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Bud endophytes of Scots pine produce adenine derivatives and other compounds that affect morphology and mitigate browning of callus cultures.

Endophytes are found in meristematic bud tissues of Scots pine (Pinus sylvestris L.) especially prior to growth, which would suggest their involvement in growth of the bud. To test this hypothesis, production of phytohormones by two bacterial (Methylobacterium extorquens, Pseudomonas synxantha) and one fungal endophyte (Rhodotorula minuta) was studied by mass spectrometry. The most common gibberellins, auxins, or cytokinins were not detected in the fractions studied. Instead, M. extorquens and R. minuta produced adenine derivatives that may be used as precursors in cytokinin biosynthesis. A plant tissue culture medium was conditioned with the endophytes, and pine tissue cultures were started on the media. Tetracycline inhibited callus production, which was restored on the endophyte-conditioned media. In addition, conditioning mitigated browning of the Scots pine explants. However, a decrease in tissue size was observed on the endophyte-conditioned media. Addition of adenosine monophosphate in the plant culture medium restored callus production and increased growth of the tissues, but had no effect on browning. Therefore, production of adenine ribosides by endophytes may play some role in the morphological effect observed in the pine tissues.

Journal Article↗

Ancient DNA from pollen: a genetic record of population history in Scots pine.

Assessments of plant population dynamics in space and time have depended on dated records of fossil pollen synthesized on a subcontinental scale. Genetic analyses of extant populations have revealed spatial relationships that are indicative of past spatial dynamics, but lack an explicit timescale. Synthesis of these data requires genetic analyses from abundant dated fossil material, and this has hitherto been lacking. Fossil pollen is the most abundant material with which to fill this data gap. Here we report genetic analyses of fossil pollen retrieved from Holtjärnen postglacial lake sediment in Sweden and show that plastid DNA is recoverable from Scots Pine and Norway spruce pollen grains that are 100 and 10 000 years old. By sequencing clones from two short plastid PCR products and by using multiple controls we show that the ancient sequences were endogenous to the fossil grains. Comparison of ancient sequences and those obtained from an extant population of Scots pine establishes the first genetic link between extant and fossil samples in this species, providing genetic continuity through time. The finding of one common haplotype present in modern, 100-year old and 10 000-year old samples suggests that it may have persisted near Holtjärnen throughout the postglacial period. This retrieval of ancient DNA from pollen has major implications for plant palaeoecology in conifer species by allowing direct estimates of population dynamics in space and time.

Base Sequence↗

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.

Acyltransferases↗

Assessing environmental and physiological controls over water relations in a Scots pine (Pinus sylvestris L.) stand through analyses of stable isotope composition of water and organic matter.

This study investigated the influence of meteorological, pedospheric and physiological factors on the water relations of Scots pine, as characterized by the origin of water taken up, by xylem transport as well as by carbon isotope discrimination (Delta13C) and oxygen isotope enrichment (Delta18O) of newly assimilated organic matter. For more than 1 year, we quantified delta2H and delta18O of potential water sources and xylem water as well as Delta13C and Delta18O in twig and trunk phloem organic matter biweekly, and related these values to continuously measured or modelled meteorological parameters, soil water content, stand transpiration (ST) and canopy stomatal conductance (G(s)). During the growing season, delta18O and delta2H of xylem water were generally in a range comparable to soil water from a depth of 2-20 cm. Long residence time of water in the tracheids uncoupled the isotopic signals of xylem and soil water in winter. Delta18O but not Delta13C in phloem organic matter was directly indicative of recent environmental conditions during the whole year. Delta18O could be described applying a model that included 18O fractionation associated with water exchange between leaf and atmosphere, and with the production of organic matter as well as the influence of transpiration. Phloem Delta13C was assumed to be concertedly influenced by G(s) and photosynthetically active radiation (PAR) (as a proxy for photosynthetic capacity). We conclude that isotope signatures can be used as effective tools (1) to characterize the seasonal dynamics in source and xylem water, and (2) to assess environmental effects on transpiration and G(s) of Scots pine, thus helping to understand and predict potential impacts of climate change on trees and forest ecosystems.

Carbon Isotopes↗

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.

Acyltransferases↗

George Scharpe, c.1581-1637. A Scots doctor at Montpellier.

Before the eighteenth century many Scots studied medicine at the medical schools of Europe, of which Montpellier was frequently the choice. George Scharpe, an early student of the University of Edinburgh, graduated in medicine at Montpellier and joined the medical faculty, where his long career can be traced from contemporary records. The practice of Scots studying abroad is described, as is Languedoc in the early seventeenth century a region and period devastated by the religious wars of France.

France↗

Biochemical parameters as biomarkers for the early recognition of environmental pollution on Scots pine trees. II. The antioxidative metabolites ascorbic acid, glutathione, alpha-tocopherol and the enzymes superoxide dismutase and glutathione reductase.

Field investigations with Scots pine trees (Pinus sylvestris L.) were performed in eastern Germany, where ambient SO2, NOx and O3 concentrations differed significantly in 1992-99 at three sites, namely Neuglobsow (yearly mean SO2 in 1992: 9 microg m(-3)), Taura (yearly mean SO2 in 1992: 54 microg m(-3)) and Rösa (yearly mean SO2 in 1992: 73 microg m(-3)). To investigate the effects of SO2, NOx and O3 on antioxidants (superoxide dismutase, ascorbic acid, glutathione, glutathione reductase, alpha-tocopherol) and pigments including chlorophyll fluorescence as well as visible damage symptoms in the form of needle yellowing and tip necroses, needles of the 1st and 2nd age class from young and mature trees were collected at the sites every October. Eight years after the start of the field study in 1992, the ambient SO2 concentrations had decreased significantly at Neuglobsow (yearly mean SO2 in 1999: 4 microg m(-3)), Taura (yearly mean SO2 in 1999: 5 microg m(-3)) and Rösa (yearly mean SO2 in 1999: 5 microg m(-3)). NOx and O3 differed less at the three sites and showed no temporal variations. Whole needle glutathione continuously decreased, although concentrations were higher in needles of the 1st and 2nd age class from the polluted sites Taura and Rösa than the unpolluted site Neuglobsow. The activities of glutathione reductase exhibited the same site-related differences and temporal variations and were correlated with concentrations of oxidized glutathione (GSSG). In contrast, the activities of the enzyme superoxide dismutase and the concentrations of whole needle ascorbic acid remained unchanged over the period. Only at the end of the investigation period did the concentrations of oxidized ascorbic acid (dehydroascorbate) increase in six-month-old needles at the polluted sites Taura and Rösa. Despite the clear decreases in SO2, the visible symptoms of needle tip necroses remained unchanged, especially at the polluted sites Taura and Rösa, although the needles contained higher pigment concentrations than needles from the unpolluted sites. The results of measurements with antioxidants as biomarkers for SO2-mediated stress in pine needles show that the adult Scots pine trees at the polluted sites suffered from greater oxidative stress than the needles from the less polluted site.

Ascorbic Acid↗

Secondary metabolite concentrations and terpene emissions of Scots pine xylem after long-term forest fertilization.

Secondary compounds are known to be associated with the resistance of conifer xylem against insects and fungi. The effects of long-term forest fertilization with nitrogen (N) or with N, calcium (Ca), and phosphorus (P) on secondary compounds in the xylem of 50-yr-old Scots pine (Pinus sylvestris L.) trees were examined. Xylem samples were collected from trees growing in three locations in southern Finland: Vilppula, Padasjoki, and Punkaharju. Forests were fertilized every fifth (Vilppula and Padasjoki) or tenth (Punkaharju) year since the 1950s. We compared concentrations of individual and total monoterpenes and resin acids in the heartwood and sapwood of Scots pine. Terpene emissions were analyzed from the sapwood and total phenolics from the heartwood. Fertilization did not have any significant effect on the concentrations and emissions of xylem monoterpenes. Concentrations of several individual terpenes in sapwood were positively correlated with the corresponding terpene emission. The concentrations of individual resin acids (i.e., abietic and dehydroabietic) decreased significantly in Punkaharju, but increased in the sapwood of N-fertilized trees compared with control ones at Padasjoki and Vilppula. The concentrations of resin acids in the heartwood were not significantly affected by fertilization. Both fertilization treatments decreased the total phenolic concentrations in the heartwood of trees growing in Padasjoki. There was a significant positive correlation between the total phenolics and total resin acid concentration. Overall, resin acids and phenolics seemed be more responsive than monoterpenes to N treatment. These results suggest that forest fertilization might cause slight changes in secondary compound concentrations of xylem, and thus might have significance in the decay resistance of wood.

Animals↗

Early- and late-onset duodenal ulcers in Chinese and Scots.

Two unselected series of 528 Chinese and 539 Scottish patients with duodenal ulcer were compared. In both races, early-onset patients (symptoms began before age 30 years) in contrast to late-onset patients (symptoms began after age 30 years) constituted significantly more males, more patients with positive familial dyspepsia, more acid hypersecretors, and more gastrointestinal bleeding, but, unlike late-onset patients, there was no significant blood group O predominance, and their females did not have an older onset age than males. Among early-onset patients, Scottish patients developed symptoms at a significantly later age and had significantly more acid hypersecretors than Chinese, whereas among late-onset patients the Scots had significantly more patients with positive familial dyspepsia, in whom the frequency of acid hypersecretion was increased. These findings indicate that (i) early and late-onset duodenal ulcers are distinct subgroups, with many features common to both Chinese and Scots despite different ethnic backgrounds, suggesting the occurrence of similar pathophysiological mechanisms in the two races, (ii) for early-onset disease, these mechanisms appear to operate sooner in the Chinese, (iii) in the Scottish patients, familial late-onset duodenal ulcers with acid hypersecretion form a substantial and distinct subgroup.

Adult↗

The Mason Brown Lecture. Scots and paediatric surgery.

Scotland and Scots paediatric surgeons have been in the forefront of the development of surgical services for children. Development has occurred at different rates in sections of society. Progress or obstruction has seldom been for 'scientific' reasons but much more according to the personal interests or drive of individuals. The Scots can justifiably claim some influence in the overall evolution of paediatric surgery. It is no wonder that Henley wrote after his visit to Walter Scott in Edinburgh, "The conceit of these bloody Scotsmen is atrocious".

General Surgery↗

Growth responses of scots pine seedlings grown in peat-based media amended with natural zeolite.

Zeolite has many good features that makes it very attractive for nursery use as a growing media over others. This study was designed to investigate influence of different growing media and their mixtures (with zeolite and without zeolite) on morphological characters of scots pine seedlings. Twenty-one treatments of varying amounts of peat, fine pumice, coarse pumice, river sand, perlite and river sand were established and were sown with scots pine seeds. At the end of first growing period, 30 seedlings from each treatment were harvested and measured for height (SH), root collar diameter (RCD), root dry weight (RDW), stem dry weight (SDW) and total dry weight (TSDW). These parameters varied significantly among treatments and were lower in zeolite added media. SH negatively correlated with Na and K content and C/N ratio of growing media but positively correlated with Mn content of media. SDW and TSDW had positive correlation with N, Fe, Mn, total porosity and loss of ignition, and had negative correlation with pH, Ca, Na and K content.

Culture Media↗

Coaction of blue light and light absorbed by phytochrome in control of glutamine synthetase gene expression in Scots pine (Pinus sylvestris L.) seedlings.

The level of plastidic glutamine synthetase (GS; EC 6.3.1.2) in the cotyledonary whorl of the Scots pine (Pinus sylvestris L.) seedling was previously reported to be regulated by light. In the present paper we report on the control by light of the GS transcript level. A full-length GS cDNA clone of Scots pine was isolated (pGS1), sequenced and employed to measure GS transcript levels. Using dichromatic light treatments it was found that the transcript level is regulated by phytochrome. The strong specific effect of blue light is to be attributed to an increase of the responsiveness to phytochrome. Since no direct correlation between the transcript level and the rate of GS protein synthesis was observed, it was concluded that GS gene expression is only coarsely regulated at the level of transcript accumulation. Synthesis of GS protein is by itself light-dependent (light-mediated fine tuning of gene expression). This control at the translational level is also exerted via phytochrome with blue light determining the responsiveness of the process toward phytochrome. If the level of the far-red absorbing form of phytochrome (Pfr) is kept very low, blue light is not capable of bringing about synthesis of GS protein.

Amino Acid Sequence↗

Active oxygen scavengers during cold acclimation of Scots pine seedlings in relation to freezing tolerance.

Freezing injury of plants may be caused by the deleterious reactions of active oxygen species, and free-radical scavenging systems may be important in the alleviation of freezing stress. To test the feasibility of this hypothesis, enzymes and metabolites that cooperatively scavenge O2 and H2O2 were analyzed in Scots pine (Pinus sylvestris L.) seedlings during a stepwise cold acclimation procedure. Elevated levels of enzymatic scavengers such as ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were found, along with increased freezing tolerance during cold acclimation, supporting the hypothesis. Induction of the scavenging systems during acclimation is discussed in relation to freezing tolerance.

Acclimatization↗

Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants.

Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.

Acyltransferases↗

Gene expression of nitrite reductase in Scots pine (Pinus sylvestris L.) as affected by light and nitrate.

A partial cDNA clone (PSnir) encoding the C-terminal region of nitrite reductase was isolated from a lambda gt11 library of the gymnosperm Pimus sylvestris (L.). Nucleotide sequence analysis showed that PSnir contains a reading frame encoding 105 amino acid residues. The amino acid sequence revealed a homology to NiR of 63-68% to dicotyledoneous and of 57-59% to monocotyledoneous species. The protein region implicated to be involved in binding of the prosthetic group is highly conserved between the NiR of the gymnosperm and of angiosperms. In all organs (cotyledonary whorls, hypocotyls, roots) the pattern of NiR gene expression in response to nitrate and light is the same at the level of transcript accumulation and at the enzyme level. This suggests that regulation of NiR gene expression in the Scots pine seedling is predominantly at the level of transcript accumulation. The highest NiR appearance was observed in roots and hypocotyls. In the cotyledonary whorls only small amounts of NiR were found. In roots and hypocotyls the accumulation of NiR mRNA and the appearance of NiR protein is mainly controlled by nitrate, whereas the regulation of NiR gene expression in the whorls is strongly affected by light and the inducive effect of nitrate is only weak.

Amino Acid Sequence↗

Nearly complete genetic maps of Pinus sylvestris L. (Scots pine) constructed by AFLP marker analysis in a full-sib family.

We have constructed nearly complete linkage maps of Pinus sylvestris (L.) using AFLP markers based on a two-way pseudo-testcross strategy in a full-sib family founded in an advanced breeding program. With 39 primer combinations, a total of 737 markers (320 from the mother and 417 from the father) segregated in a 1:1 ratio, corresponding to DNA polymorphism: heterozygous in one parent and null in the other. In the maternal parent, 188 framework markers were mapped in 12 linkage groups, equivalent to the Pinus haploid chromosome number, with a total coverage of 1,695.5 cM. In the paternal parent, 245 framework markers established a map with 15 linkage groups, spanning a genome length of 1,718.5 cM. The estimated total map length was L(F) = 1,681 cM for the female and L(M) = 1,645 cM for the male using a modified method-of-moment estimator. Combining these values with those estimated from the observed map lengths in both parents, we estimated the genome length in Scots pine to be between 1,600 and 2,100 cM. Our genome coverage was estimated to be more than 98% with a framework marker interval of 20 cM for both parents. Most of the female and male linkage groups were associated through the analysis of the intercross markers.

Chromosome Mapping↗

Vitality fertilization of Scots pine stands growing along a gradient of heavy metal pollution: short-term effects on microbial biomass and respiration rate of the humus layer.

In 1992 forest vitality fertilization experiments were established on a heavy metal deposition gradient with four treatments in three replications at distances of 0.5, 4 and 8 km from a Cu-Ni smelter in order to estimate their impact on the disturbed forest ecosystem. The increase in Cu concentration in the humus (F/H) layer of the Calluna site type Scots pine (Pinus sylvestris) stands from ca. 300 to 8000 mg kg(-1) d.m. (dry matter) along the 8 km long transect towards the smelter resulted in declining soil microbial biomass and soil respiration activity. Three independent measurements of microbial biomass: C(mic)-FE (fumigation extraction), C(mic)-SIR (substrate induced respiration), and ATP have been used together with an indicator of fungal biomass (ergosterol) and microbial activity (soil respiration). Within this Cu pollution range, all the measured microbial biomass levels declined to 10%-28% of the control plot values and activity assessed by respiration was lowered to 16%. Liming has increased the C(mic)-SIR and respiration rate. Treatments with test fertilizer, made from grounded apatite, did not result in different microbial biomass and respiration rate values compared to the respective controls along the whole gradient. Nitrogen + lime treatments resulted in similar changes to lime alone. No changes, as compared to the respective control, could be detected with nitrogen fertilization at the less polluted end of the gradient.

Journal Article↗

Seasonal responses of photosynthetic electron transport in Scots pine (Pinus sylvestris L.) studied by thermoluminescence.

The potential of photosynthesis to recover from winter stress was studied by following the thermoluminescence (TL) and chlorophyll fluorescence changes of winter pine needles during the exposure to room temperature (20 degrees C) and an irradiance of 100 micromol m(-2) s(-1). TL measurements of photosystem II (PSII) revealed that the S(2)Q(B)(-) charge recombinations (the B-band) were shifted to lower temperatures in winter pine needles, while the S(2)Q(A)(-) recombinations (the Q-band) remained close to 0 degrees C. This was accompanied by a drastically reduced (65%) PSII photochemical efficiency measured as F(v)/ F(m,) and a 20-fold faster rate of the fluorescence transient from F(o) to F(m) as compared to summer pine. A strong positive correlation between the increase in the photochemical efficiency of PSII and the increase in the relative contribution of the B-band was found during the time course of the recovery process. The seasonal dynamics of TL in Scots pine needles studied under field conditions revealed that between November and April, the contribution of the Q- and B-bands to the overall TL emission was very low (less than 5%). During spring, the relative contribution of the Q- and B-bands, corresponding to charge recombination events between the acceptor and donor sides of PSII, rapidly increased, reaching maximal values in late July. A sharp decline of the B-band was observed in late summer, followed by a gradual decrease, reaching minimal values in November. Possible mechanisms of the seasonally induced changes in the redox properties of S(2)/S(3)Q(B)(-) recombinations are discussed. It is proposed that the lowered redox potential of Q(B) in winter needles increases the population of Q(A)(-), thus enhancing the probability for non-radiative P680(+)Q(A)(-) recombination. This is suggested to enhance the radiationless dissipation of excess light within the PSII reaction center during cold acclimation and during cold winter periods.

Acclimatization↗