Glutathione status and glutathione reductase activity in spruce needles of healthy and damaged trees at two mountain sites

Levels of glutathione, in both reduced and oxidized form, and glutathione reductase activity were monitored in needles of healthy and damaged spruce trees (Picea abies (L.) Karst.) during the course of four vegetation periods at two natural sites. The glutathione content and glutathione reductase activity showed a pronounced annual rhythm in undamaged trees, whereas damaged spruce trees deviated significantly from this course. In comparison with undamaged trees, damaged trees showed markedly increased levels of glutathione during the test period of 1989-1991. However, glutathione reductase activity differed in damaged and undamaged trees, only in 1989-1990. The ratio of reduced to oxidized glutathione (GSH/GSSG ratio) was slightly higher in damaged trees, and the highest levels were found during the winter months. In the case of damaged trees, a correlation between GSH/GSSG ratio and current ozone levels at the sites could be clearly established. The present results indicate that damaged trees suffer from increased oxidative stress, especially in the period from June to October.     

No general ozone-specific indicator among the hexane- and dichloromethane-soluble components of Picea abies needles exposed to ozone in open-top chambers

The effect of ozone (< 10, 200 or 400 microg m(-3) on hexane- and dichloromethane-soluble components of Picea abies needles was determined by fumigating potted grafts from mature trees. The trees (>55 and 125 years, 2.5 m high), representing six clones of Norway spruce, were fumigated in open-top chambers at two locations in Norway for one growth season. The needles were extracted with hexane and dichloromethane; 142 compounds from the hexane extract and 164 silylated compounds from the dichloromethane extract were analysed by gas chromatography although no identifications were made. The concentration of four of the compounds from the hexane extract changed with ozone dose in a way that made them promising as indicators, but the present analytical method could not verify this possibility. None of the other 302 compounds qualified as a general indicator of ozone stress in Norway spruce, as none changed its concentration with ozone dose consistently in all romets of all clones. Most of the variation in the experiment is mainly attributable to genetic variation and to climate.     

Monoterpene patterns of different tissues and plant parts of Norway spruce (Picea abies L. Karst.)

Norway spruce saplings (Picea abies L. Karst.) were exposed for five years to controlled concentrations of ozone and/or sulphur dioxide in open-top chambers. The monoterpenes of needles, twigs (bark and wood), bark and buds harvested from these trees were qualitatively and quantitatively analysed by combined gas chromatography-mass spectrometry. Different tissues and plant parts, showed notable differences in their percentage distribution. However, comparison of the monoterpene patterns of corresponding plant material obtained from trees which had been exposed to different fumigation regimes showed no significant effects of these pollutants.     

Contents of free amino acids in needles of Norway Spruce trees in relation to novel forest decline. Studies on trees from a site in the northern Black Forest

The free amino acid content in needles of Norway spruce trees (about 45-year-old) was determined by means of HPLC. The studied trees have been growing at a forestry site in the Black Forest which is characterized by a high impact of ozone and magnesium deficiency. Measurements were carried out on visibly healthy and on damaged trees on several dates during two vegetation periods and during the course of a day. The amino acids occurring at the highest concentrations were glutamate, arginine, aspartate, and glutamine. The typical seasonal changes in the content of free amino acids, with their minimum during summer, were disturbed in the needles of the damaged trees. Particularly in summer and autumn the damaged trees showed increased levels of total amino acids and of most single amino acids. During the course of a day, the needles of the damaged trees showed a reduced amplitude in diurnal variations with the absolute level almost always higher in comparison to that of the undamaged trees. The results suggest that a shift in protein turnover towards enhanced degradation has taken place.     

Biochemical and molecular studies on declining and decline-resistant spruce in the north-east of France

In declining forests of the Vosges mountains (northeast of France), we previously observed that the yellowing of spruce (Picea abies L. cv. Karsten) needles was associated with impairment of the free radical scavenging capacity of the cells and coincided with chronic exposure to ozone. Chloroplasts of yellow needles were characterized by an abnormal accumulation of photosystem II (PSII) D1-protein in the thylakoids. Further experiments carried out on declining and decline-resistant individual spruce trees characterized in previous studies showed that needle yellowing was associated with impairment of the overall anti-oxidative defense in both the cytosol and the chloroplasts. Both enzymic (peroxidases) and non-enzymic (carotenoids) oxidant scavengers were shown to be affected in the declining spruce. PSII D1-protein accumulation seemed to result from a stabilization of the polypeptide, which led us to hypothesize that oxidative processes might interfere with the specific degradation of this protein in declining spruce, with destructive consequences for the photosystems.     

Photolysis of polycyclic aromatic hydrocarbons adsorbed on spruce [Picea abies (L) Karst] needles under sunlight irradiation

Photolysis of polycyclic aromatic hydrocarbons (PAHs) sorbed on surfaces of spruce [Picea abies (L.) Karst.] needles under sunlight irradiation was investigated. PAHs were produced by combustion of polyvinyl chloride (PVC), wood, high-density polyethylene (HDPE), and styrene in a stove. The factors of sunlight irradiation on the surfaces of spruce needles were taken into consideration when investigating the kinetic parameters. The photolysis of the 18 PAHs under study follows first-order kinetics. The photolysis half-lives range from 15 h for dibenzo(a,h)anthracene to 75 h for phenanthrene. Photolysis of some PAHs on surfaces of spruce needles may play an important role on the fate of PAHs in the environment.     

Chemical and morphological characteristics of key tree species of the Carpathian Mountains

Concentrations of Al, B, Ca, Cu, Fe, K, Mg, Mn, N, Na, P, S and Zn in the foliage of white fir (Abies alba), Norway spruce (Picea abies) and common beech (Fagus sylvatica) from 25 sites of the Carpathian Mts. forests (Czech Republic, Poland, Romania, Slovakia and Ukraine) are discussed in a context of their limit values. S/N ratio was different from optimum in 90% of localities when compared with the European limit values. Likewise we found increase of Fe and Cu concentrations compared with their background levels in 100% of locations. Mn concentrations were increased in 76% of localities. Mn mobilization values indicate the disturbance of physiological balance leading to the change of the ratio with Fe. SEM-investigation of foliage waxes from 25 sites in the Carpathian Mts. showed, that there is a statistically significant difference in mean wax quality. Epistomatal waxes were damaged as indicated by increased development of net and amorphous waxes. The most damaged stomata in spruce needles were from Yablunitsa, Synevir and Brenna; in fir needles from Stoliky, and in beech leaves from Malá Fatra, Morské Oko and Beregomet. Spruce needles in the Carpathian Mts. had more damaged stomata than fir needles and beech leaves. Spruce seems to be the most sensitive tree species to environmental stresses including air pollution in forests of the Carpathian Mountains. Foliage surfaces of three forest tree species contained Al, Si, Ca, Fe, Mg, K, Cl, Mn, Na, Ni and Ti in all studied localities. Presence of nutrition elements (Ca, Fe, Mg, K and Mn) on foliage surface hinders opening and closing stomata and it is not physiologically usable for tree species.     

Analyses of enzyme activities and other metabolic criteria after five years of fumigation

Enzymatic activity (peroxidase, glutamate dehydrogenase, glutamine synthetase), foliage buffering capacity, soluble protein and nitrogen content were measured in current and previous year needles from young spruce (Picea abies) and fir (Abies alba). The trees were exposed to low levels of SO(2) and/or O(3) and simulated acidic precipitation (pH 4.0) in open-top chambers from 1983 through 1988. Needle samples were taken during March 1988 at the end of the five-year fumigation period. Exposure to SO(2) substantially increased sulphur content in both needle age classes of spruce and fir, and concomitantly reduced the foliage buffering capacity index (BCI), whereas the combined fumigation with SO(2) and O(3) had no effect on BCI. Peroxidase activity was markedly higher in year-old needles compared to current-year needles. However, trees from the SO(2) and SO(2) + O(3) treatments exhibited statistically significant stimulated peroxidase activities. Similarly, changes in the activities of the nitrogen-metabolizing enzymes indicated an altered cellular function of the trees after the long-term pollution stress. Levels of activity of both glutamate dehydrogenase and glutamine synthetase were increased by exposure to SO(2), especially in spruce. Although glutamate dehydrogenase in spruce was affected by all treatments, such changes in activity were found in fir only with the SO(2) treatment. The highest activity of glutamine synthetase, however, occurred in the older needles of trees exposed to SO(2) + O(3). Total nitrogen concentration was either unaffected by the pollutant treatments or decreased in spruce compared to the controls. No statistically significant changes due to the fumigation were found in soluble protein concentrations. Results indicated that chronic exposure to air pollutants lead to alterations in metabolic processes in conifer needles, detectable either by changes in typical stress indicating values or by increases in ammonium assimilation capacity.     

Root and shoot growth, assimilate partitioning and cell proliferation in roots of Sitka spruce (Picea sitchensis) grown in filtered and unfiltered chambers

Rooted cuttings of clonal Sitka spruce (Picea sitchensis (Bong.) Carr.) were grown from April to October in 1 m long tubes sunk into the ground inside open top chambers. The same experiment was repeated in each of two consecutive years using a different clone of Sitka spruce each year. Air was either passed directly into the chambers (ambient air) or passed over charcoal filters which removed the majority of gaseous pollutants before entering the chambers (filtered air). Ambient pollution did not appear to influence the growth of Sitka spruce at least over the experimental period used. No significant differences were found between plants exposed to ambient or filtered air in terms of shoot and root dry mass, needle dry mass, root length, carbohydrate content of roots and needles, and in the percentage of meristematic cells close to the apex in each phase or interphase or undergoing mitosis.     

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

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

Regional variation in surface properties of Norway spruce and scots pine needles in relation to forest decline

Measurements of leaf wettability (contact angle), amounts of epicuticular wax and of surface dust are reported for Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L) Karst) trees growing at 12 sites in Europe from SW Germany to NE Scotland. At each site, three year classes (current year, 1 and 2-year-old) of needles were sampled from the mid-crown of up to 12 trees. Trees were selected at random from two strata, those showing visible decline symptoms (i.e. loss of needles or discoloration) and those apparently healthy. Needles for analysis were taken from apparently healthy (green) shoots in both cases. There were no significant differences between 'declining' and 'healthy' trees within sites, suggesting that leaf surface properties reflect environmental exposure rather than plant response. There were significant differences between sites, particularly for Norway spruce, which may be related to environmental factors including air pollution. Contact angles were consistently smaller at low-altitude sites in Britain and The Netherlands than at high-altitude sites in Germany where forests show decline symptoms. Leaf wettability decreased (contact angles increased) with wax amount and increased with dust amount. Leaf surface properties integrate environmental influences over long periods, and may be useful in identifying sites 'at risk' of developing decline symptoms, but causal relationships cannot be deduced without further direct experimentation.     

Role of climate, crown position, tree age and altitude in calculated ozone flux into needles of Picea abies and Pinus cembra: a synthesis

Ozone (O(3)) flux into Norway spruce (Picea abies) and cembran pine (Pinus cembra) needles was estimated under ambient conditions at six rural sites between 580 and 1950 m a.s.l. We also assessed age-related differences in O(3) flux by examining changes in leaf conductance across the life span of Norway spruce. At the leaf level O(3) flux into the needles was effectively controlled by stomatal conductance and, hence by factors such as temperature, irradiance and humidity, which control stomatal conductance. Seasonal variations in O(3) flux were mainly attributed to the course of the prevailing temperature. During the growing season, however, data have emphasised leaf-air vapour pressure difference as the environmental factor most likely to control stomatal conductance and O(3) flux into the needles. In the sun crown stomatal conductance averaged over the growing season decreased with increasing tree age from 42.0+/-3.5 mmol O(3) m(-2) s(-1) in 17-year-old trees to 7.1+/-1.0 mmol O(3) m(-2) s(-1) in 216-year-old trees, indicating that O(3) concentration in the substomatal cavities is higher in young than in old trees. Independent from tree age stomatal conductance and O(3) flux were approximately 50% lower in shade needles as compared to sun-exposed needles. Stomatal conductance was also greater in the current flush (24+/-5.6 mmol O(3) m(-2) s(-1)) and in 1-year old needles (16+/-4 mmol O(3) m(-2) s(-1)) than in older needle age classes (12+/-1 mmol O(3) m(-2) s(-1), averaged across the four older needle age classes). In trees similar in age (60-65 years old) average O(3) flux into sun needles increased from 0.55+/-0.36 nmol m(-2) s(-1) at the valley floor to 0.9 nmol m(-2) s(-1) in 1950 m a.s.l. Cumulative O(3) uptake during the vegetation period increased from 11.4+/-1.7 mol m(-2) in the valley to 14 mol m(-2) at the alpine timberline. Although stomatal conductance provides the principal limiting factor for O(3) flux, additional field research is necessary in order to improve our understanding concerning the quantitative 'physiological threshold dose' which internally can be active and can have adverse effects of O(3) on forest trees.     

Anatomical and physiological responses of Colorado blue spruce to vehicle exhausts

In order to examine whether the leaves of the Colorado blue spruce (Picea pungens) are damaged or not by traffic pollution, the traits of the anatomy and physiology of its leaves are investigated by exposure to vehicle exhausts in a laboratory experiment lasting 30 days. The results show that both the anatomical structures and physiological traits of the leaves are significantly affected by vehicle exhausts. The anatomical structures, including epidermis, cuticle, palisade, and spongy parenchyma are modified when exposed to the high concentrations (≥0.4 mg/m³) of vehicle exhausts. However, physiological traits such as total chlorophyll content are not changed when exposed to different concentrations of vehicle exhaust. Unlike the total chlorophyll content, the electrical conductivities increased, whereas the POD activities decreased when presented in vehicle exhausts. The present study indicates that the Colorado blue spruce changes its anatomical structures and physiological traits to avoid possible damage by vehicle exhausts.     

Photodegradation of PCDD/Fs adsorbed on spruce (Picea abies (L.) Karst.) needles under sunlight irradiation

Spruce (Picea abies (L.) Karst.) needles were exposed to exhaust gas containing polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) generated by combustion of polyvinyl chloride, wood, high-density polyethylene, and styrene. Photodegradation of PCDD/Fs adsorbed on spruce needles under sunlight irradiation was studied. The photodegradation of PCDD/Fs follows pseudo-first-order reaction kinetics, with photolysis half-lives ranging between 40 and 100 h. The photolysis rates of PCDF congeners are higher than PCDD congeners with the same chlorinated substitutions. Higher chlorinated PCDD/Fs tend to photolyze slowly. The wax components in spruce needles may act as proton donors and accelerate the photolysis rate. C-Cl cleavage through the addition of protons to PCDD/F molecules may be an important route for PCDD/Fs photodegradation.     

Shoot growth of mature Fagus sylvatica and Picea abies in relation to ozone

Epidemiological analysis of sequential growth data may be a tool in assessing ozone sensitivity of mature trees. Annual shoot growth of mature Fagus sylvatica in 83 Swiss permanent forest observation plots and of Picea abies in 61 plots was evaluated for 11 and 8 consecutive years, respectively, using branches harvested every 4 years. The data were assessed as annual deviation from average growth and related to fructification, ozone, meteorological parameters, and modelled soil water content using a mixed linear model. In beech, a significant association between ozone and shoot growth was observed which corresponded to a 7.4% growth reduction between 0 and 10 ppm h AOT40 (accumulated ozone over threshold 40). This is in the same order of magnitude as the response observed in experiments with seedlings. No interaction was found between ozone and drought parameters. In Norway spruce, shoot growth was neither associated with ozone nor with drought.     

The effect of salinity on the emergence and seedling growth of Picea mariana, Picea glauca, and Pinus banksiana

Mining operations in areas of the boreal forest have caused salinity issues to be a major concern for reclamation. One of the factors determining successful reclamation is the ability of species to self-propagate. The effects of salinity on the seedling emergence and early growth of three boreal forest conifers: Picea mariana, Picea glauca, and Pinus banksiana were determined. Seeds were planted in sand moistened with solutions of various concentrations of sodium chloride or sodium sulfate. Seedling emergence was monitored on a daily basis and growth parameters assessed after 6 weeks. The emergence of Pinus banksiana seedlings was least affected by salinity, and at certain concentrations, emergence even appeared to be stimulated by the presence of salt. Picea glauca was the most sensitive of the species studied. Hypertrophia was observed in all species at high concentrations of Na2SO4, and an increase in salt levels caused a corresponding reduction in seedling height and weight, root length and number of lateral roots.     

Ozone-induced nitrate formation in needles and leaves of Picea abies, Fagus sylvatica and Quercus robur

Much attention has been paid to ozone as a major cause of novel forest decline in Europe. In combination with acidic mist, O(3) has been observed to increase ion leaching. Besides cations lake Mg(2+), Ca(2+), K(+), NH(4)(+), considerable amounts of nitrate were found to be leached by acidic mist from needles of Norway spruce. Controlled fumigation experiments, with 100, 300, and 600 microg O(3)m(-3) over 22 days continuously, have led to a nitrate accumulation of 94.1 +/- 14.8, 119.4 +/- 28.7 and 198.9 +/- 14.9 microg NO(3)(-1) g(-1) FW, respectively, in leaves of Quercus robur. Similar values were found in leaves of Fagus sylvatica and current and previous year needles of Picea abies. Nitrate levels of controls receiving charcoal filtered air were well below 40 microg NO(3)(-) g (-1) FW. Statistically significant elevated nitrate levels were observed after only 48 h of continuous fumigation with 600 microg O(3)m(-3), in all tree species tested, and after 144 h in the 100 microg O(3)m(-3) treatment. In another experiment, trees of Picea abies were kept in two charcoal (C) and two Purafil plus charcoal (P/C) ventilated chambers, and fumigated with O and 500 microg O(3)m(-3) in cabinets of each filter-type in order to eliminate NO(x) from chamber air. After 29 days of continuous ozone fumigation, NO(3)(-) accumulation in needles amounted to 102.0 +/- 37.7 and 137.4 +/- 40.5 microg g(-1) FW in P/C and C-filtered chambers, respectively. Nitrate contents of controls were below 30 microg NO(3)(-)g(-1) FW at the end of the experiment. No significant differences in NO(3)(-) accumulation between filter treatments were observed. Since NO(x) was reduced by more than 95% in the Purafil/charcoal versus the charcoal treatment, NO(3)(-) accumulation in needles can be attributed predominantly to the influence of ozone and not to direct NO(2) uptake of needles by the possible oxidation of NO to NO(2) in the presence of ozone.     

Trends in needle and soil chemistry of Norway spruce and Scots pine stands in South Sweden 1985-1994

The nutrient status of the trees and soil in 42 stands of Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.) in Scania, South Sweden was followed from 1985 to 1994. Samples from needles taken in winter 1985, 1987, 1990, and 1994, and soils in 1988 and 1993 were analyzed. Concentrations, as well as ratios to N, of K and Cu in needles of both species decreased by approximately 40% from 1985 to 1994. Soil analyses indicate ongoing soil acidification and leaching of mineral nutrients from the soil profile. Together with deposition data and corroboration from modeled scenarios, these data support the recent contention that one consequence of enhanced deposition of N and S will be the development of nutrient imbalances in trees growing in southern Sweden.     

Responses of spruce seedlings (Picea abies) to exhaust gas under laboratory conditions--II. Ultrastructural changes and stomatal behaviour

This study examines the effects of exhaust gas exposure on the epistomatal wax structure and mesophyll ultrastructure in needles of Norway spruce (Picea abies (L.) Karst.) seedlings. Stomatal diffusive resistance was also measured. Two independent exhaust gas fumigations were performed: 100 and 200 ppb measured as NO(x), for 10 days and 50, 100 and 200 ppb NO(x) for 19 days. The obstructive effect of exhaust gas exposure on epistomatal wax tubules was apparent. The stomata became covered by flat and solid wax resulting from the structural degradation of the wax crystalloids. Increasing the exhaust gas concentration in the chamber atmosphere exacerbated the degradation of the wax structure. Exhaust gas exposure induced aggregation and electron translucence of plastoglobuli, swelling of thylakoids, increase of cytoplasmic lipids and slight increase of vesiculation of cytoplasm in mesophyll cells of current and previous year needles. These changes were exemplified in current year needles. Damage to the epicuticular waxes and mesophyll ultrastructure of spruce needles most likely reflects the NO(x) and volatile hydrocarbon fraction. The alterations in epicuticular waxes and mesophyll ultrastructure can be related to accelerated senescence of the youngest, photosynthetically active, needle generation. The exhaust gas also resulted in decreased diffusive stomatal resistance at night which indicates that the exhaust gas exposure disturbed the gas exchange of spruce seedlings. The results show that even relatively short-term exposure to realistic concentrations of exhaust gas in the atmosphere can induce rather severe injuries to the needle surface structure as well as ultrastructure at the cellular level.     

Uptake,translocation and fate of trichloroacetic acid in a Norway spruce/soil system

Trichloroacetic acid (TCA) is a secondary atmospheric pollutant formed by photooxidation of chlorinated solvents in the troposphere--it has, however, recently been ranked among natural organohalogens. Its herbicidal properties might be one of the factors adversely affecting forest health. TCA accumulates rapidly in conifer needles and influences the detoxification capacity in the trees. The aim of the investigations--a survey of which is briefly given here--was to elucidate the uptake, distribution and fate of TCA in Norway spruce. For this purpose young nursery-grown plants of Norway spruce (Picea abies (L.) Karst.) were exposed to [1,2-14C]TCA and the fate of the compound was followed in needles, wood, roots, soil and air with appropriate radio-indicator methods. As shown by radioactivity monitoring, the uptake of TCA from soil by roots proceeded most rapidly into current needles at the beginning of the TCA treatment and was redistributed at later dates so that TCA content in older needles increased. The only product of TCA metabolism/biodegradation found in the plant/soil-system was CO(2) (and corresponding assimilates). TCA biodegradation in soil depends on TCA concentration, soil humidity and other factors.