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.     

Microsatellite loci polymorphism of chloroplast DNA of scots pine (Pinus sylvestris L.) in Asia and eastern Europe

The variability of four microsatellite loci of chloroplast DNA was studied in 38 populations of Pinus sylvestris in the European part of Russia, in Siberia, Kazakhstan, Transbaikalia and Mongolia. High variability was observed in all regions. In total, 152 haplotypes were identified. The level of population differentiation R _ST was 2.1%. The differentiation of three geographical groups of populations (European, Siberian-Kazakhstan and Transbaikalian-Mongolian) was insignificant (R _CT = 0.004). At the same time, some rare haplotypes were found to be specific for a certain geographical region. Distribution of the rare haplotypes, which differentiated European populations from Asian populations and Mongolian and Transbaikalian populations from the Siberian, showed the independence of the history of these regions. This corresponds more to the hypothesis that the modern area of Pinus sylvestris originated via settlement from many origins than to the hypothesized single center of the post-glacial recolonization. The distribution of the pairwise differences between the individual specimens corresponded to the model of sudden population growth. The assessments of the age of this event for Pinus sylvestris (4.5–4.7 million years), which were obtained on the basis of this model, significantly exceeded the age of the Quaternary. Therefore, the revealed population growth is hardly due to the changes in flora related with the glaciation, but rather mirrors the moment of the species formation.     

Catabolism of 3-indole acetic acid in protoplasts from etiolated seedlings of scots pine (Pinus sylvestris L.)

Protoplasts preparated from dark grown seedlings of Pinus sylvestris L. were incubated with 3-indole (1-¹⁴C) acetic acid and 3-indole (2-¹⁴C) acetic acid (IAA). Three catabolites were consistently produced in the (2-¹⁴C) IAA feeds, one of which co-chromatographed with 3-indole methanol on reversed phase high performance liquid chromatography (HPLC). Protoplasts feed with (1-¹⁴C) IAA produced only one labelled catabolite. The non-decarboxylated compound formed was highly polar on reversed phase HPLC, both in the ion suppression and the ion pair mode. The substance was not hydrolysable at pH 11 and 100° indicating that it is not a conjugated form. Effects of time of incubation, pH and the cofactors hydrogen peroxide and 2,4-dichlorphenol on the catabolic rate of IAA are discussed.Abbreviations BSA bovine serum albumin - DCP 2,4-dichlorophenol - HPLC high performance liquid chromatography - IAA 3-indole acetic acid - IAld 3-indole carboxaldehyde - ICA 3-indole carboxylic acid - IM 3-indole methanol - MES 4-morpholineethane sulfonic acid - MO 3-methyl-oxindol - MnO 3-methylene-oxidol - OxIAA 3-oxindole acetic acid - PEG polyethylene glycol     

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.     

Contrasting climate signals across a Scots pine (Pinus sylvestris L.) tree-ring network in the Middle Volga (European Russia)

Since the late-19th century, the Middle Volga has played a major role in the supplying grains and other agricultural products to European Russia. The study area is located in the south of sub-boreal forest in the north and in the forest-steppe in the south. Due to large seasonal differences in rainfall, agriculture in the region, especially in its southern part, strongly depends on hydroclimate variability. According to climate model forecasts, the frequency and intensity of droughts in the Middle Volga are expected to increase due to ongoing warming. Here we introduce 16 new Scots pine tree ring width (TRW) chronologies (Pinus sylvestris L.) from the region and use a dendroclimatological approach to determine what climatic factors drive radial growth. Our analysis revealed contrasting climate signals across the network of sites with chronologies from the north showing weak correlation with May temperature and precipitation (r = −0.27 and r = 0.28, respectively), while the southern sites demonstrated stronger relationships with climate in the first half of the vegetation season (May to July temperature, r = −0.26 to −0.43; May and July precipitation, r = 0.29–0.35). The northern sites did not demonstrate a strong growth response to the self-calibrated Palmer drought severity index (scPDSI) whereas the southern group was more drought sensitive had a strong drought response and positively correlates with scPDSI for the period from previous July to the current October (r = 0.27–0.56). Based on this strong relationship between southern TRW and scPDSI we reconstruct June-September scPDSI using the most sensitive sites (T04S, T06S, T08S) for the period from 1830 to 2014. The model explains 31% of variance. Our reconstruction shows droughts in 19th century: in 1831–33, 1851, 1853, 1859, 1863–65, 1880, 1891–92, 1897–98 and in 20–21th centuries: in 1906, 1921, 1936, 1939, 1967, 1975, 1996, 2010.     

The postglacial history of Scots pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial DNA variation

The geographical structure of mitochondrial (mt)DNA variants (mitotypes) was investigated in 38 western European populations of Scots pine Pinus sylvestris using restriction fragment length polymorphism (RFLP) analysis of total DNA and a homologous cox1 probe. Three major mitotypes (designated a, b and d ) were detected. Within Spain all three major mitotypes were found, gene diversity was high, H_T = 0.586, and this diversity was distributed predominantly among rather than within populations (F_ST(M) = 0.813 for the seven Spanish populations). Mitotype d was present only in the most southerly population from the Sierra Nevada . Elsewhere in Europe, populations showed little or no mtDNA diversity within regions, but there were marked differences between regions. Italian populations were fixed for mitotype b ; populations from northern France, Germany, Poland, Russia and southern Sweden were fixed for mitotype a ; while populations in northern Fennoscandia were fixed for mitotype b . The isolated Scottish populations were predominantly of mitotype a , but mitotype b was present in three of the 20 populations scored. In Scotland, UK gene diversity (H_T = 0.120) and genetic differentiation among populations (F_ST(M) = 0.37) was much lower than in Spain. When interpreted in the light of complementary data from pollen analysis and nuclear genetic markers, the results suggest that present-day populations of P. sylvestris in western Europe have been derived from at least three different sources after glaciation.     

Among population differentiation at nuclear genes in native Scots pine (Pinus sylvestris L.) in Scotland

In the Scottish Highlands, Scots pine is at the north-western extreme of its wide natural distribution. Here, the remaining native populations are patchily distributed in highly variable environments, from the more continental, drier eastern Highlands to the milder, wetter Atlantic Ocean coast. As these pinewoods are the remnants of a naturally established forest, they form a valuable system for analysis of genetic and adaptive variation in heterogeneous environments. Using samples from across the Scottish population, we analysed data from nuclear and mitochondrial genes to assess patterns of within and between population genetic variation. Within population diversity levels were high, and significant genetic differentiation among pairs of Scottish populations at relatively small spatial scales was present at several nuclear loci. At these loci, no differentiation had been found among continental populations, even those separated by large geographic distances. Overall, no clear clustering of Scottish samples was found in population structure analysis suggesting that geographically distant populations with high intra-population nucleotide diversity are not strongly isolated or diverged from each other. Scottish populations lacked a mitotype that is widespread in eastern and north-eastern Europe, indicating that pines from that area may not have participated in the most recent colonisation of the British Isles.     

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.     

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.     

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.     

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.     

Assessment of scots pine (Pinus sylvestris L.) respiration at culmination stage of its current growth in forest-steppe zone of Pre-Baikal area

Respiration and growth of vegetative organs of the Scotch pine model trees have been studied at the culmination stage of its current growth in forest-steppe zone of Pre-Baikal area during vegetation periods in 1976–2005. The presence of a close relationship between vegetative organ respiration and the temperature and processes of their growth is the basis of the assessment of respiration, a calculation of the intensity of which is performed per the surface and absolute dry mass (a.d.m.) of these organs. Regardless of the basis for calculating the respiration rate in the studied organs, its value decreases from apical meristems towards the base of organs. The ratio between the total respiration of over- and underground pine organs during its calculation per the surface and a.d.m. was 3: 2 and almost 2: 1, respectively. The pine respiration increased approximately two times from the beginning to the end of the observation period (regardless of the base of calculation its intensity), while its average value calculated per the surface and a.d.m. during this period was 32.8 and 36.9 kg CO₂, respectively.     

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.     

Gene-pool variation in caledonian and European Scots pine (Pinus sylvestris L.) revealed by chloroplast simple-sequence repeats.

We have used polymorphic chloroplast simple-sequence repeats to analyse levels of genetic variation within and between seven native Scottish and eight mainland European populations of Scots pine (Pinus sylvestris L.). Diversity levels for the Scottish populations based on haplotype frequency were far in excess of those previously obtained using monoterpenes and isozymes and confirmed lower levels of genetic variation within the derelict population at Glen Falloch. The diversity levels were higher than those reported in similar studies in other Pinus species. An analysis of molecular variance (AMOVA) showed that small (3.24-8.81%) but significant (p < or = 0.001) portions of the variation existed between the populations and that there was no significant difference between the Scottish and the mainland European populations. Evidence of population substructure was found in the Rannoch population, which exhibited two subgroups. Finally, one of the loci studied exhibited an allele distribution uncharacteristic of the stepwise mutation model of evolution of simple-sequence repeats, and sequencing of the PCR products revealed that this was due to a duplication rather than slippage in the repeat region. An examination of the distribution of this mutation suggests that it may have occurred fairly recently in the Wester Ross region or that it may be evidence of a refugial population.     

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.     

Free amino acids production by ectomycorrhizal fungi of pine (Pinus sylvestris L.)

Studies on free amino acids production by five species of ectomycorrhizal fungi (Amanita muscaria, Suillus granulatus, Suillus luteus, Suillus bovinus and Rhizopogon luteolus) show that all the fungi produced mainly: glutamic acid, leucine, lysine, ornithine, arginine and an unidentified ninhydrin-positive compound X3. Both the quality and quantity of amino acids released was different in the fungal species studied. The predominant amino acids in post-culture liquids in general did not exceed 1.5 micrograms/mg dry mass.     

Seasonal changes in antioxidant level of scots pine (Pinus sylvestris L.) needles exposed to industrial pollution. I. ascorbate and thiol content

Current and previous year needles from three 16 years-old populations of Scots pine (Pinus sylvestris L.) trees were seasonally collected at the three experimental areas: Luboń- close to the phosphate fertiliser factory, Głogów — close to the copper foundry and Kórnik — control site. Głogów is the most polluted site, where at 1998 monthly mean daily concentrations of different pollutants were: SO₂ - 17 μg·m⁻³, NO_x - 12 μg·m⁻³ and dust containing heavy metals as Cu, Pb, Cd - 29 μg·m⁻³. Trees growing in Luboń were influenced for many years by high concentration of SO₂ and fluor compounds. A few years ago emissions were markedly reduced, but low pH of soil and high concentration of aluminium ions still influence the growth of trees. Seasonal changes of ascorbate and thiol content were observed in each needle class and population, with the maximum in the winter and minimum in the summer. In needles from trees growing on polluted sites higher level of ascorbic acid and thiols comparing to control site was observed. Significant differences appeared in each population of Scots pine growing under higher pollution stress in the Głogów site. In needles from trees growing in Luboń significant differences in ascorbic acid and thiols content were evidently less numerous. Needles from polluted sites in some seasons contained significantly more malondialdehyde (MDA) and those was more frequent in Głogów than in Luboń. The results indicated that in the Głogów site trees are more influenced by pollution stress than in Luboń and the defense reaction measured as an increase of the antioxidant level is more evident.