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PubMed1982

Enzymic synthesis of lignin precursors. Purification and properties of 4-coumarate:CoA ligase from cambial sap of spruce (Picea abies L.).

4-Coumarate:CoA ligase was purified from cambial sap of spruce (Picea abies). A 1627-fold purification of the enzyme with a yield of 37% was achieved by a six-step procedure including dye-ligand chromatography. Isozymes of the ligase were not detected. The enzyme has an Mr of about 63 000 and is a single polypeptide chain. Ferulic, 4-coumaric and caffeic acids are efficient substrates for the ligase. In contrast to some ligases from angiosperms, the ligase from spruce (gymnosperm) does not activate sinapic acid. The substrate specificity of the ligase is consistent with the lignin composition of spruce.

Coenzyme A Ligases↗

PubMed1978

Comparative study on the effect of some chlorinated hydrocarbon insecticides on the formation of ectotrophic mycorrhizae in seedlings of Picea abies (L.) Karst.

Pot experiments were conducted to determine the effect of lindane and toxaphene on the ectotrophic mycorrhizae in Picea abies (L.) Karst. seedlings. The concentrations investigated gave variable results. Quantitative reductions were found in mycorrhizal short roots and intensity. In general the initiated lateral roots were mainly infected by mycorrhizae, even in the presence of insecticidal injuries in the root system.

Basidiomycota↗

PubMed2005

Proteome analysis of early somatic embryogenesis in Picea glauca.

Forestry is a valuable natural resource for many countries. Rapid production of large quantities of genetically improved and uniform seedlings for restocking harvested lands is a key component of sustainable forest management programs. Clonal propagation through somatic embryogenesis has the potential to meet this need in conifers and can offer the added benefit of ensuring consistent seedling quality. Although in commercial use, mass production of conifers through somatic embryogenesis is relatively new and there are numerous biological unknowns regarding this complex developmental pathway. To aid in unravelling the embryo developmental process, two-dimensional electrophoresis was employed to quantitatively assess the expression levels of proteins across four stages of somatic embryo maturation in white spruce (0, 7, 21 and 35 days post abscisic acid treatment). Forty-eight differentially expressed proteins have been identified, which display a significant change in abundance as early as day 7 of embryo development. These proteins are involved in a variety of cellular processes, many of which have not previously been associated with embryo development. The identification of these proteins was greatly assisted by the availability of a substantial expressed sequence tag (EST) resource developed for white, sitka and interior spruce. The combined use of these spruce ESTs in conjunction with GenBank accessions for other plants improved the rate of protein identification from 38% to 62% when compared with GenBank alone using automated, high-throughput techniques. This underscores the utility of EST resources in a proteomic study of any species for which a genome sequence is unavailable.

Cell Line↗

PubMed2004

Effect of Chalara longipes on decomposition of humic acids from Picea abies needle litter.

The effect of the saprotrophic ascomycete Chalara longipes on the decomposition of humic acids was determined in a cultivation experiment. The fungus was incubated in liquid cultures in a full-strength (F system) and an organic nitrogen-free medium (F-N system), both amended with pure humic acids isolated from spruce forest litter. Fungal biomass production was highest in the F system with humic acids and lowest in the F-N system, the effect of organic nitrogen and humic acids being significant (p < 0.05). The presence of organic nitrogen seems to be essential for growth. The fungus utilized humic acids; molar mass distribution in media obtained by gel-permeation chromatography showed decrease in the humic acids fraction. Decolorization of the media reached 75 and 64 % in the F and F-N systems, respectively. The molar mass of humic acids probably also decreased as estimated from the increase in the absorbance A465/A665 (A4/A6) ratio. It is assumed that the utilization of humic acids may be mediated by the production of organic acids (as their components) and production of some oxidative enzymes.

Ascomycota↗

PubMed2004

Robust simple sequence repeat markers for spruce (Picea spp.) from expressed sequence tags.

Traditionally, simple sequence repeat (SSR)markers have been developed from libraries of genomic DNA. However, the large, repetitive nature of conifer genomes makes development of robust, single-copy SSR markers from genomic DNA difficult. Expressed sequence tags (ESTs), or sequences of messenger RNA, offer the opportunity to exploit single, low-copy, conserved sequence motifs for SSR development. From a 20,275-unigene spruce EST set, we identified 44 candidate EST-SSR markers. Of these, 25 amplified and were polymorphic in white, Sitka, and black spruce; 20 amplified in all 3 spruce species tested; the remaining five amplified in all except one species. In addition, 101 previously described spruce SSRs (mostly developed from genomic DNA), were tested. Of these, 17 amplified across white,Sitka, and black spruce. The 25 EST-SSRs had approximately 9% less heterozygosity than the 17 genomic-derived SSRs (mean H=0.65 vs 0.72), but appeared to have less null alleles, as evidenced by much lower apparent inbreeding (mean F=0.046 vs 0.126). These robust SSRs are of particular use in comparative studies,and as the EST-SSRs are within the expressed portion of the genome, they are more likely to be associated with a particular gene of interest, improving their utility for quantitative trait loci mapping and allowing detection of selective sweeps at specific genes.

Base Sequence↗

PubMed2006

Raman imaging to investigate ultrastructure and composition of plant cell walls: distribution of lignin and cellulose in black spruce wood (Picea mariana).

A detailed understanding of the structural organization of the cell wall of vascular plants is important from both the perspectives of plant biology and chemistry and of commercial utilization. A state-of-the-art 633-nm laser-based confocal Raman microscope was used to determine the distribution of cell wall components in the cross section of black spruce wood in situ. Chemical information from morphologically distinct cell wall regions was obtained and Raman images of lignin and cellulose spatial distribution were generated. While cell corner (CC) lignin concentration was the highest on average, lignin concentration in compound middle lamella (CmL) was not significantly different from that in secondary wall (S2 and S2-S3). Images generated using the 1,650 cm(-1) band showed that coniferaldehyde and coniferyl alcohol distribution followed that of lignin and no particular cell wall layer/region was therefore enriched in the ethylenic residue. In contrast, cellulose distribution showed the opposite pattern-low concentration in CC and CmL and high in S2 regions. Nevertheless, cellulose concentration varied significantly in some areas, and concentrations of both lignin and cellulose were high in other areas. Though intensity maps of lignin and cellulose distributions are currently interpreted solely in terms of concentration differences, the effect of orientation needs to be carefully considered to reveal the organization of the wood cell wall.

Acrolein↗

PubMed2004

Characterisation of different clones of Picea abies (L.) Karst using head-space sampling of cortical tissues combined with enantioselective capillary gas chromatography for the separation of chiral and non-chiral monoterpenes.

Head-space sampling (HS) has been combined with enantioselective gas chromatography (GC) for the analysis of chiral and non-chiral monoterpenes present in the cortical tissues of five different Norway spruce clones. (1S)-(-)-alpha-Pinene, (1S,5S)-(-)sabinene, (1S)-(-)-beta-pinene, and (4S)-(-)limonene dominated over (1R)-(+)-alpha-pinene, (1R,5R)-(+)-sabinene, (1R)-(+)-beta-pinene, and (4R)-(+)-limonene. Results showed a large variation in the enantiomeric composition of cortical tissues between different clones. The development of HS-GC greatly increased the speed of precise analyses of chiral monoterpenes in small samples and therefore offer excellent opportunities in studies on the ecophysiological and chemotaxomic roles of these chiral components.

Chromatography, Gas↗

PubMed2005

Effects of simultaneous ozone exposure and nitrogen loads on carbohydrate concentrations, biomass, and growth of young spruce trees (Picea abies).

Spruce saplings were grown under different nitrogen fertilization regimes in eight chamberless fumigation systems, which were fumigated with either charcoal-filtered (F) or ambient air (O3). After the third growing season trees were harvested for biomass and non-structural carbohydrate analysis. Nitrogen had an overall positive effect on the investigated plant parameters, resulting in increased shoot elongation, biomass production, fine root soluble carbohydrate concentrations, and also slightly increased starch concentrations of stems and roots. Only needle starch concentrations and fine root sugar alcohol concentrations were decreased. Ozone fumigation resulted in needle discolorations and affected most parameters negatively, including decreased shoot elongation and decreased starch concentrations in roots, stems, and needles. In fine roots, however, soluble carbohydrate concentrations remained unaffected or increased by ozone fumigation. The only significant interaction was an antagonistic effect on root starch concentrations, where higher nitrogen levels alleviated the negative impact of ozone.

Air Pollutants↗

PubMed2005

The effect of osmoticum on ascorbate and glutathione metabolism during white spruce (Picea glauca) somatic embryo development.

Water stress is an important factor which regulates organized development of both zygotic and somatic embryos. Somatic embryos of white spruce were cultured in the presence of polyethylene glycol (PEG), a non-plasmolyzing agent which increases embryo quality and number, and mannitol, a plasmolyzing agent. The effects of these two compounds on both ascorbate and glutathione metabolism were investigated at different stages of embryo development. Compared to control and mannitol-treated embryos, embryos treated with PEG accumulated higher levels of endogenous ascorbate (ASC) in its reduced form, especially during the first half of the maturation period. This increase, also observed in immature seeds, was mainly the result of two different processes: activation of the de novo ASC machinery, and recycling of ASC from ascorbate free radicals (AFR) which was modulated by the activity of ascorbate free radical reductase (AFRR, EC. 1.6.5.4). The activity of this enzyme increased during the early phases of development in both PEG-treated somatic embryos and seeds. Compared to control somatic embryos, mannitol and PEG were shown to change the levels of reduced (GSH) and oxidized glutathione (GSSG). In particular, a constant decline in the GSH/GSSG ratio was observed in the presence of PEG. This pattern was also observed in maturing white spruce seeds. Overall, these data indicate that applications of non-plasmolyzing agents in the culture medium of spruce somatic embryos result in seed-like fluctuations of the ascorbate-glutathione metabolism, which may have a positive effect on embryo yield.

Ascorbic Acid↗

PubMed2001

Investigation of uptake, translocation and fate of trichloroacetic acid in Norway spruce (Picea abies/L./Karst.) using 14C-labelling.

[1,2-14C]TCA of a high specific activity (3.7 GBq/mmol) and appropriate radioindicator techniques were used, to study the effect of trichloroacetic acid (TCA) on conifers. Easy uptake of TCA from soil through spruce roots and its further translocation by the transpiration stream up to the needles (where damage of the photosynthetic apparatus occurs) has been proved. During the growth period, after one-shot load of TCA, the uptake was most intensive in current-year needles at first; over an extended period a decrease in the level of [1,2-14C]TCA-derived radioactivity was found in the current-year needles while in older needles (C + 2), the level rose. Symptoms of TCA biodegradation and/or metabolism were found in the plant/soil system under study. During an eight-week exposure significant losses of radioactivity into the atmosphere were noticed, at least a part of them in the form of carbondioxide. The results of these more or less preliminary experiments demonstrated the suitability and advantages of the radioisotopic technique used.

Carbon Radioisotopes↗

PubMed2006

Kutznerides 1-4, depsipeptides from the actinomycete Kutzneria sp. 744 inhabiting mycorrhizal roots of Picea abies seedlings.

Bioassay-guided fractionation of culture supernatants of the actinomycete Kutzneria sp. 744 resulted in the isolation of four new depsipeptides (1-4). Structure analysis revealed the general structure: cyclo[2-(1-methylcyclopropyl)-D-glycine-(2S,3aR,8aS)-6,7-dichloro-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid-3-hydroxy-D-glutamic acid-O-methyl-L-serine-L-piperazic acid-(S)-2-hydroxy-3,3-dimethylbutanoic acid]. The 3-hydroxy-d-glutamic acid was present as its threo-isomer in 1 and 2 and as its erythro-isomer in 3 and 4. The piperazic acid was modified to its (R)-4-chloro analogue in 2 and to its C-5/N unsaturated analogue in 4. Compounds 1-4 displayed moderate spore germination inhibiting activity against several common root-rotting fungi.

Actinobacteria↗

PubMed2002

Spatial autocorrelation and linkage of Mendelian RAPD markers in a population of Picea abies Karst.

The spatial clustering of single- and di-locus genotypes in a natural, continuous population of Norway spruce was investigated using 69 Mendelian Random Amplified Polymorphic DNA (RAPD) markers that covered about 15 of the species genome, and whose linkage relationships were known. Spatial autocorrelation techniques and randomization tests, applied to both single- and di-locus genotypes, revealed a weak, though significant, spatial structure at the scale 0-200 m (5 of single-locus and 7 of di-locus genotypes). To assess the relative importance of isolation by distance and linkage between markers on their spatial genetic structuring, we grouped joins between sampled trees into equivalence categories expected to show similar, specific patterns of spatial distribution under isolation by distance. Results from both single- and di-locus analyses were consistent with the existence of patches of like homozygotes (about 8 and 11 of loci at the single- and di-locus level, respectively) surrounded by a mix of like heterozygotes. Similar structuring has been predicted by simulation models under isolation by distance and selective neutrality. Overall, linkage between markers accounted for an increase of spatial clumping of di-locus genotypes involving tightly linked loci with recombination fractions up to 0.1, a consequence of limited, stochastic spread of single-locus genotypes in space. Our results support the hypothesis that isolation by distance and linkage have a small, though significant, effect even within continuous forest tree populations. In general, the spatial distribution of multilocus genotypes within populations should be interpreted with caution when linkage relationships among the markers used are unknown.

DNA↗

PubMedDate not supplied

The DAL10 gene from Norway spruce (Picea abies) belongs to a potentially gymnosperm-specific subclass of MADS-box genes and is specifically active in seed cones and pollen cones.

Transcription factors encoded by different members of the MADS-box gene family have evolved central roles in the regulation of reproductive organ development in the flowering plants, the angiosperms. Development of the stamens and carpels, the pollen- and seed-bearing organs, involves the B- and C-organ-identity MADS-box genes. B- and C-type gene orthologs with activities specifically in developing pollen- and seed-bearing organs are also present in the distantly related gymnosperms: the conifers and the gnetophytes. We now report on the characterization of DAL10, a novel MADS-box gene from the conifer Norway spruce, which unlike the B- and C-type conifer genes shows no distinct orthology relationship to any angiosperm gene or clade in phylogenetic analyses. Like the B- and C-type genes, it is active specifically in developing pollen cones and seed cones. In situ RNA localization experiments show DAL10 to be expressed in the cone axis, which carry the microsporophylls of the young pollen cone. In contrast, in the seed cone it is expressed both in the cone axis and in the bracts, which subtend the ovuliferous scales. Expression data and the phenotype of transgenic Arabidopsis plants expressing DAL10 suggest that the gene may act upstream to or in concert with the B- and C-type genes in the establishment of reproductive identity of developing cones.

Base Sequence↗

PubMed2003

Reaction of detoxification mechanisms in suspension cultured spruce cells (Picea abies L. Karst.) to heavy metals in pure mixture and in soil eluates.

UNLABELLED: INTENTION, GOAL, BACKGROUND: The widespread and unconcerned use of chemicals in the past has led to an accumulation of pollutants in our environment. Numerous sites are polluted with a mixture of organic chemicals and heavy metals. The future use of these sites and the safe consumption of groundwater from these areas depends on our ability to assess risk by determining the bioavailability of trace levels of pollutants in the respective soil solutions. Soil eluates containing heavy metals in mixture as well as pure heavy metals in aqueous solution were added to a spruce cell culture to set up such a test system. OBJECTIVE: The present study aims at evaluating the response of cultured spruce cells to heavy metals in aqueous solution, and at characterizing these basic cellular responses as potential biomarkers. METHODS: In order to characterize cell reactions toward heavy metals, spruce cell cultures were incubated with CdSO4 (50 to 500 microM), Na2HAsO4 (1.5 to 80 microM) or PbCl2 (10 to 150 microM). Alternatively, the cells were incubated with a standard heavy metal mixture containing 80 microM Na2HAsO4, 150 microM CdSO4 and 150 microM PbCl2 in medium and with aqueous original soil eluates. Measurement of oxidative stress, antioxidants and basic detoxification enzymes involved in plant defence reactions were performed with the treated cells. RESULTS AND DISCUSSION: After 5 hrs of incubation, the onset of a strong oxidative burst was observed. H2O2 concentrations exceeded 40 microM in the culture media after 20 hrs. Concomitantly, glutathione levels showed drastic changes indicating the influence of the metals and/or the H2O2 on antioxidative systems. Following cadmium treatment, GSH and GSSG were elevated by 50 and 200% above controls. Whereas arsenic doubled GSSG levels, treatment with lead did not cause significant changes. However, a mixture of the metals decreased both metabolites by 50%. The effect of the metals was concentration-dependent and disappeared at high concentrations. Furthermore, strong induction of glutathione S-transferase (GST) subunits was observed and, although no novel subunit was expressed, the rise of a new GST isoform occurred. The most potent inducer of plant defence reactions is cadmium, followed by arsenate and lead in descending order of effectiveness. Counter ions seem to play an important role, e.g. lead chloride influenced the investigated parameters much more than lead acetate. CONCLUSIONS: The investigated metals activate gene expression through signal transduction pathways previously not associated with these metals, which points to new end points for resistance and toxicity testing. Especially a monitoring of GST subunit behaviour together with quantifying the oxidative burst seem to be promising for a biomonitoring concept. The close regulation of plant answers observed may facilitate the setup of an integrated biotest for heavy metal pollution that could be based on enzymological as well as proteome data. RECOMMENDATIONS AND OUTLOOK: Heavy metals cause stress to plant cells and elicit a whole range of answers, although specific for individual metal species. The differences observed in plant answers are suitable to distinguish between metals bioavailable in soil eluates and water samples, however only at concentrations in the microM range. It will be necessary to evaluate the effects on the RNA and transcript level. We recommend that similar plant metabolic end points and enzyme reactions be screened for their suitability as biotest systems.

Biomarkers↗

PubMed2003

Bidirectional exchange of amino compounds between phloem and xylem during long-distance transport in Norway spruce trees (Picea abies [L.] Karst).

14C-Gln, (14)C-Asp, (15)N-Gln, and (15)N-Asp were fed to cut tips of 2- or 3-year-old needles of spruce twigs, still attached to the tree. After incubation, distribution of the radiolabel and (15)N enrichment was studied in needles, bark and wood tissues of girdled twigs and untreated controls. Analysis of the twig tissues showed that between 22% and 26% of the total amount of the tracers applied had been taken up. Since export out of the application segment and distribution between needles, bark and wood was comparable for (14)C and (15)N tracer, it was concluded that, mainly the amino compounds that had been fed were subject to long- distance transport within the plant and supplied the new sprout with nitrogen. Asp was exported to a greater extent to developing needles compared with Gln. This difference in export between the two amino compounds applied may be explained by the different pool sizes of Gln and Asp in xylem and phloem or differences in xylem and phloem loading. Girdling of the stem showed that the transport of reduced nitrogen compounds from older needle generations to current-year needles proceeded in both xylem and phloem. In addition, an intensive bidirectional exchange of Gln and Asp between xylem and phloem was observed during long-distance transport.

Aspartic Acid↗

PubMed2003

Expression of Chia4-Pa chitinase genes during somatic and zygotic embryo development in Norway spruce (Picea abies): similarities and differences between gymnosperm and angiosperm class IV chitinases.

The developmental pathway of somatic embryogenesis in Norway spruce involves proliferation of proembryogenic masses (PEMs), PEM-to-somatic embryo transition and further development of the somatic embryos. It has previously been shown that extracellular signal molecules, including arabinogalactan proteins, lipo-chitooligosaccharides and chitinases, regulate somatic embryogenesis. The Chia4-Pa1 gene from Norway spruce is described here. The Chia4-Pa1 encodes a typical basic class IV chitinase, although the intron-exon organization of this gymnosperm chitinase is different from that in angiosperm class IV chitinases. The Chia4-Pa1 belongs to a small gene family with highly similar members, and the expression pattern of Chia4-Pa1 cannot be distinguished from that of other Chia4-Pa members. Upon withdrawal of plant growth regulators, i.e. during a treatment that stimulates PEM-to-somatic embryo transition and massive programmed cell death, a significant increase in transcription and translation of Chia4-Pa genes takes place. The expression pattern analysis revealed that Chia4-Pa genes are expressed in a subpopulation of proliferating cells and at the base of the somatic embryo. Furthermore, in seeds, Chia4-Pa genes are expressed in the megagametophyte in the single cell-layered zone surrounding the corrosion cavity. Taken together these results suggest that the Chia4-Pa expressing cells have a megagametophyte signalling function and that CHIA4-Pa stimulates programmed cell death and promotes PEM-to-somatic embryo transition.

Base Sequence↗

PubMed2004

The effect of reduced glutathione on morphology and gene expression of white spruce (Picea glauca) somatic embryos.

Inclusions of reduced glutathione (GSH) in the maturation medium increased the conversion frequency of white spruce somatic embryos without the need of a partial drying treatment (PDT). This beneficial effect was the result of major alterations in morphology and gene expression during the maturation period. Compared with control embryos, GSH-treated embryos showed a differential accumulation of storage products, i.e. preferential deposition of starch, the reduced formation of protein bodies, and increased vacuolation of cells. These morphological changes correlated with extensive alterations of gene expression occurring throughout the maturation period. The transcript profiles of stage-specific embryos matured with or without GSH were analysed using a DNA microarray containing 2 178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has previously been documented. The results indicate that several genes involved in a variety of signal regulatory pathways were differentially expressed in developing GSH- treated embryos. The transcript levels of many genes involved in carbohydrate metabolism and protein synthesis were altered by the presence of GSH and denoted differences in physiology between treatments. Extensive changes in the expression of genes participating in hormone synthesis, nucleotide metabolism, and meristem formation were also observed and related to the post-embryonic performance of the embryos.

Desiccation↗

PubMed2005

Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca).

In white spruce, an improvement of somatic embryo number and quality can be achieved through experimental manipulations of the endogenous levels of reduced (GSH) and oxidized (GSSG) glutathione. An optimal protocol for embryo production included an initial application of GSH in the maturation medium, followed by replacement with GSSG during the remaining maturation period. Under these conditions, the overall embryo population more than doubled, and the percentage of fully developed embryos increased from 22% to almost 70%. These embryos showed improved post-embryonic growth and conversion frequency. Structural studies revealed remarkable differences between embryo types, especially in storage product deposition pattern and organization of the shoot apical meristem (SAM). Compared with their control counterparts, glutathione-treated embryos accumulated a larger amount of starch during the early stages of development, and more protein and lipid bodies during the second half of development. Differences were also noted in the organization of SAMs. Shoot meristems of control embryos were poorly organized and were characterized by the presence of intercellular spaces, which caused separation of the subapical cells. Glutathione-treated embryos had well-organized meristems composed of tightly packed cells which lack large vacuoles. The improved organization of the shoot apical meristems in treated embryos was ascribed to a lower production of ethylene. Differences in meristem structure between control and treated embryos were also related to the localization pattern of HBK1, a shoot apical meristem 'molecular marker' gene with preferential expression to the meristematic cells of the shoot pole. Expression of this gene, which was localized to the apical cells in control embryos, was extended to the subapical cells of treated embryos. Overall, it appears that meristem integrity and embryo quality are under the direct control of the glutathione redox state.

Germination↗