Determination of reactive oxygen species generated in laccase catalyzed oxidation of wood fibers from Chinese fir (Cunninghamia lanceolata) by electron spin resonance spectrometry

The aim of the present study was to determine whether the radical reaction intermediates--reactive oxygen species (ROS) were formed during the laccase-catalyzed oxidation of wood fibers from Chinese fir (Cunninghamia lanceolata) and to quantify tentatively its production with electron spin resonance (ESR) spectrometry. To investigate the activation pathways triggered by laccase, ESR spin-trapping techniques using N-tert-butyl-alpha-phenylnitrone (PBN) as spin trap followed by ethyl acetate extraction were employed to identify and quantify the free radical intermediates. ROS such as the superoxide and hydroxyl radical was detected and quantified in the laccase catalyzed oxidation of wood fibers, suggesting that ROS is the main free radical intermediates for laccase reaction. Based on the findings of the presence of ROS and previous literature on the free radical reaction of laccase oxidation of wood fibers, a possible reaction mechanism involving ROS-mediated attack on the domains of lignin which is not directly accessible for the enzyme and solubilized low-molecular mass lignins which function as reactive compounds like adhesives and may cling back to the fiber surface, could accordingly describe laccase-catalyzed oxidation of Chinese fir wood fibers.     

Plant-mediated nitrous oxide emissions from beech (Fagus sylvatica) leaves

Nitrous oxide (N2O) emission estimates from forest ecosystems are based currently on emission measurements using soil enclosures. Such enclosures exclude emissions via tall plants and trees and may therefore underestimate the whole-ecosystem N2O emissions. Here, we measured plant-mediated N2O emissions from the leaves of potted beech (Fagus sylvatica) seedlings after fertilizing the soil with 15N-labelled ammonium nitrate (15NH4(15)NO3), and after exposing the roots to elevated concentrations of N2O. Ammonium nitrate fertilization induced N2O + 15N2O emissions from beech leaves. Likewise, the foliage emitted N2O after beech roots were exposed to elevated concentrations of N2O. The average N2O emissions from the fertilization and the root exposure experiments were 0.4 and 2.0 microg N m(-2) leaf area h(-1), respectively. Higher than ambient atmospheric concentrations of N2O in the leaves of the forest trees indicate a potential for canopy N2O emissions in the forest. Our experiments demonstrate the existence of a previously overlooked pathway of N2O to the atmosphere in forest ecosystems, and bring about a need to investigate the magnitude of this phenomenon at larger scales.     

Storage and remobilisation of sulphur in beech trees (Fagus sylvatica)

Three-year-old beech trees were fed ³⁵S-sulphate in August 1993 via a flap in a mature leaf of an upper branch. Harvest of beech trees was performed 24 h after feeding ³⁵S-sulphate, before leaf senescence, after leaf abscission, in early winter (January 1994). in late winter (March 1994). before bud break and after bud break. Twenty-four h after feeding ³⁵S-sulphate, 0.7 ± 0.5% of the ³⁵S-radioactivity taken up was exported out of the fed leaf. When trees were analysed 2 months later, i.e., before leaf senescence, this value had increased to 22 ± 7%. The exported ³⁵S-radioactivity was located in the branch containing the fed leaf (2.8 ± 13%). in basipetal parts of the trunk (41 ± 77%) and in the main rool (21 ± 6%). Leaves and apical parts of the trunk were no sink organs for the exported sulphur. Along the tree axis the main proportion of the radiolabel was located in the wood, predominantly in the acid soluble fraction. In the bark the greater portion of the radiolabel was found in the acid insoluble fraction. In both tissues the bulk of the ³⁵S of the soluble fraction was sulphate together with small amounts of glutathione. This pattern did not change until bud break. After bud break, basipetal parts of the trunk lost part of its ³⁵S-radioactivity. Of the ³⁵S-radioactivity which had been exported out of the fed leaf during the previous autumn, 16 ± 2% remained in the trunk, whereas 47 ± 7% of the ³⁵S was found in branches, mainly in the newly developed leaves. The present results show that sulphur, mainly in the form of sulphate, is stored along the tree axis in both bark and wood of beech trees and is re-mobilised during leaf development in spring.     

An electron spin resonance study of free radical intermediates in the oxidation of indole acetic acid by horseradish peroxidase

The oxidation of indole-3-acetic acid by horseradish peroxidase was studied using the spin traps t-nitrosobutane and 5,5-dimethyl-1-pyrroline N-oxide to trap free radical intermediates. The major free radical metabolite of indole acetic acid was unambiguously determined by the use of indole-3-[2,2-2H2]acetic acid to be the skatole carbon-centered free radical. In the presence of oxygen, superoxide was also trapped.     

Direct electron spin resonance detection of free radical intermediates during the peroxidase catalyzed oxidation of phenacetin metabolites

The oxidation of the phenacetin metabolites p-phenetidine and acetaminophen by peroxidases was investigated. Free radical intermediates from both metabolites were detected using fast-flow ESR spectroscopy. Oxidation of acetaminophen with either lactoperoxidase and hydrogen peroxide or horseradish peroxidase and hydrogen peroxide resulted in the formation of the N-acetyl-4-aminophenoxyl free radical. Totally resolved spectra were obtained and completely analyzed. The radical concentration was dependent on the square root of the enzyme concentration, indicating second-order decay of the radical, as is consistent with its dimerization or disproportionation. The horseradish peroxidase/hydrogen peroxide-catalyzed oxidation of p-phenetidine (4-ethoxyaniline) at pH 7.5-8.5 resulted in the one-electron oxidation products, the 4-ethoxyaniline cation free radical. The ESR spectra were well resolved and could be unambiguously assigned. Again, the enzyme dependence of the radical concentration indicated a second-order decay. The ESR spectrum of the conjugate base of the 4-ethoxyaniline cation radical, the neutral 4-ethoxyphenazyl free radical, was obtained at pH 11-12 by the oxidation of p-phenetidine with potassium permanganate.     

Strip-cankering of beech (Fagus sylvatica): Pathology and distribution of symptomatic trees

The pathology and distribution of European beech trees bearing elongated bark lesions (strip-cankers) were investigated. Two types of canker were recognized: those on small trees (<40 cm diameter at breast height (dbh): 1·4 m above ground level) which bore fruit bodies of the xylariaceous ascomycete Biscogniauxia nummularia (Bull.) O. Kuntze, and those on larger specimens (>40 cm dbh) which were consistently associated with the diatrypaceous ascomycete Eutypa spinosa (Pers.) Tul. & C. Tul. All cankers were strictly annual, having formed during single growing seasons following periods of low water availability. The regional and local distribution of trees bearing lesions also appeared to be correlated with environmental conditions, being most severe where low rainfall or high temperatures had occurred. Population studies of the associated fungi, generally considered as saprotrophs, indicated the presence of unique genotypes within individual cankered trees and provided no evidence for the existence of pathotypes within either species. Within the decay columns which underlay canker surfaces, both B. nummularia and E. spinosa formed longitudinally extensive genets, implying non-mycelial spread in colonization. Suppression of both inter- and intraspecific incompatibility between fungi occurred in regions of canker decay columns with elevated water contents. The possible significance of coexistence between the ascomycetes B. nummularia and ' Hypoxylon purpureum ' ( sensu Sharland & Rayner, 1989 b ) and of the formation of heterokaryons by E. spinosa is discussed.     

Simulation study to determine necessary sample sizes for image analysis-based quantitative wood anatomy of vessels of beech (Fagus sylvatica)

Quantitative analysis of wood anatomy is a powerful dendroecological tool to provide new insights into environmental signals encoded in tree-rings. Nevertheless, studies with long time series or large sample sizes are not very common for diffuse-porous tree species due to the laborious sample preparation and image analysis. Finding a compromise between sample size and accuracy of sample estimators is therefore of crucial importance. In this simulation study, we present a subsampling method, which can make the quantitative analysis of wood anatomy of European beech more efficient. Based on our study material we demonstrate that for the parameters vessel size, vessel density, total vessel area and accumulated hydraulic conductivity the environmental signals are captured with high trueness (deviation < 5%) when analysing only a 1 mm wide radial stripe along the increment core. Nevertheless, when information about vessel grouping indices is required, for high trueness of the results a wider radial stripe of ∼2.2 mm needs to be analysed.     

Dormancy release in beech buds (Fagus sylvatica) requires both chilling and long days

Chilling and daylength requirements for dormancy release and budburst in dormant beech ( Fagus sylvatica L.) buds have been studied in cuttings flushing in controlled environments after different durations of outdoor chilling. Non-chilled buds sampled in mid October required long days (LD) only for budburst. Buds chilled until March still required LD for normal budburst, whereas buds sampled in November and December were unable to sprout regardless of daylength conditions and would do so only after a substantial period of chilling. Four ecotypes of distant latitudinal and altitudinal origin responded very similarly with a typical quantitative photoperiodic response. In fully chilled shoots sampled in March only 13 to 40% budburst took place in 8-h SD and only after three times as long time as in continuous light. It is concluded that this dual dormancy control system ensures optimum winter stability in trees under conditions of climatic warming. In the closely related Carpinus betulus L. budburst was unaffected by daylength.     

Influence of aluminium on phosphorus and calcium localization in roots of beech (Fagus sylvatica)

Beech plants ( Fagus sylvatica L. provenance Maramures) were grown in nutrient solution at low pH (4.2) and exposed to different concentrations of AlCl₃. Uptake and leakage of Ca²⁺(⁴⁵Ca²⁺) and H²PO₄-(³²P) were studied. A high external aluminium concentration (1.0m M ) reduced the uptake and export to the shoot of both calcium and phosphate, while 0.1 m M Al increased the phosphorus level in the roots. To determine the impact of aluminium on the localization of calcium and phosphate, leakage of the elements from both intact plants and plants frozen prior to the leakage experiment was studied. The leakage of Ca²⁺ from intact plants was not affected by prior exposure to 0.1 m M Al. Freezing of the beech plants before the leakage experiment increased leakage of calcium slightly more from roots of control plants than for roots exposed to 0.1 m M Al, indicating that even low concentrations of alminium may impede the influx of calcium across the plasma membrane in the roots. The patterns of Ca²⁺ leakage from roots previously exposed to 1.0 m M Al indicated that very little Ca²⁺ was located extracellularly. The extracellular fraction of phosphate increased with increasing Al concentration in the nutrient solution. Low Al concentration (0.1 m M ) only reduced the intracellular phosphate concentration to a minor extent, while 1.0 m M Al profoundly decreased it. It is concluded that 0.1 m M AlCl₃ has a limited effect upon the localization of Ca²⁺ and phosphate in the roots. At higher levels of Al, 0.1–1.0 m M , there is a more dramatic change in nutrient localization in the free space and uptake over the plasma membrane.     

Displacement of frequently occurring heacy metals in autumn leaves of beech (Fagus sylvatica)

Summary By utilizing energy-dispersive X-ray microanalysis the heavy metals manganese, iron, zinc and lead were determined in different tissues of beech (Fagus sylvatica L.) autumn leaves. It was observed that all four metals were accumulated in sieve element-companion cell complexes of the leaf veins in the period from the green (2 October) to the brown (23 October) leaf coloration. Concomitantly, the concentration of these metals decreased in certain tissues of the stems subtending the leaves. The major reservoirs of heavy metals in the stems were periderm, cortex, pith and xylem rays. Since sieve element-companion cell complexes of the stems showed increasing metal concentrations during the transition from the green to the brown leaf stage, and since this temporary increase also occurred in the petiole phloem, it is inferred that symplastic transport can be used in addition to apoplastic transport for the displacement of heavy metals. It is assumed that the accumulation of heavy metals in the veins of autumn leaves indicates the deposition of surplus ions which are excluded when leaves are cast off.Supported by Deutsche Forschungsgemeinschaft and by Bundesministerium für Forschung und Technologie, Federal Republic of Germany, Grant PBE 373607.     

Extraordinary drought of 2003 overrules ozone impact on adult beech trees (Fagus sylvatica)

The extraordinary drought during the summer of 2003 in Central Europe allowed to examine responses of adult beech trees (Fagus sylvatica) to co-occurring stress by soil moisture deficit and elevated O₃ levels under forest conditions in southern Germany. The study comprised tree exposure to the ambient O₃ regime at the site and to a twice-ambient O₃ regime as released into the canopy through a free-air O₃ fumigation system. Annual courses of photosynthesis (A _max), stomatal conductance (g _s), electron transport rate (ETR) and chlorophyll levels were compared between 2003 and 2004, the latter year representing the humid long-term climate at the site. ETR, A _max and g _s were lowered during 2003 by drought rather than ozone, whereas chlorophyll levels did not differ between the years. Radial stem increment was reduced in 2003 by drought but fully recovered during the subsequent, humid year. Comparison of AOT40, an O₃ exposure-based risk index of O₃ stress, and cumulative ozone uptake (COU) yielded a linear relationship throughout humid growth conditions, but a changing slope during 2003. Our findings support the hypothesis that drought protects plants from O₃ injury by stomatal closure, which restricts O₃ influx into leaves and decouples COU from high external ozone levels. High AOT40 erroneously suggested high O₃ risk under drought. Enhanced ozone levels did not aggravate drought effects in leaves and stem.     

Scavenging effect of Indian grape polyphenols on 2,2'-diphenyl-1-picrylhydrazyl(DPPH) radical by electron spin resonance spectrometry

Antioxidant potency of Indian grape cultivars varying in their skin color, seed and polyphenol content (Bangalore blue, Pandhari sahebi, Sharad seedless and Thompson seedless) and their components (whole grapes, pulp with skin and seeds) was examined as 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity using electron spin resonance spectrometry. The total polyphenols in Indian grapes ranged between 3-51%. Extracted polyphenols caused a concentration dependent and significant loss in DPPH radical signal, similar to known antioxidants-Vitamin C, catechin and procyanidin B3 used as references. Among seedless cultivars, polyphenols from Sharad was more potent as antioxidant than Thompson, showing IC50 values of 1250 +/- 30 and 2650 +/- 125 microg/ml, respectively. The inhibitory effect of polyphenols from seedless grape cultivars was as effective as that of seeded variety. The results indicate that polyphenols extracted from Indian grapes/ components (with /without seeds) exhibited free radical scavenging activity and their chemopreventive properties need to be exploited by in vivo model system.     

Seasonal variation in ammonia compensation point and nitrogen pools in beech leaves (Fagus sylvatica)

Ammonia (NH₃) fluxes between beech leaves (Fagus sylvatica) and the atmosphere were investigated in a 90-year-old forest canopy and related to leaf nitrogen (N) pools and glutamine synthetase (GS) activities. The stomatal ammonia compensation point, ?? _NH3, was measured by both a twig cuvette and bioassay techniques involving measurements of pH and ammonium (NH ₄ ⁺ ) concentration in the leaf apoplastic solution. The ?? _NH3 determined on the basis of the gas exchange measurements followed a seasonal variation with early-season peaks during leaf expansion (9.6 nmol NH₃ mol^?1 air) and late-season peaks during leaf senescence (7.3 nmol NH₃ mol^?1 air). In the mid-season, the ?? _NH3 of mature green leaves was much lower (around 3 nmol NH₃ mol^?1 air) and dropped below the NH₃ concentration in the ambient atmosphere. For comparison, ?? _NH3 obtained by the apoplastic bioassay were 7.0, 3.7 and 6.4 nmol NH₃ mol^?1 air in early-, mid-, and late -season, thus agreeing reasonably well with ?? _NH3 values derived from the gas exchange measurements. Potential NH₃ emission fluxes during early and late season were 1.31 and 0.51 nmol m^?2 leaf surface area s^?1, respectively, while leaves were a sink for NH₃ during mid-season. During leaf establishment and senescence, both apoplastic and bulk tissue NH ₄ ⁺ concentrations were relatively high coinciding with low activities of glutamine synthetase, which is a key enzyme in leaf N metabolism. In conclusion, the exchange of NH₃ between beech leaves and the atmosphere followed a seasonal variation with NH₃ emission peaks being related to N mobilization during early leaf establishment and remobilization during late leaf senescence.     

Involvement of Phytophthora species in the decline of beech Fagus sylvatica in Wallonia (Belgium)

During the last decade, typical symptoms of Phytophthora diseases were observed in beech stands of several European countries. The main symptoms were the presence of bleeding cankers on the stem, a low crown density as well as the yellowing of foliage and the small size of leaves. Several species of Phytophthora, such as Phytophthora citricola, P. cambivora and P. cactorum, were reported as the causal agents. In order to evaluate the implication of the different Phytophthora species in beech decline in the southern part of Belgium (Wallonia), a monitoring was undertaken with the help of managers of public and private forests. Phytophthora strains isolated from beech of different stands as well as from soil were characterized through morphological and molecular analyses (PCR-RFLP of ITS). All the isolated strains were identified as P. cambivora, except for one strain whose identification is ongoing. Molecular analysis was also directly applied to necrosed tissues of bleeding beeches and enabled the detection of additional cases. All positive cases exhibited a profile characteristic of the species P. cambivora, except for one of the sampled trees showing a different Phytophthora profile also corresponding to the unidentified isolated strain. Identification of the Phytophthora species linked to this different RFLP profile is also ongoing. Both complementation types (A1 and A2) of P. cambivora were identified, sometimes in the same sampling site. Ornamented oogonia characteristic of this species were produced by pairing A1 and A2 strains isolated from the same site.     

Patterns of post-glacial spread and the extent of glacial refugia of European beech (Fagus sylvatica)

Aim  Establishing possible relationships between the magnitudes of the glacial distribution of the European beech, Fagus sylvatica L., and its post-glacial spread.
Location  Europe.
Methods  A database of over 400 pollen records has been used to locate Fagus populations at the end of the last glacial and during the post-glacial in Europe and to assess the areal extent of their past distribution.
Results  The rate of late-glacial and post-glacial increase in the number of pollen sites where Fagus was locally present conforms well to a logistic model of population growth. This suggests that the area occupied by beech populations expanded exponentially from the glacial refugia for a duration of over 10,000 years, until about 3500 yr bp . In the past three millennia beech populations increased at a slower rate, tending towards an equilibrium value.
Main conclusions  The conformity of the increase in beech distribution to the classical logistic model of population growth indicates that: (1) a multiplicative biological process was the main factor shaping the pattern of the post-glacial expansion of F. sylvatica in Europe, (2) climate conditions, human activity and competition may have influenced its rate of spread, and (3) beech populations did not expand with a moving closed front, but with a diffuse spread from scattered nuclei. The distribution of Fagus in Europe at the end of the last glacial appears to have been of two orders of magnitude less extensive than at present. Pleistocene refugia were likely to have been a mosaic of sparse stands of small populations scattered in multiple regions. Fagus populations appear to have increased very slowly and to a moderate extent in southern Europe, where they are now declining slightly. The central European populations increased quickly and extensively, reaching northern Europe, and are now approaching their carrying capacity.     

Effects of ozone and Phytophthora citricola on non-structural carbohydrates of European beech (Fagus sylvatica) saplings

A lysimeter study was performed to monitor long term effects of chronic ozone enrichment on saplings of European beech (Fagus sylvatica L). After 3 years of ozone exposure a root infection with Phytophthora citricola Swada was established in the fourth year to study the interaction between elevated ozone and the root infection on the carbon budget of beech saplings. By using quantitative PCR no differences in root infection with P. citricola were observed between the ozone treatments. In contrast to the first 3 years of ozone exposure, sucrose and starch concentrations in leaves were diminished in ozone treated plants in the fourth year. The root infection reduced sucrose concentrations in leaves. Starch reserves of the heterotrophic biomass were not affected by any treatments. Thus 4 years of ozone exposure and 1 year of P. citricola root infection had only limited effect on carbohydrate metabolism in beech saplings.     

Competition for nitrogen sources between European beech (Fagus sylvatica) and sycamore maple (Acer pseudoplatanus) seedlings

To investigate the short‐term consequences of direct competition between beech and sycamore maple on root N uptake and N composition, mycorrhizal seedlings of both tree species were incubated for 4 days (i.e. beech only, sycamore maple only or both together) in an artificial nutrient solution with low N availability. On the fourth day, N uptake experiments were conducted to study the effects of competition on inorganic and organic N uptake. For this purpose, multiple N sources were applied with a single label. Furthermore, fine roots were sampled and analysed for total amino acids, soluble protein, total nitrogen, nitrate and ammonium content. Our results clearly show that both tree species were able to use inorganic and organic N sources. Uptake of inorganic and organic N by beech roots was negatively affected in the presence of the competing tree species. In contrast, the presence of beech stimulated inorganic N uptake by sycamore maple roots. Both the negative effect of sycamore maple on N uptake of beech and the positive effect of beech on N uptake of sycamore maple led to an increase in root soluble protein in beech, despite an overall decrease in total N concentration. Thus, beech compensated for the negative effects of the tree competitor on N uptake by incorporating less N into structural N components, but otherwise exhibited the same strategy as the competitor, namely, enhancing soluble protein levels in roots when grown under competition. It is speculated that enhanced enzyme activities of so far unknown nature are required in beech as a defence response to inter‐specific competition.