Peroxisome proliferation in Norway spruce induced by ozone

Summary Catalase (EC 1.11.1.6) activity (both total and specific activity) of particulate fractions of needles of Norway spruce [Picea abies (L.) Karst.] was elevated approximately 2-fold following exposure of trees to 60–70 g/m³ of ozone during the growing season compared to trees receiving charcoal filtered air (about 15 g/m³ ozone). Measurements were from homogenates fractionated into particulate and soluble (supernatent) activities. In contrast, the catalase activity of the supernatant was unchanged in response to ozone treatment. Catalase activity declined as the needles aged comparing current, 1-, and 2-year needles but the ozone-induced increment remained constant. Electron microscope cytochemistry using peroxidatic coupling with 3,3-diaminobenzidine carried out in parallel, revealed catalase-containing peroxisomes both in situ and in the particulate fractions analyzed for catalase activity. The tissue volume occupied by peroxisomes in response to needle age and ozone appeared to vary approximately in proportion to the measured catalase activity. Overall cytochemical reactivity for catalase declined with needle age, but, for all years, was greater in needles of trees receiving air supplemented with ozone compared to those of trees receiving charcoal filtered air.Abbreviations DAB 3,3-diaminobenzidine tetrahydrochloride     

Relationships among foliar chemistry, foliar polyamines, and soil chemistry in red spruce trees growing across the northeastern United States

Forest trees are constantly exposed to various types of natural and anthropogenic stressors. A major long-term goal of our research is to develop a set of early physiological and biochemical markers of stress in trees before the appearance of visual symptoms. Six red spruce (t Picea rubens Sarg.) stands from the northeastern United States were selected for collection of soil and foliage samples. All of the chosen sites had soil solution pH values below 4.0 in the Oa horizon but varied in their geochemistry. Some of these sites were apparently under some form of environmental stress as indicated by a large number of dead and dying red spruce trees. Samples of soil and needles (from apparently healthy red spruce trees) were collected from these sites four times during a two-year period. The needles were analyzed for perchloric acid-soluble polyamines and exchangeable inorganic ions. Soil and soil solution samples from the Oa and B horizons were analyzed for their exchange chemistry. The data showed a strong positive correlation between Ca and Mg concentrations in the needles and in the Oa horizon of the soil. However, needles from trees growing on relatively Ca-rich soils with a low exchangeable Al concentration and a low Al:Ca soil solution ratio had significantly lower concentrations of putrescine and spermidine than those growing on Ca-poor soils with a high exchangeable Al concentration and a high Al:Ca soil solution in the Oa horizon. The magnitude of this change was several fold higher for putrescine concentrations than for spermidine concentrations. Neither putrescine nor spermidine were correlated with soil solution Ca, Mg, and Al concentrations in the B horizon. The putrescine concentrations of the needles always correlated significantly with exchangeable Al (r²=0.73, t p0.05) and soil solution Al:Ca ratios (r²=0.91, t p0.01) of the Oa horizon. This suggests that in conjunction with soil chemistry, putrescine and/or spermidine may be used as a potential early indicator of Al stress before the appearance of visual symptoms in red spruce trees.     

Incorporation of atmospheric 15NO2-nitrogen into free amino acids by Norway spruce Picea abies (L.) Karst.

During spring and autumn 1991, potted 6-yearold spruce trees (Picea abies (L.) Karst.) were fumigated with 60 nl·1^{–1 15}NO₂ for 4 days under controlled conditions in constant light. Current and previous flush needles, the bark and the fine roots were analysed for total ¹⁵N content and incorporation of ¹⁵N into the -amino nitrogen of free amino acids. In addition, in vitro nitrate reductase activity and stomatal conductance of the needles were measured. Nitrate reductase activity was significantly higher in the needles of fumigated trees compared to control trees exposed to filtered air. With an average of 9.1% ¹⁵N, free glutamate was the pool with the most label. Taking into account the time-course of the labelling of this pool, this figure can be taken as an estimate of the minimum contribution of NO₂ to the N nutrition of the needles. ¹⁵N-labelled amino acids were also detected in the bark and the roots, indicating export from the needles.     

Carbon and nitrogen isotope ratios in different compartments of a healthy and a declining Picea abies forest in the Fichtelgebirge,NE Bavaria

Summary Natural carbon and nitrogen isotope ratios were measured in different compartments (needles and twigs of different ages and crown positions, litter, understorey vegetation, roots and soils of different horizons) on 5 plots of a healthy and on 8 plots of a declining Norway spruce (Picea abies (L.) Karst.) forest in the Fichtelgebirge (NE Bavaria, Germany), which has recently been described in detail (Oren et al. 1988a; Schulze et al. 1989). The ¹³C values of needles did not differ between sites or change consistently with needle age, but did decrease from the sun-to the shade-crown. This result confirms earlier conclusions from gas exchange measurements that gaseous air pollutants did no long-lasting damage in an area where such damage was expected. Twigs (¹³C between-25.3 and-27.8) were significantly less depleted in ¹³C than needles (¹³C between-27.3 and-29.1), and ¹³C in twigs increased consistently with age. The ¹⁵N values of needles ranged between-2.5 and-4.1 and varied according to stand and age. In young needles ¹⁵N decreased with needle age, but remained constant or increased in needles that were 2 or 3 years old. Needles from the healthy site were more depleted in ¹⁵N than those from the declining site. The difference between sites was greater in old needles than in young ones. This differentiation presumably reflects an earlier onset of nitrogen reallocation in needles of the declining stand. ¹⁵N values in twigs were more negative than in needles (-3.5 to-5.2) and showed age- and stand-dependent trends that were similar to the needles. ¹⁵N values of roots and soil samples increased at both stands with soil depth from-3.5 in the organic layer to +4 in the mineral soil. The ¹⁵N values of roots from the mineral soil were different from those of twigs and needles. Roots from the shallower organic layer had values similar to twigs and needles. Thus, the bulk of the assimilated nitrogen was presumably taken up by the roots from the organic layer. The problem of separation of ammonium or nitrate use by roots from different soil horizons is discussed.     

Supply and compartmentalization of potassium in mesophyll cells of the needles of spruce,Picea abies (L.) Karst.

Summary The water and potassium content and the relative vacuolar volume ( = V_vacuole/V_cell) of mesophyll cells of the needles of healthy 21-yearold spruce trees [Picea abies (L.) Karst.] were determined. In 5-year-old needles was 0.626 ± 0.178 (ovx ± SD). Potassium concentrations in the bulk tissue water ranged from about 65 to 105 mM. Simulations were made using this information and a simple two-compartmental model of the cell with the bulk cytoplasm and the vacuole and assuming that the minimum cytoplasmic and vacuolar K⁺ concentrations are 100–150 mM and 10–15 mM respectively. It is shown that a K⁺ content of needles below 50 mmol/1 tissue water would be precarious for maintenance of normal physiological and metabolic performance.     

Effects of ozone on organic acids in needles of Norway spruce and Scots pine

Summary The effect of ozone, needle age, and season on the pH of homogenate and acid contents of Scots pine and Norway spruce needles is presented. In addition enzyme activities of cytochrome C-oxidase (cyt. C-ox), phosphoenolpyruvate-carboxylase (PEPC), shikimic acid-dehydrogenase (SHDH) and malate-dehydrogenase (MDH) were measured in Scots pine needles. In freshly sprouted spruce needles the level of quinic acid is high and the pH of the needle homogenate is low. Shikimic acid starts at low levels, increases with increasing needle age and becomes dominant, whereas the quinic acid content decreases. Malic acid has a marked seasonal trend; no trend was found in citric acid. Ozone (200 g/m³) decreased shikimic acid and quinic acid, whereas pH, malic acid and citric acid increased. Ozone (100 g/m³) had a similar effect, except in the current-year spruce needles. In Scots pine needles ozone led to increased enzymatic activities of cyt. C-ox, PEPC and SHDH, and a decrease in the activity of MDH. This effect was more pronounced in summer than in autumn, but the visible damage was greater in autumn. These effects can be found with other stresses and are not specific for ozone.     

Chronic environmental pollution alters adenylate levels in needles and fine roots of Scots pine

Contents of ATP, ADP, AMP, inorganic phosphate, and values of ATP/ADP ratio, adenylate energy charge (AEC), phosphorylation potential (PP) and adenylate kinase activity were analysed in needles and fine roots of Scots pine trees grown at the polluted and control (free of acute air pollution) site. Also chemical properties of the soil and mineral elements in needles from both sites were analysed. In comparison with the control, developing needles from the polluted site contained less ATP, the same amount of ADP and more AMP, and had lower values of ATP/ADP, AEC and PP. In one-year-old needles from the polluted site no change or a decrease in ATP was recorded, while ADP decreased, AMP increased, AEC did not change, and ATP/ADP ratio and PP were higher. In fine roots from the polluted site AMP level was higher, while ATP, ADP, ATP/ADP ratio, PP and AEC were lower than in the control.     

Monitoring uptake and contents of Mg,Ca and K in Norway spruce as influenced by pH and Al,using microprobe analysis and stable isotope labelling

In a model system using intact spruce trees (Picea abies [L.] Karst.) we followed the path of magnesium, calcium and potassium during uptake into the root and during long-range transport into the shoot, by multiple stable isotope labelling. The roots of two- and three-year-old spruce trees originating from soil culture were removed from the soil and, in part or in toto, exposed to labelling solutions containing the stable isotopes ²⁵Mg or ²⁶Mg, ⁴¹K and ⁴²Ca or ⁴⁴Ca. Optical-emission-spectroscopy (ICP-OES) of plant fractions and labelling solutions was combined with the quantitative analysis of stable isotope ratios in sections of shock frozen, cryosubstituted material using the laser-microprobe-mass-analyser (LAMMA). This combination allowed us to distinguish, both in bulk samples and on the cellular level between (i) the fraction of elements originally present in the plant before the start of the labelling, (ii) the material taken up from the labelling solution into the plant and (iii) any material released by the plant into the labelling solution.In single-root labelling experiments, roots of three-year-old spruce trees, grown in nursery soil, were exposed to various pH conditions. The exchange of Mg and Ca with the labelling solution was nearly 100% in the cell walls of the mycorrhized finest roots. This exchange was only slightly affected by a step down to pH 3.5. The absolute Mg and Ca content in the cell walls was moderately reduced by incubation at pH 3.5 and strongly reduced in the presence of Al at this pH. After a pH 3.5 and 2 mM Al treatment we found Al in the xylem cell walls and the cortex cell lumina at elevated concentrations. To analyse the combined effect of high Al and high proton concentrations on the long-range transport, we used a split-root system. The root mass of an intact two-year-old spruce tree, grown in mineral soil, was divided into even parts and both halves incubated in solutions with two sets of different stable isotopes of Mg and Ca (side A: no Al, ²⁵Mg and ⁴²Ca; side B: +Al, ²⁶Mg and ⁴⁴Ca) and ⁴¹K on both sides. We observed a large uptake of Mg, Ca and K into the plant and a pronounced release. The net uptake of all three elements was lower from the Al-doted solution. In cross-sections of the apical shoot we found after seven-day labelling period about 60–70% of the Mg and Ca and 30% of the K content in the xylem cell walls originating from both labelling solutions. The clear majority of the Mg and Ca label originated from the Al-doted side.     

Nitrate reductase in needles,roots and trunk wood of spruce trees [Picea abies (L.) Karst.]

Summary Nitrate reductase (EC 1.6.6.2) activity (NRA), as measured by an in vivo assay, is present in needle leaves and mycorrhizal fine root tips of adult Norway spruce [Picea abies (L.) Karst.] in at least equal amounts on a fresh weight basis, in both adult and 5-year-old trees. NRA could also be demonstrated in trunk wood of deroted trees after fertilization with 5 mM , exhibiting a longitudinal profile in the trunk. Inducibility in needles can more efficiently be achieved by NO₂ (100 g·m^-3) than by 5 mM nitrate, which is effective only in root-amputated trees. A remarkably high level of needle-NRA in unfertilized trees, which are characterized by a very low level of nitrate in the xylem sap, suggests that NRA in spruce needles may in part be constitutive. Organic-N is a major nitrogen source for the needles even in root-amputated trees, indicating pronounced exchange processes between ray parenchyma and trunk xylem, which in turn are modified by the nitrogen source fed to the trunk stump. Intact trees exhibit a very similar amino acid composition of the xylem sap, regardless of whether or has been fed. The amino acid pattern of the needles is not thrown out of balance by flooding with and , which occurs in fertilized derooted trees. This indicates a distinct potential for homoeostasis of nitrogen entrance-metabolism (i.e. NRA and glutamine synthetase activity) in the needles. In the ectomycorrhiza/fine root-system (EMC), marked differences in NRA were observed depending on root-tip diameter and along the longitudinal profile of the fine roots. EMC-nitrate reductase is strongly enhanced by . Needle-NRA exhibits a circannual rhythm. An early summer maximum is followed by a December minimum. This activity pattern matches well the transitory increase of soluble nitrogen in spring and the total protein maximum in winter. In an indirect way assimilatory NRA may well contribute to nitrogen overfertilization (by consumption of NO_X) as one possible cause of the contemporary decline of spruce populations.     

The gating of nucleotide-sensitive K+ channels in insulin-secreting cells can be modulated by changes in the ratio ATP4−/ADP3− and by nonhydrolyzable derivatives of both ATP and ADP

Summary The³¹P-NMR technique has been used to assess the intracellular ratios and concentrations of mobile ATP and ADP and the intracellular pH in an insulin-secreting cell line, RINm5F. The single-channel current-recording technique has been used to investigate the effects of changes in the concentrations of ATP and ADP on the gating of nucleotide-dependent K⁺ channels. Adding ATP to the membrane inside closes these channels. However, in the continued presence of ATP adding ADP invariably leads to the reactivation of ATP-inhibited K⁺ channels, even at ATP^4–/ADP^3– concentration ratios greater than 71. Interactions between ATP^4– and ADP^3– seem competitive. An increase in the concentration ratio ATP^4–/ADP^3– consistently evoked a decrease in the open-state probability of K⁺ channels; conversely a decrease in ATP^4–/ADP^3– increased the frequency of K⁺ channel opening events. Channel gating was also influenced by changes in the absolute concentrations of ATP^4– and ADP^3–, at constant free concentration ratios. ADP-evoked stimulation of ATP-inhibited channels did not result from phosphorylation of the channel, as ADP--S, a nonhydrolyzable analog of ADP, not only stimulated but enhanced ADP-induced activation of K⁺ channels, in the presence of ATP. Similarly, ADP was able to activate K⁺ channels in the presence of two nonhydrolyzable derivatives of ATP, AMP-PNP and methylene ATP.     

Production,standing biomass and natural abundance of <Superscript>15</Superscript>N and <Superscript>13</Superscript>C in ectomycorrhizal mycelia collected at different soil depths in two forest types

Nutrient uptake by forest trees is dependent on ectomycorrhizal (EM) mycelia that grow out into the soil from the mycorrhizal root tips. We estimated the production of EM mycelia in root free samples of pure spruce and mixed spruce-oak stands in southern Sweden as mycelia grown into sand-filled mesh bags placed at three different soil depths (0–10, 10–20 and 20–30 cm). The mesh bags were collected after 12 months and we found that 590±70 kg ha^–1 year^–1 of pure mycelia was produced in spruce stands and 420±160 kg ha^–1 year^–1 in mixed stands. The production of EM mycelia in the mesh bags decreased with soil depth in both stand types but tended to be more concentrated in the top soil in the mixed stands compared to the spruce stands. The fungal biomass was also determined in soil samples taken from different depths by using phospholipid fatty acids as markers for fungal biomass. Subsamples were incubated at 20°C for 5 months and the amount of fungal biomass that degraded during the incubation period was used as an estimate of EM fungal biomass. The EM biomass in the soil profile decreased with soil depth and did not differ significantly between the two stand types. The total EM biomass in the pure spruce stands was estimated to be 4.8±0.9×10³ kg ha^–1 and in the mixed stands 5.8±1.1×10³ kg ha^–1 down to 70 cm depth. The biomass and production estimates of EM mycelia suggest a very long turnover time or that necromass has been included in the biomass estimates. The amount of N present in EM mycelia was estimated to be 121 kg N ha^–1 in spruce stands and 187 kg N ha^–1 in mixed stands. The ¹³C value for mycelia in mesh bags was not influenced by soil depth, indicating that the fungi obtained all their carbon from the tree roots. The ¹³C values in mycelia collected from mixed stands were intermediate to values from pure spruce and pure oak stands suggesting that the EM mycelia received carbon from both spruce and oak trees in the mixed stands. The ¹⁵N value for the EM mycelia and the surrounding soil increased with soil depth suggesting that they obtained their entire N from the surrounding soil.     

Biocide manipulation of N flow to investigate root/microbe competition in forest soil

Root/microbe competition was investigated as a mechanism controlling fertilizer N uptake by coniferous forest trees. Combinations of biocides both with and without ¹⁵N labelled urea, were applied to microcosms containing Sitka spruce seedlings, to selectively inhibit target microbial groups which may be competing with roots for N. After 1 growing season, concentrations of fertilizer N and total N in the trees, as well as populations of microbes and animals, were determined. Biocidal inhibition of microbial populations, particularly of fungi, was associated with significant increases in concentraions of fertilizer N and total N in Sitka spruce seedlings. Application of the fungicide benlate, for example, increased the concentration of fertilizer-derived N in spruce needles by one order of magnitude, and was associated with significant reductions in FDA-active hyphal lengths of fungi. This approach to investigating N-flow offers considerable potential for short term experiments involving competition for fertilizer/available N, where the microbial biomass represents the major sink for N in competition with roots.     

The ion concentration of dew condensed on Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.) needles

Summary Dew droplets collected with pipettes from coniferous needles were analysed for their ionic composition. Almost all samples of dew taken from Scots pine trees (Pinus sylvestris) showed significantly higher ion concentrations than those taken from Norway spruce trees (Picea abies). This can be explained by the micromorphology of the needle surface. The higher microscale roughness of the wax layer of a pine needle causes a more efficient flux of atmospheric aerosol particles compared to the spruce needle surface. Dew on coniferous needles is shown to be capable of maintaining pH values below 3 for several hours.     

Effects of Cd and Zn on the development of annual xylem rings of young Norway spruce (Picea abies) plants

Three-year-old spruce (Picea abies) saplings were planted and cultivated for 2 years in pots with 3 1 substrate, consisting of a homogenized mixture of sand, peat and forest soil with a high organic content (volume ratio 11.52). This substrate was amended with 10–180 mol Cd [kg soil dry weight (DW)]^–1, 50–7500 mol Zn (kg soil DW)^–1 (determined with 1 M ammonium acetate extracts) or combinations of both elements. Annual xylem growth rings in stems of plants treated with 50 mol Cd (kg soil DW)^–1 or 7500 mol Zn (kg soil DW)^–1 were significantly narrower than in control plants. Growth reductions were more pronounced in the second year of the experiment. The contents of Cd and Zn in stem wood and needles were positively correlated with the substrate concentrations. The Mg contents of the spruce needles were inversely correlated with soil concentrations of Cd and Zn. Root development was impeded at moderate concentrations of Cd (50 mol kg^–1) or Zn (1000 mol kg^–1) in the substrate. The adverse effects of potentially toxic trace elements, like Cd or Zn, on xylem growth of spruce plants are discussed with regard to possible growth reductions in forest trees under field conditions.     

The effects of various soils,different provenances and air pollution on root tip chromosomes in Norway spruce

The classification of chromosomal aberrations was used to characterize different factors affecting chromosomes in the root meristem of Norway spruce [Picea abies (L.) Karst.] trees. It is important to know the most significant factor affecting the chromosomes in the root meristem of plants at natural sites. The results suggest that an intensive site effect is more significant than the soil or the provenance of the individual. This cytogenetic plant test system was also used to investigate 5-year-old spruce trees exposed in environmental chambers to elevated concentrations of carbon dioxide (750 cm³m^–3) and ozone (0.08 cm³m^–3) as single variables or in combination, and then transferred to a field for observation of a memory effect. The fumigated variants showed an increased number of chromosomal aberrations compared to the controls, which carried on as a memory effect in the root meristems far beyond the fumigation period.     

Effects of ozone on starch accumulation in Norway spruce (Picea abies)

Summary Fumigation with 100 g/m³ and 200 g/m³ ozone in closed-top fumigation chambers induced starch accumulation in chloroplasts of Norway spruce. This accumulation was probably due to a partial inhibition of the starch translocation at night. The intensity of the effect was dependent on the season and the age of the needles. The accumulation was reversed in winter. It is therefore unlikely that such an effect has much significance for plant health.     

Mineral nutrient imbalances and arginine concentrations in needles of Picea abies (L.) Karst. from two areas with different levels of airborne deposition

Summary This study evaluated the utility of free arginine concentrations as a possible alternative to mineral nutrient concentrations as an indicator of mineral nutrient imbalances in Norway spruce [Picea abies (L.) Karst.]. The concentrations of mineral nutrients and arginine were measured in the needles of spruce trees from two areas in Sweden, one with high (15–30 kg ha^–1 year^–1) airborne N deposition, and one with lower (1–4 kg ha^–1 year^–1) deposition. The spruce needles from the area with high deposition in southern Sweden had elevated concentrations of free arginine, especially on peat sites. No increase in concentrations was found in the low deposition area in northern Sweden. The arginine concentrations on different sampling occasions were consistent for each site and for individual trees. Trees on peat sites in the south seemed to suffer from P deficiency in relation to N availability. A tendency for K deficiency in needles from peat sites was also found. Needles from trees on mor plots showed acceptable levels of these nutrient elements. Sites in the northern area showed low N concentrations, but the ratios between the different mineral elements analyzed in this study and N were within ranges normally found. A low P/N ratio correlated to high free arginine concentration. The threshold for elevated arginine concentrations is crossed when P/N ratios drop below 0.07–0.08. A tendency for increased arginine levels when ratios between N and the other mineral elements are low was also found, although it was not as strong as that for the P/N ratio. The results are discussed in relation to mineral nutrient imbalances in spruce stands caused by airborne deposition.     

Acclimation of Two Distinct Plant Species,Spring Barley and Norway Spruce,to Combined Effect of Various Irradiance and CO2 Concentration During Cultivation in Controlled Environment

The short-term acclimation (10-d) of Norway spruce [Picea abies (L.) Karst] to elevated CO₂ concentration (EC) in combination with low irradiance (100 mol m^–2 s^–1) resulted in stimulation of CO₂ assimilation (by 61 %), increased total chlorophyll (Chl) content (by 17 %), significantly higher photosystem 2 (PS2) photochemical efficiency (F_v/F_m; by 4 %), and reduced demand on non-radiative dissipation of absorbed excitation energy corresponding with enhanced capacity of photon utilisation within PS2. On the other hand, at high cultivation irradiance (1 200 mol m^–2 s^–1) both Norway spruce and spring barley (Hordeum vulgare L. cv. Akcent) responded to EC by reduced photosynthetic capacity and prolonged inhibition of F_v/F_m accompanied with enhanced non-radiative dissipation of absorbed photon energy. Norway spruce needles revealed the expressive retention of zeaxanthin and antheraxanthin (Z+A) in darkness and higher violaxanthin (V) convertibility (yielding even 95 %) under all cultivation regimes in comparison with barley plants. In addition, the non-photochemical quenching of minimum Chl a fluorescence (SV₀), expressing the extent of non-radiative dissipation of absorbed photon energy within light-harvesting complexes (LHCs), linearly correlated with V conversion to Z+A very well in spruce, but not in barley plants. Finally, a key role of the Z+A-mediated non-radiative dissipation within LHCs in acclimation of spruce photosynthetic apparatus to high irradiance alone and in combination with EC was documented by extremely high SV₀ values, fast induction of non-radiative dissipation of absorbed photon energy, and its stability in darkness.     

Wood ash treatment affects seasonal N fluctuations in needles of adult<Emphasis Type="Italic"> Picea abies</Emphasis> trees: a <Superscript>15</Superscript>N-tracer study

A ¹⁵N-tracer experiment was carried out in a stand of adult spruce trees [Picea abies (L.) Karst.] located on the Swiss Plateau in order to investigate the effects of wood ash treatment on seasonal nitrogen fluctuations in fine roots and needles. Treatments included irrigation (W), liquid fertilization (LF) and wood ash (A) application. ¹⁵N fluctuation in fine roots and current to 3-year-old needles was studied after one ¹⁵N pulse for 2 consecutive years (1999, 2000). ¹⁵N tracer was rapidly incorporated into the fine roots of adult trees, and ¹⁵N values reached similar levels in all treatments 2 months after the pulse. In the needles, the largest increase in ¹⁵N was observed in those of the current year. Following the initial peak during spring growth, ¹⁵N values in needles of control trees showed an oscillating pattern through the season. This oscillation is attributed to the increased use of internal N sources, as soon as the roots can no longer meet the increased N demand during the sprouting phase. However, W-, LF- and A-treated trees no longer showed the oscillation in ¹⁵N. Additional water (W and LF) as well as fertilizer (A and LF) may have induced shifts in the microbial flora, thus increasing the unlabelled N release from the soil. The strongest dampening was observed for the A treatment, indicating sufficient N availability from the soil, and making intensive use of the internal N sources unnecessary. Treatment with wood ash thus resulted in a similar fertilizer response to liquid fertilization.     

Substrate level versus oxidative phosphorylation in the generation of ATP in Thiobacillus denitrificans

Particulate fractions of Thiobacillus denitrificans catalyse the phosphorylation of ADP to ATP during the oxidation of various inorganic sulphur compounds or NADH via an electron transport chain. On the other hand, a soluble cell-free fraction synthesized ATP from APS and inorganic phosphate.The production of ATP was verified either by the firefly luciferin-luciferase enzyme system or by the incorporation of ³²Pi into ATP. During the oxidation of sulphide, sulphite and NADH the production of ATP from ADP by particulate fractions is inhibited by compounds that inhibit electron transfer and by uncouplers of oxidative phosphorylation. However, these compounds had little effect on the production of ATP from AMP during the oxidation of sulphite by the soluble fraction. NADH was the most effective electron donor for oxidative phosphorylation. The soluble fraction contained high activities of ATP sulphurylase, inorganic pyrophosphatase and adenylate kinase but ADP sulphurylase activity was relatively low. The effects of inhibitors on ATP production from APS and Pi are compared with those on adenylate kinase and ATP sulphurylase.Abbreviations APS adenosine-5-phosphosulphate - DNP 2,4-dinitrophenol - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide