did not trigger any change in phenolic metabolites in Arnica. In contrast, a pronounced increase in the ratio of B-ring ortho-diphenolic (quercetin) compared to B-ring monophenolic (kaempferol) flavonols resulted from a decrease in temperature by 5 degrees C in the applied climate regime. In conclusion, enhanced UV-B radiation is probably not the key factor triggering shifts in the phenolic composition in Arnica grown at higher altitudes but rather temperature, which decreases with altitude.

There is limited information on the impact of present-day ultraviolet-B (UV-B) radiation on a reprogramming of gene expression in crops. Summer wheat was cultivated in controlled environmental facilities under simulated realistic climatic conditions. We investigated the effect of different regimes of UV-B radiation on summer wheat (Triticum aestivum L.) cultivars Nandu, Star and Turbo. Until recently, these were most important in Bavaria. Different cultivars of crops often show great differences in their sensitivity towards UV-B radiation. To identify genes that might be involved in UV-B defence mechanisms, we first analyzed selected genes known to be involved in plant defence mechanisms. RNA gel blot analysis of RNA isolated from the flag leaf of 84-day-old plants showed differences in transcript levels among the cultivars. Flag leaves are known to be important for grain development, which was completed at 84 days post-anthesis. Catalase 2 (Cat2) transcripts were elevated by increased UV irradiation in all cultivars with highest levels in cv. Nandu. Pathogenesis-related protein 1 (PR1) transcripts were elevated only in cv. Star. A minor influence on transcripts for phenylalanine ammonia-lyase (PAL) was observed in all three cultivars. This indicates different levels of acclimation to UV-B radiation in the wheat cultivars studied. To analyze these responses in more detail, UV-B-exposed flag leaves of 84-day-old wheat (cv. Nandu) were pooled to isolate cDNAs of induced genes by suppression-subtractive hybridization (SSH). Among the initially isolated cDNA clones, 13 were verified by RNA gel blot analysis showing an up-regulation at elevated levels of UV-B radiation. Functional classification revealed genes encoding proteins associated with protein assembly, chaperonins, programmed cell death and signal transduction. We also studied growth, flowering time, ear development and yield as more typical agricultural parameters. Plant growth of young plants was reduced at increased UV-B radiation. Flowering and ear development were delayed concomitantly, whereas total grain weight was not influenced at any of the UV-B irradiation regimes.

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.

Plant growth largely depends on microbial community structure and function in the rhizosphere. In turn, microbial communities in the rhizosphere rely on carbohydrates provided by the host plant. This paper presents the first study on ozone effects in the plant-rhizosphere-bulk soil system of 4-year-old beech trees using outdoor lysimeters as a research platform. The lysimeters were filled with homogenized soil from the corresponding horizons of a forest site, thus minimizing field heterogeneity. Four lysimeters were treated with ambient ozone (1 × O3) and four with double ambient ozone concentrations (2 × O3; restricted to 150 ppb). In contrast to senescence, which was almost unaffected by ozone treatment, both the photochemical quantum yield of photosystem II (PSII) and leaf gas exchange were reduced (11 - 45 %) under the elevated O3 regime. However, due to large variation between the plants, no statistically significant O3 effect was found. Even though the amount of primary metabolites, such as sugar and starch, was not influenced by elevated O3 concentrations, the reduced photosynthetic performance was reflected in leaf biochemistry in the form of a reduction in soluble phenolic metabolites. The rhizosphere microbial community also responded to the O3 treatment. Both community structure and function were affected, with a tendency towards a lower diversity and a significant reduction in the potential nutrient turnover. In contrast, litter degradation was unaffected by the fumigation, indicating that in situ microbial functionality of the bulk soil did not change.

The present study was conducted to investigate the effect of decomposition site and plant litter species on the colonizing microbial communities. For this, litter bag technique using beech and spruce litter was combined with RNA-based fingerprinting and cloning. Litter bags were incubated for 2 and 8 weeks in the Ah horizon of beech and beech-spruce mixed forest sites. Although sugars and starch were rapidly lost, lignin content increased by more than 40% for beech and more than doubled for spruce litter at both soil sites at the end of the experiment. Denaturing gradient gel electrophoresis analysis of 16S and 18S rRNA RT-PCR products was used for screening of differences between bacterial and fungal communities colonizing the two litter types. Development of the microbial community over time was observed to be specific for each litter type and decomposition site. RT-PCR products from both litter types incubated in beech-spruce mixed forest site were also cloned to identify the bacterial and fungal colonizers. The 16S rRNA clone libraries of beech litter were dominated by gamma-proteobacterial members, whereas spruce libraries were mainly composed of alpha-, beta-, and gamma-proteobacterial members. Ascomycota members dominated the 18S rRNA clone libraries. Clones similar to Zygomycota were absent from spruce, whereas those similar to Basidiomycota and Glomeromycota were absent from beech libraries. Selective effects of litter quality were observed after 8 weeks. The study provides an insight into the bacterial and fungal communities colonizing beech and spruce litter, and the importance of litter quality and decomposition site as key factors in their development and succession.