UV-B induction of flavonoid biosynthesis in Scots pine (Pinus sylvestris L . ) seedlings

 Cultivation of Scots pine (Pinus sylvestris L.) seedlings under simulated global radiation including the UV-B band (280 – 320 nm; 220 mW m^–2 UV-B_BE) led to increased formation of the diacylated flavonol glucosides 3″,6″-di-p-coumaroyl-astragalin and 3″,6″-di-p-coumaroyl-isoquercitrin in primary and cotyledonary needles, respectively. 3″,6″-Di-p-coumaroyl-astragalin was also the main constitutive diacylated flavonol glucoside in both needle types. This compound predominantly accumulated in primary needles upon UV-B irradiation, and reached concentrations of 2.4 μmol g^–1 fresh weight (fw). Its concentration was only weakly affected in cotyledonary needles. 3″,6″-Di-p-coumaroyl-isoquercitrin was mainly induced in cotyledonary needles with maximum concentrations of 0.8 to 0.9 μmol g^–1 fw, but was virtually unaffected in primary needles under the same irradiation conditions. Pulse labelling with L-(U-¹⁴C)phenylalanine revealed that these metabolites were formed de novo. Phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone synthase (EC 2.3.1.74) were only slightly induced by the UV-B treatment. The results described here represent the first report on UV-B-induced flavonoid biosynthesis in a conifer species. Received: 5 December 1995 / Accepted: 20 March 1996     

Fertilization in a mature Scots pine (Pinus sylvestris L.) stand—effects on fine roots

The effects of liquid and solid fertilizers on fine-root development were studied in a 130-year-old Scots pine (Pinus sylvestris L.) stand. Ingrowth cores,viz. initially root-free mesh bags with sieved mineral soil taken outside the plots and driven to a depth of 30 cm from the soil surface, were subsequently resampled and the amount of fine roots was estimated. The total accretion of both fine-root length and dry weight was comparatively high in the liquid fertilization plot. The most substantial net accretion, however, during the 1984 period was in the control plot. The results of the study is that the ramification pattern of the fine roots was strongly influenced by fertilization. The average number of root tips per unit length was 9.9 cm⁻¹ in the control plot cm⁻¹, compared with both the solid (A and B) and liquid fertilization plots (2.3, 3.2 and 3.3 cm⁻¹, respectively) due to a greater occurrence of mycorrhizal aggregates (‘ball mycorrhiza’). The effects of fertilization on the mineral nutrient concentrations in the fine roots were limited—the strongest effects were in the liquid-fertilized area. The observed increase in the concentration of most mineral nutrients in the latter experimental area, however, may be due to a change in the growth pattern of the fine roots rather than an effect of the fertilizers themselves.     

Stem emissions of monoterpenes,acetaldehyde and methanol from Scots pine (Pinus sylvestris L.) affected by tree–water relations and cambial growth

Tree stems are an overlooked source of volatile organic compounds (VOCs). Their contribution to ecosystem processes and total VOC fluxes is not well studied, and assessing it requires better understanding of stem emission dynamics and their driving processes. To gain more mechanistic insight into stem emission patterns, we measured monoterpene, methanol and acetaldehyde emissions from the stems of mature Scots pines (Pinus sylvestris L.) in a boreal forest over three summers. We analysed the effects of temperature, soil water content, tree water status, transpiration and growth on the VOC emissions and used generalized linear models to test their relative importance in explaining the emissions. We show that Scots pine stems are considerable sources of monoterpenes, methanol and acetaldehyde, and their emissions are strongly regulated by temperature. However, even small changes in water availability affected the emission potentials: increased soil water content increased the monoterpene emissions within a day, whereas acetaldehyde and methanol emissions responded within 2–4 days. This lag corresponded to their transport time in the xylem sap from the roots to the stem. Moreover, the emissions of monoterpenes, methanol and acetaldehyde were influenced by the cambial growth rate of the stem with 6–10-day lags.     

Does the lichen mat alleviate the effects of wet deposited nickel on soil microorganisms and Scots pine (Pinus sylvestris L.) seedlings?

A field experiment was conducted in a dry heath forest dominated by Scots pine (Pinus sylvestris L.) and a mat-forming lichen (Cladina stellaris (Opiz) Brodo) to assess the effect of wet-deposited nickel (Ni) on pine seedlings and soil microorganisms, and to explore whether an intact lichen mat could act as a buffer against heavy metal deposits. Pine seedlings were planted in quadrats covered by a natural lichen layer and in quadrats from which the lichen layer had been completely removed. The quadrats were exposed to four levels of Ni deposition: 0 (i.e., distilled water), 10, 100 and 1000 mg m^–2 year^–1 in two growing seasons. Increasing Ni deposition led to an increase in the Ni content of the needles, roots and the soil organic layer. The lichen mat reduced Ni flow to the organic soil layer, but had no significant, reducing effect on needle or root Ni concentration. The most severe Ni treatment had detrimental effects on seedling growth and increased peroxidase activity in the previous years needles. Removal of the lichen layer did not increase susceptibility of seedlings to Ni. Values of maximal carbon use efficiency (Max) and metabolic quotient (qCO₂) of the soil microorganisms indicated protective value of the lichen mat to soil microorganisms at the highest Ni treatment. Skimming per se decreased basal respiration, qCO₂ and concentrations of potassium in the soil and also increased the lag period of the microorganisms as a response to in situ substrate addition.     

Development of needle retention in Scots pine (Pinus sylvestris) in 1957–1991 in northern and southern Finland

The needle trace method was used to study retrospectively the long-term latitudinal variation in needle retention in Scots pine (Pinus sylvestris L.) in Finland. The mean annual summer needle retention (ANR) along the main stem varied from 3.4 to 6.0 needle sets during the period 1957–1991. The lowest values were observed in southern and the highest in northern Finland. The length of the growing season, expressed as the thermal sum (threshold value +5 °C), was negatively correlated with the mean ANR (r=-0.96). The geographical needle retention pattern (NRP) for the period 1957–1991 showed a clearly increasing trend from 1957 to 1969 (southern Finland) and to 1975 (northern Finland); thereafter, the NRP tended to decrease close to its minimum value recorded in 1991. The general level of the NRP was approximately 5.0 needle sets in northern Finland and 3.5–4.0 needle sets in southern Finland. The NRP, with its 6–12 year cycle for southern Finland, was clearly periodical. Differences in the NRP among the ten stands in southern Finland were small, whereas the said periodicity was missing and the differences were high among the stands in northern Finland. The results indicate that variation in the number of needle sets, viz. defoliation of pines, is a normal phenomenon. The role of net carbon assimilation as a regulator of the number of needle sets is discussed.     

Sequence variation patterns along a latitudinal cline in Scots pine (Pinus sylvestris): signs of clinal adaptation?

Adaptive clinal variation is abundant in nature, and its genetic basis is of great interest. The polygenic nature of this variation poses a challenge for identifying the causative loci underlying these adaptations. Here, we have examined the patterns of sequence variation in ten candidate genes for timing of bud set and cold tolerance, traits that form strong latitudinal clines in Scots pine (Pinus sylvestris). A set of reference genes was used for comparison and to infer a simplified demographic background model with approximate Bayesian computation methods. Against the resulting bottleneck model, the neutrality tests show little signs for local adaptation, but species-wide selection is suggested in some of the studied genes. In line with the theoretical expectations, we see little evidence for adaptive differentiation with F _ST methods. Instead, allele frequency clines were found in three genes (dhn1, ftl2, and prr1). Our results add to the discussion on which molecular signals best characterize a gene subject to clinally varying selection. This will be especially relevant when these kinds of observations can be examined in parallel with association study results.     

Emission of hydrogen sulfide by twigs of coniferes — acomparison of Norway spruce (Picea abies (L.) Karst.), Scotch pine (Pinus sylvestris L.) and Blue spruce (Picea pungens Engelm.)

The emission of reduced volatile sulfur compounds from twigs of Norway spruce (Picea abies (L.) Karst.) was measured in the field by cryosampling and gaschromatographic analysis. Trees were growing in the Erzgebirge (E-Germany) at Oberbärenburg and at the Kahleberg and at a third stand in NW-Bavaria (S-Germany). Emission rates were also measured for Scotch pine (Pinus sylvestris L.) and Blue spruce (Picea pungens Engelm.) at the Kahleberg. Twigs still attached to the trees were enclosed in a flow-through gas exchange cuvette. H₂S was detected as the predominant reduced sulfur compound emitted from the twigs. The mean H₂S emission rate from twigs of Norway spruce varied between 0.04 pmol kg^-1 dw s^-1 at Würzburg and 6.21 pmol kg^-1 dw s^-1 at the Kahleberg. Comparing different species at the Kahleberg, the mean H₂S emission rate was almost the same from twigs of Norway spruce (6.2 pmol kg^-1 dw s^-1) and Blue Spruce trees (5.9 pmol kg^-1 dw s^-1) but it was approximately 18 times higher for Scotch pine (110 pmol kg^-1 dw s^-1). The percentage of SO₂-exclusion via H₂S-emission of the tree species investigated at the Kahleberg is calculated on the basis of data on SO₂ fluxes. It is very small for Norway spruce and Blue spruce. However, for Scotch pine, H₂S emission contributes about 10% to the detoxification of SO₂.     

Speciation history of three closely related pines Pinus mugo (T.), P. uliginosa (N.) and P. sylvestris (L.)

Nucleotide polymorphisms at genomic regions including 17 nuclear loci, two chloroplast and one mitochondrial DNA fragments were used to study the speciation history of three pine species: dwarf mountain pine (Pinus mugo), peat‐bog pine (P. uliginosa) and Scots pine (P. sylvestris). We set out to investigate three specific speciation scenarios: (I) P. uliginosa is a homoploid hybrid between the other two, (II) the species have evolved without gene flow after divergence and (III) there has been substantial gene flow between the species since their divergence. Overall, the genetic data suggest that P. mugo and P. uliginosa share the same gene pool (average net divergence of 0.0001) and that the phenotypic differences (e.g. growth form) are most likely due to very limited areas of the genome. P. mugo and P. uliginosa are more diverged from P. sylvestris than from each other (average net divergence of 0.0027 and 0.0026, respectively). The nucleotide patterns can best be explained by the divergence with migration speciation scenario, although the hybrid speciation scenario with small genomic contribution from P. sylvestris cannot be completely ruled out. We suggest that the large amount of shared polymorphisms between the pine taxa and the lack of monophyly at all loci studied between P. sylvestris and P. mugo–P. uliginosa can largely be explained by relatively recent speciation history and large effective population sizes but also by interspecific gene flow. These closely related pine taxa form an excellent system for searching for loci involved in adaptive variation as they are differentiated in phenotype and ecology but have very similar genetic background.     

Did the ambient ozone affect stem increment of Scots Pines (Pinus sylvestris L.) on territories under regional pollution load? Step III of Lithuanian studies

This study aimed to explore if changes in stem increment of Scots pines (Pinus sylvestris L.) could be related to changes in ambient ozone concentration when the impact of tree dendrometric parameters (age, diameter) and crown defoliation are accounted for. More than 200 dominant and codominant trees from 12 pine stands, for which crown defoliation had been assessed since 1994, were chosen for increment boring and basal area increment computing. Stands are located in Lithuanian national parks, where since 1994-95 Integrated Monitoring Stations have been operating. Findings of the study provide statistical evidence that peak concentrations of ambient ozone (O3) can have a negative impact on pine tree stem growth under field conditions where O3 exposure is below phytotoxic levels.     

Revisiting protein heterozygosity in plants��nucleotide diversity in allozyme coding genes of conifer Pinus sylvestris

Allozyme variation has been and continues to be a major source of information on the level of genetic variation among plant species. Deciphering the molecular basis of electrophoretic variation is essential for understanding the forces affecting the protein level variation. In this study, the relationship between allozyme heterozygosity and nucleotide diversity in plants is investigated among and within species. Allozyme and nucleotide diversity in 27 plant species was reviewed. At the multilocus level, the two methods are congruent: a clear correlation between the two measures of genetic diversity among plant species was observed, strengthening the view that effective population size is the major determinant of genome-wide diversity. Nucleotide diversity at six allozyme coding genes (6pgdB, aco, gdh, gotC, mdhA, and mdhB) in conifer Pinus sylvestris was investigated jointly with electrophoretic data. Single non-synonymous charge-changing mutations were found together with electrophoretic alleles that consequently were mutationally unique. Synonymous site nucleotide diversity (point estimate of θ _W—0.009 per bp) and silent site divergence from Pinus pinaster at allozyme coding loci were at comparable levels with other loci in the species. Linkage disequilibrium was extensive compared to earlier estimates from P. sylvestris and other trees, spanning several kilobases. Allozyme coding genes had an excess of closely related haplotypes whose frequency has recently increased possibly as a result of partial selective sweeps or balancing selection, but complex demographic effects cannot be excluded.     

Expression of recombinant human IFN-γ protein in soybean (Glycine max L.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Globular androgenic haploid embryos of TV21 and TV19 cultivars of Camellia ssp., obtained on embryo induction medium (EIM), Murashige and Skoog medium...     

Structural and functional properties of antimicrobial protein L5 of Lysоbacter sp. XL1

The Gram-negative bacterium Lysobacter sp. XL1 secretes into the extracellular space five bacteriolytic enzymes that lyse the cell walls of competing microorganisms. Of special interest are homologous lytic proteases L1 and L5. This work found protein L5 to possess Gly-Gly endopeptidase and N-acetylmuramoyl-l-Ala amidase activities with respect to staphylococcal peptidoglycan. Protein L5 was found to be capable of aggregating into amyloid-like fibril structures. The crystal structure of protein L5 was determined at a 1.60-Å resolution. Protein L5 was shown to have a rather high structural identity with bacteriolytic protease L1 of Lysobacter sp. XL1 and α-lytic protease of Lysobacter enzymogenes at a rather low identity of their amino acid sequences. Still, the structure of protein L5 was revealed to have regions that differed from their equivalents in the homologs. The revealed structural distinctions in L5 are suggested to be of importance in exhibiting its unique properties.

     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

A dendroecological reconstruction of use by Saami of Scots Pine (Pinus sylvestris L.) inner bark over the last 350 years at Sädvajaure,N. Sweden

The historical use of Scots Pine (Pinus sylvestris L.) inner bark by the Saami near Lake Sädvajaure, N. Sweden was studied with dendro-ecological methods. Damming of the lake for hydroelectric power enabled destructive sampling of all pines with scars from bark peeling in an area of 870 ha. A total of 111 dead and live pines with 136 bark peelings were found. Stem-sections were taken for cross-dating to determine the precise peeling year and season. The oldest peeling was dated to 1618, which is the oldest reported evidence of Saami use of inner bark. No bark peelings were made after 1870 in the studied area which coincides with a shift to more extensive reindeer herding, and with colonization by Swedish farmers in the area. The regular use of inner bark over time and the absence of peeling peaks in known agrarian famine years support the hypothesis that inner bark was used regularly, and not only as an emergency food. Changes in spatial peeling activity around the lake is interpreted as temporal changes in nomadic fishing activity. We conclude that tree ring studies can provide valuable information about former mobile use of natural resources by Saami in boreal forest landscapes.     

Germination and phenology of 1-year-old maritime pine (Pinus pinaster Aït.) seedlings under continuous light

Summary Nine half-sib families of maritime pine (Pinus pinaster Aït.), of known vigor and growth pattern (monocyclic vs polycyclic), were grown under continuous light in two temperature regimes. Results of germination and development to the beginning of the second flush are reported here. Families significantly differed for mean date of germination and date of first budset. After 18 weeks of continuous light most of the seedlings had set a bud. High temperature (25° C vs 25° C/20°C) hastened first budset by approximately 5 days. However, 3 families had not reached 75% budset at the end of the experiment. Variation in date of first budset was almost exclusively explained by variation in the period between initiation of primary needles and budset. Further, 2 developmental stages could be distinguished within this period, the boundary between them being the emergence of the first secondary needles. Variation in height at first budset was mostly due to variation in growth rate, not in duration. Possible causes of early budset and implication for selection are discussed.     

Purification and properties ofα-amylase from chicken (Gallus Gallus L.) pancreas

Summary Amylase from chicken pancreas was purified by an affinity method involving filtering a crude extract from pancreas through a Sepharose-wheat albumin column and eluting the retained enzyme with maltose. The purified amylase showed two active bands upon polyacrylamide electrophoresis in an alkaline buffer system and only one band in an acidic buffer system. The enzyme is a Ca²⁺—glycoprotein which behaves as a typical-amylase. It consists of a single polypeptide chain with molecular weight 53,000 and contains 5.3 moles of reducing sugars per mole of protein. Optimal conditions of pH and temperature for the enzymic activity are 7.5 and 37°C. The enzyme is irreversibly inactivated by removal of Ca²⁺ by exhaustive dialysis and is activated by the presence in the assay mixture of Cl^–; other halides are less effective than Cl^– in activating the enzyme.     

Identification of seed sources and parents of Pinus sylvestris L. using visible–near infrared reflectance spectra and multivariate analysis

The potential of visible (VIS) and near infrared (NIR) spectroscopy for identifying seed sources and parents of Pinus sylvestris L. was studied. Seeds of a single family (clones AC1005 × BD1178) collected from three localities in Sweden—Sävar (north), Röskär (central) and Degeberga (south)—and seeds from four maternal (clone no. BD1032, AC1014, BD1178 and AC1005) and four paternal (Y3020, BD1178, AC1014 and BD1032) parents were used to evaluate the method. VIS and NIR reflectance spectra were recorded on individual seeds using a NIRSystems Model 6500 spectrometer from 400 to 2,498 nm with a resolution of 2 nm. The VIS + NIR spectroscopic data were pre-treated with multiplicative signal correction, and analysed by soft independent modelling of class analogy (SIMCA) and partial least squares-discriminant analysis (PLS-DA). The computed models were later applied to classify samples in the external test sets. The results show that seed sources were identified with 100% classification accuracy using PLS-DA models in the VIS + NIR, VIS and NIR regions. The average classification accuracy for maternal parents ranged from 92% to 96%, while paternal parents were identified with 91.2–96% accuracies. The classification accuracy using the SIMCA approach was relatively low for seed sources as well as maternal and paternal parents. It can be concluded that VIS + NIR spectroscopy could be employed as a rapid and non-destructive method for monitoring putative seed sources. The result underscores the prospect of the technique for characterizing seeds based on genotype, thereby serving as a tool in tree improvement and breeding.     

Influence of substituent effects on spectroscopic properties and antimicrobial activity of 5-(4��-substituted phenylazo)-2-thioxothiazolidinone derivatives

5-(4??-substituted phenylazo)-2-thioxothiazolidinone derivatives (HL_n) have been synthesized and characterized by elemental analysis, spectra (IR, electronic and ¹H NMR). The IR spectral data indicate that the compounds can exist in two resonance structures. The synthesized ligands were screened for their antimicrobial activity against four bacterial species, two Gram positive bacteria (Bacillus cereus and Staphylococcus aureus) and two Gram negative bacteria (Escherichia coli and Klebsiella pneumoniae) as well as against some species various of fungi; Aspergillus niger, Penicillium chrysogenum, Penicillium italicum and Fusarium oxysporium. The results showed that most these ligands are good antibacterial agents against B. cereus and S. aureus and antifungal agents against A. niger and F. oxysporium. HL₃ was found to be the most effect compound against all tested microorganisms. The size of clear zone were ordered as follows p-(OCH₃?<?CH₃?<?H?<?Cl?<?NO₂) as expected from Hammett??s constant ??^R.     

Self-compatibility of ��Katy�� apricot (Prunus armeniaca L.) is associated with pollen-part mutations

Apricot (Prunus armeniaca L.) cultivars originated in China display a typical S-RNase-based gametophytic self-incompatibility (GSI). ‘Katy’, a natural self-compatible cultivar belonging to the European ecotype group, was used as a useful material for breeding new cultivars with high frequency of self-compatibility by hybridizing with Chinese native cultivars. In this work, the pollen-S genes (S-haplotype-specific F-box gene, or SFB gene) of ‘Katy’ were first identified as SFB ₁ and SFB ₈, and the S-genotype was determined as S ₁ S ₈. Genetic analysis of ‘Katy’ progenies under controlled pollination revealed that the stylar S₁-RNase and S₈-RNase have a normal function in rejecting wild-type pollen with the same S-haplotype, while the pollen grains carrying either the SFB ₁ or the SFB ₈ gene are both able to overcome the incompatibility barrier. However, the observed segregation ratios of the S-genotype did not fit the expected ratios under the assumption that the pollen-part mutations are linked to the S-locus. Moreover, alterations in the SFB ₁ and SFB ₈ genes and pollen-S duplications were not detected. These results indicated that the breakdown of SI in ‘Katy’ occurred in pollen, and other factors not linked to the S-locus, which caused a loss of pollen S-activity. These findings support a hypothesis that modifying factors other than the S-locus are required for GSI in apricot.