Advances of air pollution science: from forest decline to multiple-stress effects on forest ecosystem services.

Over the past 20 years, the focus of forest science on air pollution has moved from forest decline to a holistic framework of forest health, and from the effects on forest production to the ecosystem services provided by forest ecosystems. Hence, future research should focus on the interacting factorial impacts and resulting antagonistic and synergistic responses of forest trees and ecosystems. The synergistic effects of air pollution and climatic changes, in particular elevated ozone, altered nitrogen, carbon and water availability, must be key issues for research. Present evidence suggests air pollution will become increasingly harmful to forests under climate change, which requires integration amongst various stressors (abiotic and biotic factors, including competition, parasites and fire), effects on forest services (production, biodiversity protection, soil protection, sustained water balance, socio-economical relevance) and assessment approaches (research, monitoring, modeling) to be fostered.

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.

A cyclophilin gene marker confirming geographical differentiation of Norway spruce populations and indicating viability response on excess soil-born salinity.

A newly identified cyclophilin-encoding cDNA clone was used to design a codominant inherited EST-PCR marker in Norway spruce. The study of the current minor polymorphism revealed a geographically structured differentiation pattern across 17 test populations, showing a slight clinal variation south-north through Europe. Based on the frequency of alleles, isolation-by-distance analysis and the Ewens-Watterson test, we conclude that a selectively neutral random-drift mutation recently occurred within the Alpine population group, thus being responsible for the genetic variation detected. Analysis of tolerant and susceptible subsets of two adjacent Bavarian spruce populations affected by soil-born NaCl pollution, revealed that the cyclophilin marker locus also confirms biased genotype frequencies. Considering an unlinked PCR marker of a ribosomal protein-encoding EST clone, deviations between pooled tolerant subsets and pooled sensitive subsets were proven to be more significant for two-locus homozygous genotypes than for each locus alone. We suggest that both loci are linked to adaptive genomic regions. Their potential to test the feasibility of marker-assisted selection of both NaCl-tolerant and drought resistant tree populations is discussed.

Selection effects of air pollution on gene pools of Norway spruce, European silver fir and European beech.

The effects of industrial pollution on allelic and genotypic structures of Norway spruce. European silver fir and European beech were investigated by means of isozyme analysis. In a mixed Norway spruce-silver fir forest stand in an area heavily polluted by sulphur dioxide and heavy metals in the region of Spis (eastern Slovakia), pairs of neighbouring damaged and apparently healthy trees were selected in two replicates (44 and 69 pairs in a heavily and moderately damaged stand, respectively). Pairwise sampling of trees with contrasting vitality was applied to reduce potential effects of site heterogeneity on the vitality of sampled trees. No significant differences in allelic and genotypic frequencies were found between sets of healthy and declining trees. There were differences in the single-locus heterozygosities, but these were not consistent between the replicates. However, the set of damaged trees exhibited higher levels of genetic multiplicity and diversity, possibly due to the deleterious effect of rare alleles under the conditions of air pollution. Consequently. following the decline of pollutant-sensitive trees, the remaining stand will be depleted of a part of alleles with unknown adaptive value to future selection pressures.

Reproductive systems in conifer seed orchards : 3. Female and male fitnesses of individual clones realized in seeds of Pinus sylvestris L.

In order to quantify female and male fitness values of clones in a Pinus sylvestris L. seed orchard, multilocus-genotypes of parental clones were compared with those of open pollinated seeds in the bulked orchard crop. Female and male contributions to individual seeds were distinguished by observing enzyme gene loci active in both endosperm and embryo tissue. Seed probes from two successive flowering periods were surveyed. The female and male fitnesses of five parental clones measured relative to the population mean were derived. The contributions of four clones were found to be sexually asymmetric. One clone, for instance, made exclusively female contributions in one flowering period. Variations existed in fitness values between clones. Deviations in sex specificity occurred between flowering periods: one clone contributed asymmetrically in both periods, but in sexually reversed proportions. A method to comprehensively quantify and illustrate the observed phenomena is proposed.

Reproductive systems in conifer seed orchards : 2. Reproductive selection monitored at an LAP gene locus in Pinus sylvestris L.

In a Scots pine seed orchard the genetic structures at an enzyme gene locus active in pine seeds were compared among the parental clones and the allorchard seeds produced over a period of three years. The genotypes of the seeds were identified as ordered pairs consisting of the female and male contribution. The sexual reproductive function monitored at the studied locus differed significantly between the two sexes. This fact proves the necessity of taking sexual asymmetry into account in studies of reproductive selection. This is done by comparing the observed genotypic structures among the offspring with the corresponding multiplicative structure expected under random gametic fusion. Additionally, accounting for partial self-fertilization increased the conformity between the observed and the hypothesized model structures. The differences in female and male gametic contributions to the offspring were used to estimate female and male relative fitness components. Significant deviations between the allelic and/or genotypic structures of orchard clones and their seed, and between seed lots collected in different years, may reduce the efficiency of realizing breeding gains in seed orchards.

Aminopeptidases in seeds of picea abies (L.) Karst.: characterization of leucine aminopeptidase by molecular properties and inhibitors.

By either acrylamide or starch gel electrophoresis of Norway spruce (Picea abies) seed extracts, two prominent isoenzyme bands were obtained after staining for leucine aminopeptidase (LAP). These bands were proved to correspond to each other by reelectrophoresis in both gel media. Single endosperm studies with acrylamide gels showed clearly that, in addition to LAP, two bands are expressed after staining for alanine aminopeptidase (AAP) as well. Both the LAP and the AAP activities appeared together as a single peak between catalase and ferritin after gel chromatograhy on Sepharose. Isoelectric focusing in sucrose gradients proved the two LAP activities to have identical isoelectric points revealed that LAP, but not AAP, is detectable by standard starch gel electrophoretic procedures. The two LAP bands refer to approximate molecular weights of 71,000 and 131,000, respectively. Disaggregation studies did not conclusively determine whether these two bands represent different enzymes or not. only inhibitors succeeded in producing a definite differentiation by selective inhibition of one of the two bands. It is concluded that in both gel media the isoenzyme bands reflect the activity of two distinct leucine aminopeptidases.