Interaction between atmospheric and pedospheric nitrogen nutrition in spruce (Picea abies L. Karst) seedlings

The effect of NO₂ fumigation on root N uptake and metabolism was investigated in 3-month-old spruce (Picea abics L. Karst) seedlings. In a first experiment, the contribution of NO₂ to the plant N budget was measured during a 48 h fumigation with 100mm³m^?3 NO₂. Plants were pre-treated with various nutrient solutions containing NO₂ and NH₄⁺, NO₃^? only or no nitrogen source for 1 week prior to the beginning of fumigation. Absence of NH₄⁺ in the solution for 6d led to an increased capacity for NO₃^? uptake, whereas the absence of both ions caused a decrease in the plant N concentration, with no change in NO₃^? uptake. In fumigated plants, NO₂ uptake accounted for 20–40% of NO₃^? uptake. Root NO₃^? uptake in plants supplied with NH₄⁺plus NO₃^? solutions was decreased by NO₂ fumigation, whereas it was not significantly altered in the other treatments. In a second experiment, spruce seedlings were grown on a solution containing both NO₂ and NH₄⁺ and were fumigated or not with 100mm³m^?3 NO₂ for 7 weeks. Fumigated plants accumulated less dry matter, especially in the roots. Fluxes of the two N species were estimated from their accumulations in shoots and roots, xylem exudate analysis and ¹⁵N labelling. Root NH₄⁺ uptake was approximately three times higher than NO₃^? uptake. Nitrogen dioxide uptake represented 10–15% of the total N budget of the plants. In control plants, N assimilation occurred mainly in the roots and organic nitrogen was the main form of N transported to the shoot. Phloem transport of organic nitrogen accounted for 17% of its xylem transport. In fumigated plants, neither NO₃^? nor NH₄⁺ accumulated in the shoot, showing that all the absorbed NO₂ was assimilated. Root NO₃^? reduction was reduced whereas organic nitrogen transport in the phloem increased by a factor of 3 in NO₂-fimugated as compared with control plants. The significance of the results for the regulation of whole-plant N utilization is discussed.     

Response to three seasons of elevated ozone in the progeny of healthy and unhealthy Norway spruce trees from a plantation with the 'top dying' syndrome

We tested a hypothesis that elevated ozone was an eliciting or contributing factor in outbreaks of the 'top dying' (or 'subtop dying') syndrome in Norway spruce ( Picea abies ). Progeny were used from open-pollinated trees within a stand with the 'top dying' syndrome. The mother trees were classified in relation to the expression of the 'top dying' syndrome, and progeny from the healthiest and least healthy thirds of the population were exposed to high and low concentrations of ozone for three seasons. Elevated ozone did not affect height growth of the trees. It did not measurably affect net photosynthesis, stomatal conductance or instantaneous water use efficiency. Chlorophyll and carotenoid contents were also not significantly affected by ozone concentration. In the first year, instantaneous water use efficiency was lower in the progeny of the unhealthy mother trees than in the healthy mother trees. Furthermore, the unhealthy mother trees tended to produce longer annual shoots and showed more winter damage at the end of the experiment. None of these parameters were related to ozone concentration in the atmosphere. These results do not support a hypothesis that elevated ozone is a significant contributory factor or an eliciting factor in the development of the syndrome.     

Ribulose-l,5-bisphosphate carboxylase activity and influence of air pollution in spruce

Methods were established, which render possible a simultaneous determination of ri-bulose-l,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity and chlorophyll content of Norway spruce (Picea abies Karst.) needles from a detergent-containing aqueous crude extract. Spruce RuBP carboxylase was tentatively characterized with regard to kinetic properties. Recovery experiments employing purified wheat RuBP carboxylase proved quantitative extraction of the enzyme from spruce foliage. Five timber stands consisting of 35–62 years old spruce, two of which exhibited the typical symptoms of recent spruce decline, were compared. For the needle generations 1 to 4 the enzyme activities as well as chlorophyll and protein concentrations were determined. The results do not indicate an involvement of RuBP carboxylase in spruce decline.     

Effects of acid irrigation and liming on nitrate reduction and nitrate content of Picea abies (L.) Karst. and Oxalis acetosella L.

Nitrate reductase activities (NRA) and nitrate concentration per unit biomass in Picea abies (L.) Karst. roots from four different soil horizons and in leaves and roots of the frequent field-layer species Oxalis acetosella L. were measured on six different irrigation and liming treatments within the Höglwald project, S-Bavaria, Germany. Liming increased and acid irrigation reduced soil nitrate availability when compared to control plots. Nitrate assimilation capacities of the respective plant compartments per unit of soil volume or ground area were calculated from the NRA per unit of biomass and from the biomass distribution on the various treatments.Mean NRA per unit of biomass in Picea abies roots ranged between 0.23 and 0.09 mol NO₂^- g_-1 d.w. h_-1 without significant effects of soil horizon or treatment. Limed and non-limed treatments showed for Picea different root distributions within the soil profile, but root biomass per unit of ground area (295 to 220 g d.w. m^-2) was not affected by the various treatments. Thus, nitrate assimilation capacity of Picea roots per unit of ground area ranged between 19.5 and 11.4 mol NO₂^- m^-2 h_-1 without major treatment effects.In laminae of Oxalis acetosella mean NRA per unit of biomass ranged between 2.91 and 0.27 mol NO₂^- g_-1 d.w. h_-1 and, in contrast to Picea abies, treatment effects were found with NRA on limed plots increased and on acid irrigated plots reduced when compared to control plots. Mean leaf biomass of Oxalis per unit of ground area ranged between 9.57 and 0.66 g d.w. m^-2 and responded in a similar manner to the various treatments. Thus, for the Oxalis leaf NRA per unit of ground area (27.85 to 0.18 mol NO₂ m^-2 h_-1) a cumulative response to the variations in nitrate availability was found.The different responses of Picea abies and Oxalis acetosella to changes in soil nitrate availability are discussed with respect to their suitability to prevent soil nitrate leaching.     

Metabolism of nitrate and ammonium in seedlings of Norway spruce (Picea abies) measured by in vivo 14N and 15N NMR spectroscopy

In vivo ¹⁵N and ¹⁴N nuclear magnetic resonance spectroscopy was used to investigate the assimilation of nitrate and ammonium in seedlings of Norway spruce (Picea abies [L.] Karst.). The main objective was to study accumulation of free NH+₄ and examine to what extent the nitrogen source affects the composition of the free amino acid pools in roots, stems and needles. NH+₄ concentrations in plants growing in the presence of 0.5–50 mM ammonium were quantified using ¹⁴N NMR. The NH+₄ values in tissues ranged from 6 to 46 μmol (g fresh weight)^?1. with highest concentrations in roots and needles. The tissue NH+₄ peaked at 5.0 mM NH+₄ in the medium. and failed to increase when NH+₄ in the medium was increased to 50 mM, indicating metabolic control of the concentration of this cation in tissues. The ¹⁴N NMR spectra were used to estimate pH of the NH+₄ storage pools. Based on the pH sensitivity of the quintet of ¹⁴NH+₄ resonance, we suggest that the pH of the ammonium storage compartments in the roots and stems should be 3.7–3.8, and in needles 3.4–3.5, representing extremely low pH values of the tissue. ¹⁵N from nitrate or ammonium was first incorporated into the amide group of glutamine and then into α-amino groups, confirming that the glutamine synthetase/ glutamate synthase cycle is the major route of nitrogen assimilation into amino acids and thus plays a role in lowering the levels of NH+₄ in the cytoplasm. NH+₄ can also be assimilated in roots in plants growing in darkness. The main ¹⁵N-labelled amino acids were glutamine. arginine and alanine. Almost no ¹⁵N signals from needles were observed. Double labelling (δN + w, wN) of arginine is consistent with the operation of the ornithine cycle, and enrichment indicates that this cycle is a major sink of newly assimilated nitrogen. Nitrogen assimilation in roots in the presence of added methionine sulphoximine and glutamate indicated the catabolic action of glutamate dehydrogenase. The ¹⁵N NMR spectra of plants grown on ¹⁵N-urea showed a marked increase in the labelling of ammonium and glutamine. indicating high urease activity. Amino acids were also quantified using high pressure liquid chromatography. Arginine was found to be an important transport form of nitrogen in the stem.     

Variable growth and reproduction response of the spruce shoot aphid, Cinara pilicornis, to increasing ozone concentrations

Spruce shoot aphid, Cinara pilicornis Hartig (Homoptera: Lachnidae), is an aphid species that has shown enhanced performance on trees exposed to SO₂ or mixtures of air pollutants, whereas results with ozone have been contradictory. Using a 4-week chamber fumigation experiment, we tested how different population dynamic parameters of aphids are affected by ozone (O₃). Mean relative growth rate (MRGR), development time and reproduction rate of C. pilicornis were determined using 7h day-time O₃ concentrations of 0, 40, 80 and 160 ppb. Development period from birth to reproductive stage was fastest at 80 ppb during early shoot elongation, while reproduction and intrinsic rate of population increase was not significantly affected by ozone concentration. There was a significant negative correlation between ozone concentration and the MRGR of first instar nymphs in the third fumigation week. In feeding test performed on seedlings after fumigation, the MRGR was reduced on shoots which had been exposed to 80 ppb O₃. The results suggest that elevated O₃ concentration during early shoot elongation period may stimulate population development of C. pilicornis, but on maturing shoots, high O₃ concentration has a negative effect on aphid performance. This might be due to accelerated ageing of O₃ exposed shoots.     

The interpretation of leaf-drying curves

A theoretical expression for the rate of water loss through the surface of excised leaves has been derived so that curves fitted to experimental data can be used to infer physical properties of the leaves such as the solute concentration and the conductance for water vapour loss. The use of the equations is illustrated by reference to data from an experiment in which red spruce (Picea rubens Sarg.) and Norway spruce (P. abies(L.) Karst.) seedlings were grown under five different regimes: outdoors (±50% shade), in a greenhouse (±50% shade) and in controlled-environment chambers.     

The rate of protein synthesis in needles of Norway spruce (Picea abies): Light stimulation, regulation through photophosphorylation, stress enhancement

The incorporation of ¹⁴C-leucine into the total-protein fraction of needles of Norway spruce (Picea abies [L.] Karst.) during short time incubation was used as a measure of protein synthesis in the light and in the dark. Light saturation curves, obtained for needles of different ages (new flush and 1 and 2 years old) or at different seasons (summer-winter) followed the Michaelis-Menten algorithm, exhibiting marked differences with regard to light saturation (V_max) and the half-saturation constant (K_{5. 2}). The light saturation curves of ATP level (mg g^?1 fresh weight) and of leucine incorporation into protein (nmol mg^?1 h^?1) matched each other, suggesting that photophosphorylation may be decisive for the rate of protein synthesis in the light. This is confirmed by the action spectrum of leucine incorporation. which resembled an action spectrum of leaf photosynthesis, and also by partial inhibition of protein synthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of non-cyclic photophosphorylation. Light stimulated protein synthesis showed pronounced seasonal fluctuations with a summer maximum. Furmigation of 5 years old spruce trees for 3 months with SO₂ in combination with O₃ and/or NO₂ caused a distinct enhancement of the protein synthesis rate in the light and, at a reduced absolute level, also in the dark. A similar result was obtained for 40 to 70 years old spruce stands when healthy and sick trees were compared: the latter being afflicted by the novel type of forest decline, which is characterized by yellowish bronze discolouration of sun-exposed older needles and partial loss of older needle generations (3 to 4 years old). The 1 year old needles of the unhealthy trees showed a markedly increased ¹⁴C-leucine incorporation rate which, in the dark, was even more pronounced than in the light. Stress-physiological mechanisms, which could possibly explain this stimulation, are discussed.     

Responses of Photosynthesis,Carbohydrates and Antioxidants in Needles of Norway Spruce to Slow and Rapid Changes in Ozone

The goal of the present study was to examine the effects of slow and rapid changes of ozone (O₃) concentrations on the physiological behaviour of current-year needles of Norway spruce (Picea abies (L.) Karst.). For this purpose five-year-old spruce seedlings were exposed in growth chambers for 49 days to either charcoal-filtered air, slowly increasing O₃ concentrations from zero up to 100 nl I^?1 in weekly steps of 25 nl I^?1, or immediately to 100 nl I^?1 of O₃. During the investigation period gas exchange, carbohydrate and antioxidant contents of the current flush were measured. In needles which experienced slowly increasing O₃ concentrations, cumulative O₃ uptake was approximately 30 % lower than in needles continuously fumigated with 100 nl I^?1 of O₃. The higher 0₃ uptake in the permanent 100 nl I^?1 O₃ treatment caused a pronounced decline in net photosynthesis, in the efficiency of CO₂ uptake and in the starch content of the seedlings. Initially the ascorbate pool increased, but after 5 weeks of exposure ascorbate concentrations declined and were comparable to values obtained in charcoal-filtered controls, while the thiol contents were enhanced during fumigation with permanent 100 nl I-^?1 O₃. On the contrary, slowly increasing O₃ caused a significant increase in total needle ascorbate throughout the fumigation period, which probably prevented an O₃-induced decline in the photosynthetic machinery as photosynthesis was not affected although the thiol contents were not enhanced. Furthermore, starch content was slightly higher than in O₃-free controls. These results suggest that seedlings of Norway spruce have the possibility to acclimate to O₃ stress, as slowly increasing O₃ concentrations seemed to increase resistance and the seedlings were able to compensate.     

Diagnostic parameters for selecting against novel spruce (Picea abies) decline: II. Response of photosynthesis and transpiration to acute NOx exposures

The dose- and time-response effects of sequential 3 h+3 h NO→NO₂ day time exposures [0–9 μl l^?1 (ppm) NO, 0–7.5 μl l^?1 NO₂] followed by 3 h+3 h NO→NO₂ night-time exposures (0–9.5 μl l^?1 NO, 0–9 μl l^?1 NO₂) on photosynthesis, transpiration and dark respiration were examined for nine Carpatho-Ukrainian (‘Rachovo’) half-sib families and for two populations, one from the FRG (‘Westerhof’) and one from the GDR (‘Schmiedefeld’) of Norway spruce [Picea abies (L.) Karst.], all in their 4th growing season. In a second exposure series the exposure sequence was reversed. None of the treatments induced needle scorching. The higher NO_x (NO or NO₂) concentrations reduced photosynthesis and transpiration within 1 h. The physiology of the different spruce types was affected significantly differently, the most sensitive spruce having its photosynthesis suppressed 6.6 times and its transpiration 5.5 times more than the most tolerant. ‘Westerhof’ was more sensitive to NO₂ than the average ‘Rachovo’ half-sibs. The gradients of different photosynthesis and transpiration sensitivities among the half-sibs (and ‘Westerhof’) demonstrated a significant, positive, mutual correlation, but significant negative correlations with the gradient of novel decline symptoms among their parents growing in Danish forests. The relative photosynthesis and transpiration sensitivies may thus serve as diagnostic parameters for laboratory selection of the most resistant trees to novel spruce decline. The average NO₂ flux density was three times larger than the average NO flux density. Only for NO₂ and in light was stomatal NO_x uptake larger than the total NO_x uptake. Both night transpiration and dark respiration were stimulated by high concentrations of night NO_x, preceded by day NO_x exposures.     

The Liphook Forest Fumigation Project: an overview

The aim of the Liphook Project was to assess the effects of SO₂ and O₃, singly and in combination, on coniferous forest ecosystems. More than 4000 trees of Scots pine (Pinus sylvestris), Norway spruce (Picea abies) and Sitka spruce (P. sitchensis) were fumigated for nearly 4 years using an open-air fumigation technique especially developed for the purpose. The technique eliminated artifacts due to chambers and enabled a variety of effects of the pollutant gases on forest ecosystems to be studied. Most symptoms of forest decline did not occur, but each species reacted in a different way to SO₂ stress, providing no evidence for universal forest decline symptoms. However, some of the mechanisms hypothesized to underlie forest declines were observed as an effect of SO₂ treatment, though others were not, notably any major effect of O₃. The results are assessed against proposed regulatory standards (critical loads and levels) for the protection of forest ecosystems against pollution.