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 implications of industrial pollution stress on lipids composition in Scots pine (Pinus sylvestris L.) roots

The influence of pollution stress (SO₂, Cu²⁺, Pb²⁺, Zn²⁺ and fluoride) on composition changes in cellular membranes of roots of three European of Scots pine (Pinus sylvestris L.) populations were examined. Plant material growing in three experimental areas: Kórnik relatively free of air pollution (control), Luboń: SO₂ and HF and Głogów: SO₂ and heavy metals. Analysis of total phospholipids and their composition indicates that the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were present in lower concentration in the root tissues from both polluted sites (Luboń and Głogów). The difference in PC:PE ratio between control and both polluted sites was greater in root of Scots pine population from Russia than in the population from Slovakia. Under pollution conditions the content of lipid soluble antioxidant α-tocopherol was lower about 220 %, in comparison to the control. The action of pollution stress also lead to lowering of unsaturated:saturated ratio of total fatty acid, and lower content of polyunsaturated fatty acids, linoleic acid (18:2) and eicosatrienoic acid (20:3). We concluded that the long-term pollution stress markedly inhibited lipid biosynthesis in root tissue of Scots pine and it is probably contribute to the reduction of productivity of forests. These results also suggest that lipid composition can be used as an indicator of changes in tissue roots of Scots pine caused by air and/or soil long-term pollution.     

No evidence for depletion of carbohydrate pools in Scots pine (Pinus sylvestris L.) under drought stress

The physiological mechanisms leading to Scots pine (Pinus sylvestris L.) decline in the dry inner alpine valleys are still unknown. Testing the carbon starvation hypothesis, we analysed the seasonal course of mobile carbohydrate pools (NSC) of Scots pine growing at a xeric and a dry-mesic site within an inner alpine dry valley (750 m a.s.l., Tyrol, Austria) during 2009, which was characterised by exceptional soil dryness. Although, soil moisture content dropped to ca. 10% at both sites during the growing season, NSC concentrations rose in all tissues (branch, stem, root) until the end of July, except in needles, where maxima were reached around bud break. NSC concentrations were not significantly different in the analysed tissues at the xeric and the dry-mesic site. At the dry-mesic site, NSC concentrations in the aboveground tree biomass were significantly higher during the period of radial growth. An accumulation of NSC in roots at the end of July indicates a change in carbon allocation after an early cessation in aboveground growth, possibly due to elevated belowground carbon demand. In conclusion, our results revealed that extensive soil dryness during the growing season did not lead to carbon depletion. However, even though carbon reserves were not exhausted, sequestration of carbohydrate pools during drought periods might lead to deficits in carbon supply that weaken tree vigour and drive tree mortality.     

Flavonol 3-O-glycoside hydroxycinnamoyltransferases from Scots pine (Pinus sylvestris L.)

Flavonol 3-O-glucosides esterified with ferulic or p-coumaric acid at positions 3' and 6' are the major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles. The last steps in the biosynthesis of these compounds are catalyzed by enzymes that transfer the acyl part of hydroxycinnamic acid CoA esters to flavonol 3-O-glucosides. A newly developed enzyme assay revealed three flavonol 3-O-glucoside hydroxycinnamoyltransferases (HCTs) in Scots pine needles with specificities for positions 3', 4' or 6'. The positions of the acyl groups were identified by cochromatography with reference compounds and by NMR spectroscopy. The enzymes were characterized by molecular mass, isoelectric point, and also pH and temperature optima. Substrate specificities for flavonol glycosides and hydroxycinnamic acid CoA esters as well as kinetic properties of 3'- and 6'HCT suggested that acylation preferably occurs with glucosides and p-coumaroyl-CoA. In addition, acylation takes place in a well-defined order, beginning at position 6' followed by acylation at position 3'. These results give the first detailed characterization of flavonol 3-O-glycoside HCTs involved in the protection of plant tissues against UV-B (280-315 nm) radiation.     

Electrophoretic analysis of genetic linkage in Scots pine (Pinus sylvestris L.)

Eighty megagametophytes from each of 24 Scots pines (Pinus sylvestris L.) were subjected to horizontal starch gel electrophoresis. The trees were from crosses among widely separated provenances, and each was polymorphic for 8 to 14 loci. Evidence for linkage among 275 two-locus combinations was tested using chi-square analysis. Data from different trees were pooled to calculate map distances for the species. Nineteen of the twenty-nine loci tested were linked in one of six groups; the groups varied in size from two to seven loci. Similarities in linkage relationships among Scots pine, other pines, and other species within the Pinaceae support karyological research that suggests extensive conservation of the conifer genome.     

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.     

Long-term effects of boron and copper on phenolics and monoterpenes in Scots pine (Pinus sylvestris L.) needles

Backgroud and aims

Plant boron (B) status is known to affect plant secondary metabolites but most studies have been short termed and in controlled environments. Copper (Cu) effects on phenolics are better known at toxic than at low levels. Here, the chemistry of Scots pine (Pinus sylvestris L.) needles was studied 20 years after fertilisation with B and Cu in a long-term field experiment on a drained boreal peatland.

Methods

Phenolic compounds were analysed from three needle year classes using high performance liquid chromatography (HPLC) and condensed tannins with modified acid-butanol assay. Monoterpenes in the youngest needles were analysed by gas chromatography–mass spectrometry (GC–MS).

Results

Needle B concentrations were at deficient level in controls (5.7 μg g^?1), but at the optimum level (12 μg g^?1) still 20 years after fertilisation. Copper concentrations were low but not deficient (4.0 μg g^?1 in unfertilised, 4.8 μg g^?1 in fertilised). Needle ageing increased the concentrations of individual phenolics in most cases, but decreased the concentration of condensed tannins. The concentrations of several individual phenolics were reduced by B fertilisation compared to B-deficient control, significantly in the cases of (+)-catechin and a neolignan. The concentrations of eight compounds and the sum of small-molecule phenolics were higher in Cu fertilised trees. Condensed tannins and monoterpenes were not affected by the micronutrients.

Conclusions

Boron and copper additions affected mostly the same phenolic compounds, but B decreased while Cu increased their concentrations, Cu effects being clearer. The higher phenolic concentrations in B deficient trees were not likely large enough to explain leader dieback in B-deficient trees. The effects and interactions of these micronutrients need to be further studied in field conditions to establish firstly if the changes in phenolics are consistent among species, and secondly what mechanisms lead to the changes. Although small, the changes in phenolic concentrations may affect the interactions of the trees with their biotic and abiotic environment, when consistent over many years.     

Trends of chhemical element content in needles of Scots pine (Pinus sylvestris L.) under various natural conditions and emission load

The studies were conducted in different parts of the Baikal Region in Southern Pribaikalia, Southwestern Transbaikalia, and Northern Pribaikalia. It was shown that the chemical element content depends largely on biogeographical conditions and varies slightly between different types of forest in the same geographical region. However, the most significant factor leading to an imbalance of most elements is the emission load. It is concluded that the investigation of element trends in assimilating organs of woody trees is an important link in the system analysis of the vital status of tree stands under natural conditions and on exposure to abiotic factors.     

Photosynthetic electron transport adjustments in overwintering Scots pine (Pinus sylvestris L.)

As shown before [C. Ottander et al. (1995) Planta 197:176-183], there is a severe inhibition of the photosystem (PS) II photochemical efficiency of Scots pine (Pinus sylvestris L.) during the winter. In contrast, the in vivo PSI photochemistry is less inhibited during winter as shown by in vivo measurements of deltaA820/A820 (P700+). There was also an enhanced cyclic electron transfer around PSI in winter-stressed needles as indicated by 4-fold faster reduction kinetics of P700+. The differential functional stability of PSII and PSI was accompanied by a 3.7-fold higher intersystem electron pool size, and a 5-fold increase in the stromal electron pool available for P700+ reduction. There was also a strong reduction of the QB band in the thermoluminescence glow curve and markedly slower Q-A re-oxidation in needles of winter pine, indicating an inhibition of electron transfer between QA and QB. The data presented indicate that the plastoquinone pool is largely reduced in winter pine, and that this reduced state is likely to be of metabolic rather than photochemical origin. The retention of PSI photochemistry, and the suggested metabolic reduction of the plastoquinone pool in winter stressed needles of Scots pine are discussed in terms of the need for enhanced photoprotection of the needles during the winter and the role of metabolically supplied energy for the recovery of photosynthesis from winter stress in evergreens.     

Environmental and developmental effects on the biosynthesis of UV-B screening pigments in Scots pine (Pinus sylvestris L.) needles

The major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles are flavonol 3-o-glycosides (F3Gs) esterified with hydroxycinnamic acids at positions 3" and 6". Acylation is the last step in biosynthesis and is catalysed by position-specific hydroxycinnamoyl transferases (3" and 6"HCT). The UV-B dependence of these enzyme activities was studied in primary needles of Scots pine seedlings grown under different UV-B conditions in environmentally controlled sun simulators. 6"HCT activity was induced upon UV-B irradiation while 3"HCT activity was not induced but showed high constitutive values. To investigate the biosynthesis of diacylated F3Gs during needle development under natural conditions, the HCT activities and metabolite contents were analysed in needles of field-grown mature pine trees. Accumulation of diacylated compounds as well as of 6"HCT activity occurred transiently in the first year of needle development only. In contrast, 3"HCT activity exhibited broad maxima in two consecutive years during needle growth. The data suggest that acylated F3Gs are first formed as soluble compounds which are then translocated into the cell wall to be bound by their hydroxycinnamoyl residues.     

Removal of nitrogen during needle senescence in Scots pine (Pinus sylvestris L.)

The concentrations of arginine, protein and total nitrogen (N) and the abundance of¹⁵N were measured in 3-and 4-year-old needles of Scots pine trees fertilized with either 0 (C), 36 (N1) or 73 (N2) kg N ha^-1 year^-1 annually for 22 years (average doses of N). Remaining green needles and needles that were shed were compared and removal of N from total, protein and arginine pools was calculated. Earlier investigations had shown that high arginine concentrations are found in needles of trees that have an excessive N supply (Näsholm and Ericsson 1990). This study aimed to elucidate the fate of the accumulated arginine during needle senescence. It was speculated that a low removal of arginine during senescence would implicate that the primary function of arginine is in N detoxification and not in N storage. Moreover, litter quality would be altered if needles are shed with high concentrations of arginine and this might affect the turnover of N in forest ecosystems. In remaining green needles, the concentration of total N increased with increasing N supply. Protein N concentrations were higher in fertilized trees, but did not differ between the two N treatments. Arginine N was low in C and N1 trees but high in N2 trees. Senescent needles from C and N1 trees had about equal total N concentrations while in N2 trees this concentration was significantly higher. Protein N in senescent needles did not differ between treatments. Arginine N, however, was less than 0.1 mg g^–1 dw in C and N1 trees but was higher than 1.5 mg g^–1 dw in N2 trees. Removal of N was highest in N1 trees followed by C trees while N2 trees removed least N from senescing needles. The high concentration of total N in senescent needles from N2 trees was to a great extent explained by a high arginine concentration.The ¹⁵N value of remaining, green needles was higher (less negative) in N2 trees than in C and N1 trees. The same pattern was found for senescent needles. Comparisons of ¹⁵N values between remaining, green and senescent needles within each treatment showed a significant increase in ¹⁵N for all treatments during senescence possibly indicating losses of N as NH₃ (g) from needles during senescence. It is concluded that arginine, accumulated in response to high N supply, is retranslocated only to a small extent during needle senescence. The ecological and physiological implications of this finding are discussed.     

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.     

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.     

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.     

Bacterial endobionts in the big non-mycorrhizal roots of Scots pine (Pinus sylvestris L.)

Subsurface bacterial growth occurred in an N-free medium inoculated with interior tissues of big non-mycorrhizal roots (7 to 8 mm diameter) of 15-20 years-old Scots pine (Pinus sylvestris L.) growing on sand dunes at the Baltic Sea of Poland. The bacteria were not N2 fixers as determined by the acetylene reduction method. Light microscopic and scanning electron microscopic observations revealed massive bacterial clusters residing in the cortical cells underlying epidermis and parenchyma. The bacteria produced yellow-green pigments on King's medium, which fluoresced under ultraviolet (UV) irradiation at 366 nm wavelength, and could be a siderophore-producing Pseudomonas.     

The effects of UV-B radiation on epidermal anatomy in loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.)

The effects of enhanced UV‐B radiation on the needle anatomy of loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.) were studied in the field under supplemental UV‐B radiation supplied by a modulated irradiation system. The supplemental UV‐B levels were designed to simulate either a 16 or 25% loss of stratospheric ozone over College Park, Maryland. Enhanced UV‐B radiation caused different responses in these two species. The needles of loblolly pine had larger amounts of tannin in the lumen of epidermal cells and more wall‐bound phenolics in the outer epidermal walls of UV‐B‐treated needles, whereas the most pronounced effect on Scots pine needles was increased cutinization. In both species, the outer epidermal cell walls thickened and the needle cross‐sectional and mesophyll areas decreased (statistically significantly only in Scots pine). This suggests that more carbon may have been allocated to the protection mechanisms at the expense of photosynthetic area. The difference in response between these species suggests that the response to UV‐B radiation is not mediated by a single mechanism and that no generalization with regard to the effects of UV‐B on conifers can be made.     

Genetic improvement of the chemical composition of Scots pine (Pinus sylvestris L.) juvenile wood for bioenergy production

Chemical composition is one of the key characteristics that determines wood quality and in turn its suitability for different end products and applications. The inclusion of chemical compositional traits in forest tree improvement requires high‐throughput techniques capable of rapid, non‐destructive and cost‐efficient assessment of large‐scale breeding experiments. We tested whether Fourier‐transform infrared (FTIR) spectroscopy, coupled with partial least squares regression, could serve as an alternative to traditional wet chemistry protocols for the determination of the chemical composition of juvenile wood in Scots pine for tree improvement purposes. FTIR spectra were acquired for 1,245 trees selected in two Scots pine (Pinus sylvestris L.) full‐sib progeny tests located in northern Sweden. Predictive models were developed using 70 reference samples with known chemical composition (the proportion of lignin, carbohydrates [cellulose, hemicelluloses and their structural monosaccharides glucose, mannose, xylose, galactose, and arabinose] and extractives). Individual‐tree narrow‐sense heritabilities and additive genetic correlations were estimated for all chemical traits as well as for growth (height and stem diameter) and wood quality traits (density and stiffness). Genetic control of the chemical traits was mostly moderate. Of the major chemical components, highest heritabilities were observed for hemicelluloses (0.43–0.47), intermediate for lignin and extractives (0.30–0.39), and lowest for cellulose (0.20–0.25). Additive genetic correlations among chemical traits were, except for extractives, positive while those between chemical and wood quality traits were negative. In both groups (chemical and wood quality traits), correlations with extractives exhibited opposite signs. Correlations of chemical traits with growth traits were near zero. The best strategy for genetic improvement of Scots pine juvenile wood for bioenergy production is to decrease and stabilize the content of extractives among trees and then focus on increasing the cellulose:lignin ratio.     

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.     

Changes in fungal community of Scots pine (Pinus sylvestris) needles along a latitudinal gradient in Sweden

Conifer needles are typically long lived and can host a diverse community of fungal species with various effects on their host tree. The purpose of this study was to analyse shifts in the fungal community of Scots pine (Pinus sylvestris) needles on different spatial scales using 454 pyrosequencing. The fungal community composition changed gradually along a north-south gradient through Sweden, representing boreal to temperate vegetation zones. OTU richness and Shannon's diversity index increased with increasing latitude, but only in naturally regenerated forests. On the tree level, needles with symptoms of disease hosted a more diverse mycobiota compared to healthy needles, presumably supporting more pathogenic or saprotrophic species. This study provides a better insight into the patterns of fungal communities of Scots pine needles and highlights the need for further experimental research to identify specific environmental factors shaping the abundance of different fungal species.