Temperature and light dependent modifications of chlorophyll fluorescence kinetics in spruce needles during winter

Prompt chlorophyll a fluorescence kinetics at room temperature were measured from intact spruce needles. The fluorescence signal was recorded after varying light pretreatments. During the winter, induction curves showed characteristic changes in both the initial peak of fluorescence FV/FP (FP-FO/FP) and the steady state level F_dr (FP-FT/FP). Winter stress induced decreases in both values which showed close correlation to the light and temperature pre-history of the plants. In February changes in fluorescence induction indicative of a restoration of photosynthesis were detected and these corresponded to a rise of temperature above zero in combination with low light levels. In March increasing light intensity combined with chilling temperatures induced again decreases of both values of chlorophyll fluorescence induction suggesting the occurrence of photoinhibition.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

Physical, immunological and kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase from fir (Abies alba) and spruce (Picea abies)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from fir ( Abies alba Mill.) and spruce ( Picea abies [L.] Karst.) needles was purified to homogeneity. The enzyme was isolated from crude extracts through quantitative precipitation in 40-55% and 40-60% (NH₄)₂SO₄ for fir and spruce. respectively, followed by linear sucrose gradient centrifugation. Using two dimensional gel electrophoresis, the isoelectric points were determined. For the large subunit (LSU) it was 6.7 for both species, and for the small subunit (SSU) it was 7.1 and 7.7 for fir and spruce, respectively. Very few differences in tryptic peptides and amino acid composition of Rubisco LSU were observed between fir and spruce. By contrast, marked differences characterized the same analyses for the Rubisco SSU of the two species. Moreover, substitution of residues was observed in the sequenced N-terminal region when comparing fir and spruce SSU. The Ouchterlony technique showed no immu-nochemical difference between Rubisco of fir and spruce when a rabbit antiserum to spinach Rubisco was used. The Eadie-Hofstee plots of carboxylase activity indicated that the apparent K_m(CO₂) were 31 and 36 μ M for the fir and spruce enzymes, respectively.     

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.     

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.     

NO2‐induced nitrate reductase activity in needles of Norway spruce (Picea abies) under laboratory and field conditions

The induction of activity of the enzyme nitrate reductase (NR, EC 1.6.6.1, 1.6.6.2) in needles of Norway spruce ( Picea abies [L.] Karst.) by nitrogen dioxide (NO₂) was studied under laboratory and field conditions. In fumigation chambers an increase in nitrate reductase activity (NRA) was detected 4 h after the start of the NO₂ treatment. During the first 2 days with 100 µg NO₂ m⁻³, NRA reached a constant level and did not change during the following 4 days. At the same level of NO₂, NRA was lower in needles from trees grown on NPK‐fertilized soil than on non‐fertilized soil. After the transfer of spruce trees from fertilized soil to NPK‐rich nutrient solution, NRA was transiently increased. This effect was assigned to root injuries causing nitrate transport to the shoot and subsequent induction of NRA. Neither trees on fertilized soil nor trees transferred to NPK‐poor nutrient solution had increased NRA unless NO₂ was provided. The NO₂ gradient in the vicinity of a highway was used to test the long‐term effect of elevated levels of NO₂ on needle NRA of potted and field‐grown spruce trees. Compared with less polluted sites, permanently increased NRAs were detected when NO₂ concentrations were above 20 µg m⁻³. Controls of field measurements some 10 years after the introduction of catalytic converters in cars showed no significant change neither in NO₂ levels nor in the decreasing NRA of spruce needles with the distance from the highway.     

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.     

Diurnal regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity and its content in Norway spruce needles

Diurnal changes of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activity and its content were measured to find the mechanism of RuBPCO activity regulation in Norway spruce needles. Both initial and total RuBPCO activities as well as the activation state had a typical pattern with two peaks in the morning and afternoon, respectively, and a midday depression. On the 19 October, RuBPCO content decreased during the day from 3.1 to 1.4 g m⁻², while on the 20 October it was approximately constant both in the morning and in the afternoon (2.7 g m⁻²). Neither initial nor total activity of RuBPCO copied irradiances. Relatively low morning and evening values of total activities indicate that nocturnal inhibitor CA1P is important in Norway spruce. However, the midday depression of total activity indicates that besides CA1P there function some other inhibitors of RuBPCO. In addition, the diminution of RuBPCO content during the day may indicate repression of its gene expression.     

Purification of intact chloroplasts from spruce (Picea abies) by free-flow electrophoresis

Highly purified chloroplasts were isolated from spruce ( Picea abies [L.] Karst.) needles by free-flow electrophoresis. The chloroplast crude suspension was separated into two structurally different populations. The two populations could not be distinguished according to their protein/chlorophyll ratios and protein patterns from two-dimensional gel electrophoresis. The O² evolution, however, showed differences: the chloroplast population deflected towards the cathode contained a major part of structurally intact chloroplasts in contrast to the population deflected towards the anode. The two populations were not contaminated by endoplasmatic membranes or mitochondria.     

Seasonal profiles of sulphur, phosphorus, and potassium in Norway spruce wood

Seasonal profiles of sulphur, phosphorus, and potassium content in the wood of trees have been established for the first time. This became possible by using a novel laser ablation system coupled to HR-ICP-MS for measuring these elements in Norway spruce drill cores. This technique combines excellent spatial resolution with superior detection power, and makes it possible to measure low element concentrations even in relatively narrow annual rings. Despite its low quantity in wood, sulphur is an important macronutrient for plants and seems to display seasonal variations of its concentration, which correspond to actual theories of sulphur metabolism in plants. A similar seasonal pattern was also found for phosphorus, another crucial element in tree nutrition. This was unexpected, because it was previously assumed that the distribution of phosphorus remains constant throughout the year. Potassium, the third element measured, seems to be especially accumulated in the latewood. The profiles presented in this article suggest a seasonal variation, revealing some new aspects of Norway spruce (PICEA ABIES) metabolism.     

Ethylene accelerates the breakdown of cytokinins and thereby stimulates rooting in Norway spruce hypocotyl cuttings

Cuttings were taken from 4-week-old seedlings of Norway spruce ( Picea abies L. Karst.) raised at two different irradiation levels. Rooting experiments showed that root formation was increased by the ethylene formed by adding 1-aminocyclopropane-1-carboxylic acid ACC or Ethrel, especially in the slowly rooting cuttings grown under high light (HL). Cobaltousion. an ethylene synthesis inhibitor, delayed rooting, especially in the easily rooted cuttings grown under low light (LL).
Compounds isolated from the cuttings using immunoaffinity chromatography, on a column with antibodies against cytokinins, and separated by HPLC decreased in amount during the first week of the rooting period. An increase in ethylene production accelerated this process, especially in cuttings grown under HL, whereas cobaltous ion delayed it. We suggest that ethylene stimulates rooting by enhancing the degradation of cytokinins.     

Interaction between sublethal pollution by sulphur dioxide and drought stress. The effect on photosynthetic capacity

Five-year-old Picea abies L. plants were grown in growth cabinets in the presence (3.1 μmol m⁻³) or absence of SO₂. After 5 weeks, the photosynthetic capacity of mature needles produced in the year was the same in both conditions. Trees were then submitted in situ to drought stress by withholding water. The decline of leaf photosynthetic capacity was greatest in the presence of SO₂. Chlorophyll decreased only when trees were submitted to dehydration in the presence of SO₂; however, this al-one could not account for the large decline in photosynthetic capacity observed under that condition. Needle water content was the lowest during dehydration in the presence of SO₂. It is concluded that the critical factor in the interaction between pollution by SO₂ and drought stress is the greater dehydration of the tissue found in stressed plants grown in the presence of SO₂. The large decline in photosynthetic capacity under such conditions might be due to this greater dehydration.     

Inhibition of photosynthesis and transpiration in relation to mercury-induced root damage in spruce seedlings

Spruce seedlings [ Picea abies (L.) Karst.] were exposed in nutrient solutions to a range of concentrations of HgCl₂ and CH₃HgCl for 7 weeks. The mineral, chlorophyll and water contents of the needles, and dry weights of root and needles were then estimated. The rates of photosynthesis, transpiration and dark respiration of the intact plants were determined using a Li-cor portable photosynthesis-measuring system. CO₂ uptake decreased as the supply of both forms of Hg increased. Rates of transpiration were significantly reduced only after exposure to CH₃HgCl. Similar concentrations of Hg were found in needles independent of the form of Hg supplied. Decreased rates of CO₂ uptake at 100 n M HgCl₂ and 1 n M CH₃HgCl could be explained by lower levels of chlorophyll, and by lower levels of chlorophyll and closed stomata at all other CH₃HgCl concentrations. Only at 1000 n M HgCl₂ were other photosynthetic parameters affected.
Decreased rates of transpiration and the lower chlorophyll levels in the needles did not appear to be due to the direct action of Hg, but rather to root damage that leads to a decrease in water supply and nutrient levels in the needles.