Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.)

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-l-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20–56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.     

Food reserves of Scots pine (Pinus sylvestris L.)

Summary The amounts of starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were studied in Scots pine (Pinus sylvestris L.) during an annual cycle in current-year needles and in 1-, 2- and 3-year-old needles collected shortly after bud break. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. Newly emerging needles contained relatively large amounts of starch, but only trace amounts of fat. During autumn and winter, fat content rose, while starch content decreased; amounts of both these reserve materials were very high the next spring shortly before bud break and decreased again during shoot elongation. Concentration of intermediates in triacylglycerol biosynthesis (diacylglycerols and free fatty acids), were low in summer and high in winter. The same pattern was observed for fructose and glucose (the predominant soluble sugars), galactose/arabinose and raffinose/melibiose. In contrast, sucrose concentrations were highest in spring and in autumn. Mature needles of different ages collected in May showed significant differences only in their triacylglycerol and starch content. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance and the possibility of carbohydrate-fat interconversions.     

Long-term needle litterfall of a Scots pine Pinus sylvestris stand: relation to temperature factors

Summary Needle litterfall of a Scots pine was caught over 24 years (1962–1986) with litter-traps in a Scots pine stand in southeastern Finland. The age of the trees averaged 111 years in 1962. The stand was naturally recruited and only minor silvicultural treatments occurred during its history. Litterfall showed great year-to-year variation, the minimum being 18 g/m² (in 1968) and maximum 213 g/m² (in 1973). There was no overall trend in the amount of litterfall, and the age of the stand was thus not important in determining the needle fall. We used time domain time series analysis (ARIMA) and standard climatic data (temperature, precipitation) to investigate the relationship of litterfall to climatic factors. Mean July temperature was clearly correlated with needle litterfall. High temperature in July coincided with enhanced litterfall in the same and the next year. Litterfall enhanced litterfall in the same and the next year. Litterfall increased also after high temperatures during March–April, but only in the same year. In addition to these the litterfall had a 4-year self-dependency. This is approximately the same as the mean longevity of needles in the study area. Altogether the time series model we propose covers about 90% of the variance of the original time series.     

Ectomycorrhizal development on Scots pine (Pinus sylvestris L.) seedlings in different soils

In the present study ectomycorrhizal development of Laccaria bicolor, Rhizopogon luteolus and Suillus bovinus associated with Scots pine (Pinus sylvestris) seedings was studied as affected by primary stand humus, secondary stand humus, podsolic sandy soil or peat in perspex growth chambers. After 9 weeks, ectomycorrhizal development with S. bovinus was significantly greater in peat and primary stand humus than in secondary stand humus or podsolic sandy soil. Ectomycorrhizal development with R. luteolus in secondary stand humus was higher than in primary stand humus. Degree of ectomycorrhizal development of L. bicolor, R. lutuelus and S. bovinus on Scots pine was related to potassium concentration, organic matter content and pH of the soils suggesting that chemical composition of the soils affects ectomycorrhizal development.     

Growth and nutrient characteristics in bog and fen populations of Scots pine (Pinus sylvestris)

Nutrient content in peat and growth rate, rate of nutrient accumulation and allocation patterns in Scots pine Pinus sylvestris L. from eleven natural Swedish peatlands were examined. The peatlands studied represented a wide range of climatic conditions and mire types. Whole and even-sized pines with intact root-systems were excavated to give the whole-pine budget for growth and nutrient accumulation. All samples originated from hummock communities.Pine growth and nutrient characteristics were much more variable in the minerogenous sites than in the ombrogenous sites, which indicates a larger environmental heterogeneity within the minerogenous sites. In the ombrogenous sites, rate of pine growth was constant, approximately 1 mg day^-1, and independent of latitudinal variation. There was either no relationship between latitudinal location and growth rate in the minerogenous sites, which suggests that pine growth is largely controlled by site-specific, very local conditions. The growth rate of pines was not correlated with any peat nutrient. The pines allocated a large proportion of their nutrient-pool to the metabolically active current year's growth. This is likely a trait that enables Scots pine to occupy a wide range of peatland types in which it experience a marked imbalance and shortage of nutrients.     

The effect of ozone and season on the pool sizes of cyclitols in Scots pine (Pinus sylvestris)

Summary The effect of 100 and 200 g · m^-3 (50 and 100 ppb) ozone and ambient air on 4-year-old grafts of a Scots pine clone was tested in closedtop fumigation chambers. Ozone decreased the myo-inositol and inccreased the pinitol levels in the 1-year-old needles, whereas the effect in the current-year needles was less distinct. In neither case did ambient air lead to any detectable effects on these parameters compared with controls. However, there was a pronounced chamber effect in the levels of myo-inositol and pinitol compared with trees standing in the open field, where inositol was higher and pinitol lower. There is some evidence, based on visible symptoms as well as biochemical changes, that season is an important modifier of the effect of ozone on Scots pine.     

Daily dynamics in xylem cell radial growth of Scots pine (Pinus sylvestris L.)

Daily dynamics of radial cell expansion during wood formation within the stems of 25-year-old Scots pine trees (Pinus sylvestris L.), growing in field conditions, were studied. The samples of forming wood layers were extracted 4 times per day for 3 days. Possible variations in the growth on different sides of the stem, duration of cell development in radial cell expansion phase and dynamics of cell growth in this phase were taken into account. The perimeters of tracheid cross-sections as a reflection of primary cell wall growth were the criterion of growth in a radial direction. For the evaluation of growing cell perimeters a special system for digital processing and image analysis of tracheid cross-sections of the forming wood was used. Growth rate for certain time intervals was estimated by the change in the relation of the perimeter of each observed cell in each of ten tracheid rows in each of 12 trees to the perimeter of the xylem cell of the same row before the expansion. Temporal differences in average values of the relations were estimated by Analyses of Variance. The existence of daily dynamics of Scots pine xylem cell radial growth has been proved. Intensive growth of pine tracheids has been shown to occur at any time of the day and to depend on the temperature regime of the day and the night as well as water supply of stem tissues. Moreover, reliable differences (P = 0.95) in the increment of cell walls during tracheid radial expansion have been found. Pulsing changes of the water potentials both of the cell and the apoplast, as the reason for the fluctuations of radial cell growth rate, were discussed.     

Effect of light on abscisic acid content in photosensitive Scots pine (Pinus sylvestris L.) seed

Dormancy-breaking treatment of the photosensitive Scots pine (Pinus sylvestris L.) seed by white light incubation or a 15-min exposure to red light decreased the abscisic acid content prior to radicle protrusion. Incubation in the dark or exposure to red light followed by a 5-min far-red light irradiation did not cause as great a decrease in abscisic acid content nor was the dormancy relieved. The ability of the far-red light to keep the ABA level high and to prevent germination gradually disappeared as the length of the dark period between the red and far-red treatments was increased to 24 h. ABA was quantified on a gas chromatograph with an electron capture detector.     

Photoreactivation of the UV light effects on growth of Scots pine (Pinus sylvestris L.) seedlings

Summary Ultraviolet-B light (UV-B) and ultraviolet-A light (UV-A) at higher doses exert a strong inhibitory (toxic) effect on axis growth in Scots pine (Pinus sylvestris L.) seedlings. This effect is unrelated to control of growth rate by phytochrome. Rather, after a toxic UV dose growth of the pine seedling no longer responded to phytochrome. Both, the effect of UV-B as well as the inhibiting effect of UV-A could be photoreactivated by blue light (B). The action of UV-A was 2 fold: (i) it exerted a toxic effect which could be photoreactivated by B, and (ii) applied after UV-B it photoreactivated to some extent the toxic UV-B effect. Obviously, the UV-A range causes a toxic effect, and at the same time is capable of photoreactivating the toxic UV effect. At higher doses the toxic effect prevails.     

Food reserves of scots pine (Pinus sylvestris L.)

Summary Starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were measured in 30-year-old Scots pine (Pinus sylvestris L.) trees during an annual cycle in the sapwood (youngest ten xylem rings). The radial distribution of carbohydrates and lipids was studied in the trunkwood of 90 -to 150-year-old Scots pine trees collected at the end of the growing season. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. The amounts of glucose, fructose, sucrose, and galactose/arabinose in the sapwood were slightly higher in winter than in summer. Raffinose/stachyose increased up to 5-fold during the cold period. At the beginning of the growing season starch amounts rose, and then decreased in summer and autumn. No concentration changes were observed in the total amounts of diacylglycerols and fatty acids throughout the year. Triacylglycerol levels were slightly higher in February than in summer and autumn. Relative frequencies of individual fatty acids were similar in all lipid fractions. Glucose, fructose, sucrose, starch and triacylglycerols disappeared almost entirely at the transition zone from sapwood to heartwood. In contrast, free fatty acids and galactose/arabinose rose in centripetal direction, and diacylglycerols remained constant across trunk cross-sections. The relative amounts of individual fatty acids changed markedly in the free fatty acid fraction and in the triacylglycerols when crossing the sapwood-heartwood boundary. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance, and heartwood formation. The results are compared to those found in needles.     

Seasonal changes in the utilization and turnover of assimilation products in 8-year-old Scots pine (Pinus sylvestris L.) trees

Summary The present study aimed at a physiological understanding of the seasonal changes of the carbohydrate patterns and levels in the various tissues of 8-year-old Scots pine (Pinus sylvestris L.) trees growing under ambient climatic conditions in the botanical garden at Bayreuth. The photosynthates of selected twig sections were labelled by ¹⁴CO₂ fixation and after chase periods of 1 h up to 8 months, the distribution of radiocarbon in the whole trees was determined and the labelling of identified carbohydrates was compared with the levels of these compounds in the individual tissues. Bud break and sprouting in spring is exclusively supplied by the recent photosynthates of the previous year's needles. During summer assimilates of the old needles were utilized for secondary growth of the axial system while growth of the recent-year's shoots was supported by their own photosynthesis. In autumn, soluble carbohydrates were produced instead of starch, a major part of which in addition to recent photosynthates was utilized for root growth during the cold season. Another part of the autumnal storage material was incorporated into the cell walls of the latest xylem and phloem elements still in winter. A pronounced starch-oligosaccharide interconversion upon frost hardening, and its reversal in spring as has been described for deciduous trees, could not be observed. This was due to maintenance of photosynthetic capability even in the cold season and the replacement of consumed storage material especially in late winter and early spring by new photosynthates.     

Sensitivity to Cd or Zn of host and symbiont of ectomycorrhizal Pinus sylvestris L. (Scots pine) seedlings

Pinus sylvestris seedlings infected with either the ectomycorrhizal (ECM) fungus Paxillus involutus or Suillus variegatus were exposed to a range of Cd or Zn concentrations. This was done to investigate the relationship between the sensitivity of ECM fungi and their host plants over a wide range of concentrations. P. involutus ameliorated the toxicity of Cd and Zn to P. sylvestris with respect to root length, despite significant inhibition of ECM infection levels by Cd (Cd EC₅₀ [effective concentration which inhibits ECM infection by 50%] values were: P. involutus 3.7 μg g^-1 Cd; S. variegatus 2.3 μg g^-1 Cd). ECM infection by P. involutus also decreased Cd and Zn transport to the plant shoots at potentially toxic concentrations and also influenced the proportion of Zn transported to the roots and shoots, with a higher proportion retained in the roots of the seedlings. ECM infection did increase host biomass production, but this was not affected by the presence of Cd or Zn. Root and shoot biomass production by P. sylvestris, in both the presence and absence of ECM fungi, was unaffected by Cd and Zn at all concentrations tested. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of elevated CO2 and temperature on secondary compounds in the needles of Scots pine (Pinus sylvestris L.)

The aim of this study was to evaluate the long-term effects of elevated CO₂ concentration (doubling of ambient CO₂ concentration) and temperature (2–6°C elevation) on the concentration and content of secondary compounds in the needles of Scots pine (Pinus sylvestris L.) saplings grown in closed-top environmental chambers. The chamber treatments included (1) ambient temperature and CO₂, (2) ambient temperature and elevated CO₂, (3) elevated temperature and ambient CO₂, and (4) elevated temperature and elevated CO₂. The needle sampling and analyses of monoterpenes, HPLC-phenolics and condensed tannins in current- and 1-year-old needles were made in two consecutive years. The results showed that the effects of elevation of CO₂ and temperature were greatest on the monoterpene concentration in the needles while the concentration of HPLC-phenolics remained almost unaffected by the changed growing conditions. Most of the observed decrease in monoterpene concentration was caused by the CO₂ enrichment while the effect of elevated temperature alone was not as significant. The accumulation of condensed tannins tended to increase due to the elevation of CO₂ alone compensating the reduced carbon allocation to monoterpenes. Overall, the responses of the concentrations of secondary compounds to the elevation of CO₂ and temperature are variable and depend strongly on the properties and characteristics of each compound as well as on the interrelation between the production of these compounds and the primary production of trees.     

Triacylglycerols in the seeds of northern Scots pine,Pinus sylvestris L., and Norway spruce,Picea abies (L.) Karst.

The geographical variation in the composition of triacylglycerols in seeds of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.], grown in Finland, was analyzed. The total lipid content of pine seeds was slightly higher in the northernmost provenance (68 °50N), whereas the lipid content of spruce seeds was not affected systematically by the geographical origin of the seeds. The species studied differed in the proportions of fatty acids in their triacylglycerols, though the three most abundant components were the same, i.e. oleic acid (181 n9), linoleic acid (182n-6) and 5,9,12-octadecatrienoic acid (183 5c9c12c). These fatty acids corresponded to more than 80 mol% of the total fatty acids. According to mass spectrometric analyses, the triacylglycerols of both spruce and pine seeds consisted of the same molecular species with 52–56 acyl carbons, but in different proportions. Molecules with 54 acyl carbons represented approximately 75% of the pine and 85% of the spruce triacylglycerols, with the most abundant molecular species being 545, 546 and 547. Some minor differences in the fatty acid composition of triacylglycerols of pine seeds from different seed collecting areas were found: the proportion of linoleic acid slightly increased whereas that of 5,11,14-eicosatrienoic acid decreased towards the northern origins. Similar differences were not found in the proportions of fatty acids in spruce seed lots. Furthermore, the proportions of triacylglycerols in both pine and spruce seeds from northern and southern collecting areas were not significantly different. The higher content of total lipids in spruce seeds compared with pine seeds may be due to the structure of the seed coat, and the lipophilic layers inside it, acting as a barrier to imbibition.     

Freeze-preservation of buds from Scots pine trees

A procedure has been developed for freeze-preservation of buds of the Scots pine (Pinus sylvestris L.). Instead of liquid nitrogen, cold storage in –80°C was used. The partly dormant material used in the experiments was obtained directly from a natural stand in Northern Finland and no prefreezing or cryoprotectants for preconditioning were used. Cooling velocity was 1°C/min up to a terminal freezing temperature of –39°C, after which the buds were immersed in liquid nitrogen at –196°C for 10 minutes. The material was then transferred to a deepfreezer at –80°C and stored up to 6 months. After rapid thawing, the buds were sterilized and their viability was tested by FDA staining and by culturing meristems on 1/2 MS medium for at least two weeks. All the freezing experiments were performed during March and April. The best survival of buds (90–100%) was achieved at the beginning of April, after which a pronounced decline in survival occurred obviously due to a rise in the water content of the buds.     

Chemical composition and biochemical changes in needles of Scots pine (Pinus sylvestris L.) stands at different stages of decline in Bulgaria

Water content was decreased in the needles of Scots pines (Pinus sylvestris L.) with moderate and slight levels of stress-induced decline. In pines with moderate decline a misbalance of foliar nutritional elements was observed. In 1-year-old needles of afflicted pines, the content of potassium and calcium was decreased, but the content of phosphorus, magnesium, iron, manganese and of the heavy metals cadmium and lead was increased. In 2-year-old needles of damaged pines, the content of calcium, iron and of the heavy metal cadmium was decreased, but the content of phosphorus, magnesium, manganese and zinc was increased. Potassium deficiency and zinc levels above toxicity tolerance characterized the nutrient status of declined Scots pines. In the same pines chlorophyll a/b ratio was diminished in 2-year-old needles and the protein content was increased in 1-year-old needles but was decreased in 2-year-old needles.In the second phase of the study 25- and 40-year-old pines with slight decline were examined. An increase in the protein content of 2-year-old needles of 25-year-old declined pines and of both 1- and 2-year-old needles of older 40-year-old pines was observed. A 280% increase of peroxidase activity in 1-year-old and a 178% increase in 2-year-old needles of 25-year-old damaged pines were shown. In older 40-year-old damaged pines a slight increase only in 1-year-old needles was seen. The observed age-related decrease in peroxidase activity in older 40-year-old symptomatic pines might be responsible for the lower resistance to decline in older pines. Our results show that clear but complex chemical and biochemical changes are observed in the needles of Scots pines in Bulgaria and that these changes hold promise for use as indicators of stress.     

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.     

Elevated CO2 concentration and temperature effects on the partitioning of chemical components along juvenile Scots pine stems (Pinus sylvestris L.)

The effects of doubled ambient [CO₂] and different temperature levels on young Pinus sylvestris growing in phytotron chambers were studied. Five chambers were supplied with ~380 (‘ambient air’) and five with ~700 μmol mol⁻¹ CO₂ (‘elevated [CO₂]’). Temperature levels in the chambers ranged in increment steps of 2°C from −4°C to +4°C relative to the long-term monthly (day and night) average air temperature levels in Berlin–Dahlem. Substrate was medium fertile; soil moisture and air humidity were kept constant. After three vegetation periods twigs and stems were harvested, weighed, homogenized, and analyzed chemically. There was no significant temperature effect on wood mass accumulation, clearest positive [CO₂] effect occurred in the youngest twigs. In total, wood mass increased by 28.5% at doubled ambient [CO₂]. N-contents (percentage) decreased at elevated [CO₂] in the uppermost stem sections and not in twig wood causing wider C/N ratios in total. In response to elevated temperature, N-contents decreased slightly in twigs (~0.3%). Traces of free glucose, fructose and sucrose, which decreased from the top to the bottom, were found in stem wood, in contrast to traces of starch that increased from the top to the bottom. In response to elevated [CO₂] only a little more (0.05%) was accumulated in the top shoot and in tendency; glucose, fructose, and sucrose contents were lower at the bottom of stems as compared to the control. There was no obvious response of these non-structural carbohydrates to elevated temperature except for starch that decreased to half of the content from the lowest to the highest temperature level. Among the hemicellulose compounds, rhamnose and arabinose declined from the top shoot to the bottom of stem, whereas 4-O-methyl-d-glucuronic-acid, mannose, and xylose increased. Contents (percentage) of galactose remained approximately stable along the stem. The clearest positive effect of elevated [CO₂] along the whole stem was found for mannose with differences of 0.6–0.3%. In contrast to rhamnose and arabinose that showed a negative response to elevated [CO₂], mannose was reduced towards the uppermost stem sections. The 4-O-methyl-d-glucuronic-acid was slightly lowered at the bottom, and galactose and xylose showed no [CO₂] response. The only hemicellulose compound which reacted to temperature elevation was galactose. It increased slightly (~0.1% per 1°C). Cellulose and lignin (Klason) behaved oppositely: cellulose increased and lignin decreased from the top to the bottom. These structural components behaved reversely also in response to elevated [CO₂]. In stem parts above the bottom section, cellulose content was slightly higher at elevated [CO₂], and lignin content was slightly lower at the bottom. Lignin reacted to temperature elevation by a very slight increase on the average (~0.1% per one 1°C). Cellulose, however, decreased by ~0.2% per 1°C temperature elevation. The importance of persistent sinks of carbon in woody plant parts is discussed in respect to the greenhouse effect.     

Soil acidification as caused by the nitrogen uptake pattern of Scots pine (Pinus sylvestris)

Three-year-old Scots pine (Pinus sylvestris) trees were grown on a sandy forest soil in pots, with the objective to determine their NH₄/NO₃ uptake ratio and proton efflux. N was supplied in three NH₄-N/NO₃-N ratios, 3:1, 1:1 and 1:3, either as ¹⁵NH₄+¹⁴NO₃ or as ¹⁴NH₄+¹⁵NO₃. Total N and ¹⁵N acquisition of different plant parts were measured. Averaged over the whole tree, the NH₄/NO₃ uptake ratios throughout the growing season were found to be 4.2, 2.5, and 1.5 for the three application ratios, respectively. The excess cation-over-anion uptake value (C_a-A_a) appeared to be linearly related to the natural logarithm of the NH₄/NO₃ uptake ratio. Further, this uptake ratio was related to the NH₄/NO₃ ratio of the soil solution. From these relationship it was estimated that Scots pine exhibits an acidifying uptake pattern as long as the contribution of nitrate to the N nutrition is lower than 70%. Under field circumstances root uptake may cause soil acidification in the topsoil, containing the largest part of the root system, and soil alkalization in deeper soil layers.     

Nitrogen mineralization and H+ transfers in a Scots pine (Pinus sylvestris L.) forest soil as affected by liming

H⁺ production due to N uptake in a mature Scots pine stand subjected to high NH₄ ⁺ deposition was previously estimated to amount to approx. 2.2 kmol ha^-1 y^-1. The question whether H⁺ transfers related to N mineralization (ammonification and nitrification) offset or corroborate this proton production is investigated in the present research. To determine N mineralization, soil cores were used of which both ends were closed with layers of ion exchange resin (IER) to prevent influx and efflux of ions. The effect of liming on N mineralization and the resulting H⁺ production was investigated in 7 incubation periods of each ca. 8 wk. Because of its high mobility NO₃ accumulated in both IER layers at the expense of that in the incubated forest floor and mineral soil. Net N mineralization in the soil cores as a whole amounted to 40 and 77 kg N ha^-1 in 384 d in the control and limed plots, respectively. In both treatments ca. 65% of mineralized N was nitrified. H⁺ production due to N mineralization amounted to approx. 1.2 kmol ha^-1 y^-1 in the control and limed plots. Liming reduced the amount of C in the forest floor, but not forest floor mass, because of an increased mixing with mineral particles.