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Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydroquercetin and Flavan-3,4-cis-Diol (Leucocyanidin) to (+)-Catechin by Reductases Extracted from Cell Suspension Cultures of Douglas Fir.

Extracts of cell suspension cultures from Douglas fir (Pseudotsuga menziesii) needles catalyzed the production of (+)-catechin (2,3-trans flavan-3-o1) from the 2,3-trans-flavan,3,4-cis-diol (leucocyanidin) in a NADPH-dependent reductase reaction at pH 7.4. Catechin was also produced, along with the 3,4-cis-diol, in a double step reduction from (+)-dihydroquercetin. It was necessary to eliminate any thiol such as 2-mercaptoethanol or dithiothreitol from the extract or assay mixture because these thiols apparently formed thioethers with the 3,4-diols.

Journal Article↗

Distribution of endogenous indole-3-acetic Acid and compression wood formation in reoriented branches of douglas-fir.

Five-year-old segments of intact 7-year-old branches of Douglas-fir (Pseudotsuga meziesii [Mirb.] Franco) were reoriented to determine the relation between indole-3-acetic acid (IAA) and the formation of compression wood. Eight branches per treatment were either left at their original angle (mean of 69 degrees , the control), or bent proximal to the segment to reorient it up or down 30 degrees . Differentiating xylem tissue from the upper and lower sides of each segment was collected and extracted separately for IAA analysis by in-line fluorescence detection of free IAA and IAA methyl ester after sequential C(16) reversed-phase high performance liquid chromatography. The IAA methyl ester was confirmed by gas chromatography-mass spectroscopy. Compression wood formed on the upper side of branches reoriented up and on the lower side of controls or branches reoriented down. IAA was present in all samples. The difference in IAA concentration between upper and lower sides was either not correlated, or negatively correlated in segments reoriented down, with both the occurrence of compression wood and the rate of new tracheid production. Mean concentrations for whole branch segments were not affected by the treatments, regardless of whether IAA concentrations were expressed on a surface area, weight, or cell basis.

Journal Article↗

Modeling the haemodynamic response in fMRI using smooth FIR filters.

Modeling the haemodynamic response in functional magnetic resonance (fMRI) experiments is an important aspect of the analysis of functional neuroimages. This has been done in the past using parametric response function, from a limited family. In this contribution, we adopt a semi-parametric approach based on finite impulse response (FIR) filters. In order to cope with the increase in the number of degrees of freedom, we introduce a Gaussian process prior on the filter parameters. We show how to carry on the analysis by incorporating prior knowledge on the filters, optimizing hyper-parameters using the evidence framework, or sampling using a Markov Chain Monte Carlo (MCMC) approach. We present a comparison of our model with standard haemodynamic response kernels on simulated data, and perform a full analysis of data acquired during an experiment involving visual stimulation.

Hemodynamics↗

Mitochondrial DNA products among RAPD profiles are frequent and strongly differentiated between races of Douglas-fir.

Racial differentiation and genetic variability were studied between and within the coastal, north interior, and south interior races of Douglas-fir using RAPD and allozyme markers. Nearly half of all RAPD bands scored (13: 45%) were found to be amplified from mitochondrial DNA. They exhibited maternal inheritance among hybrids and backcrosses between the races, and were much more highly differentiated (GST = 0.62 for haplotype frequencies) than were allozymes (GST = 0.26). No evidence of hybridization or introgression was detected where the coastal and interior races come into proximity in central Oregon.

Base Sequence↗

Molecular markers reveal extensive intraspecific below-ground overlap of silver fir fine roots.

Fine roots are expected to be important determinants of plant competition, but very little is known about the extent of root system overlap. Here, we describe the application of two highly variable plastide microsatellites to study the fine root distribution of tree individuals in a silver fir forest. We demonstrate that the spread of fine roots exceeds the width of above-ground parts, and that fine root overlaps among neighbouring trees are extensive both laterally and in depth. This approach will help to improve models of below-ground competition and will facilitate estimations of fine root biomass and thus of below-ground C pools.

Abies↗

Water flow through junctions in Douglas-fir roots.

Roots are important conduits for the redistribution of water within the rooting zone. Root systems are often highly branched, and water flow between regions undoubtedly involves passage through junctions between individual roots. This study considered junctions in the roots of Douglas-fir with regard to the resistances encountered by water flow through the xylem. Flow into the root branch distally along the main root encountered much greater resistance than flow into the branch and proximally along the main root (toward the plant stem). When the main root proximal to the junction was gradually shortened, the resistance to flow in the branch root and distally along the main root increased dramatically. Thus, flow in this manner appears to depend on lateral flow within the root over many centimetres proximal to the junction and not just within the direct connection at the junction. These results suggest that the hydraulic nature of junctions is an important aspect of hydraulic redistribution of water within the soil utilizing flow through roots.

Plant Roots↗

Hydraulic redistribution in a Douglas-fir forest: lessons from system manipulations.

Hydraulic redistribution (HR) occurs in many ecosystems; however, key questions remain about its consequences at the ecosystem level. The objectives of the present study were to quantify seasonal variation in HR and its driving force, and to manipulate the soil-root system to elucidate physiological components controlling HR and utilization of redistributed water. In the upper soil layer of a young Douglas-fir forest, HR was negligible in early summer, but increased to 0.17 mm day(-1) (20-60 cm layer) by late August when soil water potential was approximately -1 MPa. When maximum HR rates were observed, redistributed water replenished approximately 40% of the water depleted from the upper soil on a daily basis. Manipulations to the soil or to the soil/plant water potential driving force altered the rate of observed HR indicating that the rate of HR is controlled by a complex interplay between competing soil and plant water potential gradients and pathway resistances. Separating roots from the transpiring tree resulted in increased HR, and sap flow measurements on connected and disconnected roots showed reversal of water flow, a prerequisite for HR. Irrigating a small plot with deuterated water demonstrated that redistributed water was taken up by small understorey plants as far as 5 m from the watering source, and potentially further, but the utilization pattern was patchy. HR in the upper soil layers near the watering plot was twice that of the control HR. This increase in HR also increased the amount of water utilized by plants from the upper soil. These results indicate that the seasonal timing and magnitude of HR was strongly governed by the development of water potential differences within the soil, and the competing demand for water by the above ground portion of the tree.

Circadian Rhythm↗

Does soil CO2 efflux acclimatize to elevated temperature and CO2 during long-term treatment of Douglas-fir seedlings?

We investigated the effects of elevated soil temperature and atmospheric CO2 on soil CO2 efflux (SCE) during the third and fourth years of study. We hypothesized that elevated temperature would stimulate SCE, and elevated CO2 would also stimulate SCE with the stimulation being greater at higher temperatures. The study was conducted in sun-lit controlled-environment chambers using Douglas-fir (Pseudotsuga menziesii) seedlings grown in reconstructed litter-soil systems. We used a randomized design with two soil temperature and two atmospheric CO2 treatments. The SCE was measured every 4 wk for 18 months. Neither elevated temperature nor CO2 stimulated SCE. Elevated CO2 increased the temperature sensitivity of SCE. During the winter, the relationship between SCE and soil moisture was negative but it was positive during the summer. The seasonal patterns in SCE were associated with seasonal changes in photosynthesis and above-ground plant growth. SCE acclimatized in the high-temperature treatment, probably because of a loss of labile soil carbon. Elevated CO2 treatment increased the temperature sensitivity of SCE, probably through an increase in substrate availability.

Acclimatization↗

Elevated CO(2) and elevated temperature have no effect on Douglas-fir fine-root dynamics in nitrogen-poor soil.

Here, we investigate fine-root production, mortality and standing crop of Douglas-fir (Pseudotsuga menziesii) seedlings exposed to elevated atmospheric CO(2) and elevated air temperature. We hypothesized that these treatments would increase fine-root production, but that mortality would be greater under elevated temperature, leading to a smaller increase in standing crop. Seedlings were grown in outdoor, sun-lit controlled-environment chambers containing native soil. They were exposed in a factorial design to two levels of atmospheric CO(2) and two levels of air temperature. Minirhizotron methods were used to measure fine-root length production, mortality and standing crop every 4 wk for 36 months. Neither elevated atmospheric CO(2) nor elevated air temperature affected fine-root production, mortality, or standing crop. Fine roots appeared to root deeper in the soil profile under elevated CO(2) and elevated temperature. Low soil nitrogen (N) levels apparently limited root responses to the treatments. This suggests that forests on nutrient-poor soils may exhibit limited fine-root responses to elevated atmospheric CO(2) and elevated air temperature.

Carbon Dioxide↗

Development and evaluation of methods to detect nucleopolyhedroviruses in larvae of the Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough).

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r(2) = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.

Animals↗

Enrichment of cadmium-mediated antibiotic-resistant bacteria in a Douglas-fir (Pseudotsuga menziesii) litter microcosm.

A set of Douglas-fir needle litter microcosms was amended with cadmium, acid, a combination of both, or neither. After 2 weeks of incubation, bacterial colony counts were made of litter homogenates inoculated onto agar media containing an antibiotic (streptomycin, chloromycetin, ampicillin, or gentamicin), cadmium, both, or neither. In all microcosms bacterial abundance was similar but the quality was very dissimiliar. Cadmium-treated microcosms had populations enriched for cadmium and gentamicin resistance and streptomycin and chloramphenicol sensitivity. Acid amendment had no consistent effect on the microcosm populations except that which could be attributed to the cadmium treatment amendment alone.

Anti-Bacterial Agents↗

Metabolic and Genotypic Fingerprinting of Fluorescent Pseudomonads Associated with the Douglas Fir-Laccaria bicolor Mycorrhizosphere.

A collection of 300 isolates of fluorescent pseudomonads was established from Douglas fir-Laccaria bicolor mycorrhizas and mycorrhizosphere and from adjacent bulk soil. These isolates were first phenotypically characterized with the Biolog method. Taxonomic identification assigned 90% of the isolates to the different biovars of Pseudomonas fluorescens, with inverted frequencies of biovars V and I from the bulk soil to the mycorrhizas, suggesting that the mycorrhizas exert a selective stimulation of the P. fluorescens bv. I and a counterselection of the P. fluorescens bv. V present in the soil. Multivariate analyses of the carbon source utilization data led to the definition of homogenous metabolic groups and to the identification of the most discriminating substrates for each group. The isolates from the mycorrhizosphere and from the mycorrhizas seem to preferentially utilize carbohydrates, in particular trehalose, which is the most abundant carbohydrate accumulated in the mycelium of L. bicolor. The results suggest that L. bicolor exerts a trehalose-mediated selection on the fluorescent pseudomonads present in the vicinity of the mycorrhizas. Isolates of P. fluorescens from the mycorrhizosphere and mycorrhizas were then genotypically characterized by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes and enterobacterial repetitive intergenic consensus-PCR DNA fingerprinting. Both methods revealed a high genetic polymorphism within the population studied, which was well correlated with the phenotypic characterization.

Journal Article↗

Structure and dynamics of experimentally introduced and naturally occurring laccaria sp. Discrete genotypes in a douglas fir plantation

Ectomycorrhizal fungi have been introduced in forest nurseries to improve seedling growth. Outplanting of inoculated seedlings to forest plantations raises the questions about inoculant persistence and its effects on indigenous fungal populations. We previously showed (M.-A. Selosse et al. Mol. Ecol. 7:561-573, 1998) that the American strain Laccaria bicolor S238N persisted 10 years after outplanting in a French Douglas fir plantation, without introgression or selfing and without fruiting on uninoculated adjacent plots. In the present study, the relevance of those results to sympatric strains was assessed for another part of the plantation, planted in 1985 with seedlings inoculated with the French strain L. bicolor 81306 or left uninoculated. About 720 Laccaria sp. sporophores, collected from 1994 to 1997, were typed by using randomly amplified polymorphic DNA markers and PCR amplification of the mitochondrial and nuclear ribosomal DNAs. All plots were colonized by small spontaneous discrete genotypes (genets). The inoculant strain 81306 abundantly fruited beneath inoculated trees, with possible introgression in indigenous Laccaria populations but without selfing. In contrast to our previous survey of L. bicolor S238N, L. bicolor 81306 colonized a plot of uninoculated trees. Meiotic segregation analysis verified that the invading genet was strain 81306 (P < 0.00058), implying a vegetative growth of 1.1 m. year-1. This plot was also invaded in 1998 by strain S238N used to inoculate other trees of the plantation. Five other uninoculated plots were free of these inoculant strains. The fate of inoculant strains thus depends less on their geographic origin than on unknown local factors.

Journal Article↗

Douglas-fir root-associated microorganisms with inhibitory activity towards fungal plant pathogens and human bacterial pathogens.

A microbial culture collection composed of 1820 bacterial strains, including 298 actinomycete strains, was established from the roots of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings harvested from conifer nurseries and forest sites. Two hundred and thirty-four strains inhibited the growth of Fusarium, Cylindrocarpon, and (or) Pythium spp. in in vitro assays. A significantly greater proportion of bacterial strains from actinomycete genera exhibited antifungal properties compared with bacterial strains from nonactinomycete genera. Eighty-nine percent of identified inhibitory strains were Streptomyces, Streptoverticillium, Bacillus, Pseudomonas, or Burkholderia species. The actinomycete species were isolated almost exclusively from forest seedlings. Recovery of inhibitory strains representing 29 microbial species was enhanced using a variety of methods to isolate microorganisms from the roots of seedlings from nursery and forest sites. Bacterial strains (including actinomycete strains) with antifungal activity were tested for in vitro growth inhibition of six clinical human bacterial pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa). Forty-eight percent of the tested strains inhibited one or more human pathogens, Inhibitory activity towards fungal and bacterial pathogens was strain specific, not species specific, and many inhibitory strains exhibited broad-spectrum activity. Strains with antifungal activity against several conifer root pathogens were also more likely to inhibit multiple species of clinical bacterial pathogens.

Actinomycetales↗

Effects of shade and root confinement on the expression of plagiotropic growth in juvenile-origin Douglas-fir rooted cuttings.

The purpose of this experiment was to determine why juvenile-origin Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) rooted cuttings, which remain plagiotropic (branchlike) when grown in containers in shaded glasshouses, become orthotropic (vertical) after they are transplanted to an outdoor environment. Plagiotropic rooted cuttings (mean angle from vertical = 45-50 degrees) from three full-sib families were transplanted into an outdoor nursery and subjected to four treatments consisting of a factorial of (1) shaded or unshaded and (2) bareroot or confined roots. After two growing seasons, treatments had significantly affected plant size and biomass in the order unshaded-bareroot > shaded-bareroot > unshaded-confined > shaded-confined, but plants in all treatments had become nearly orthotropic. It is concluded that neither shading nor root confinement is, but other glasshouse environmental conditions are, responsible for the persistence of plagiotropic growth.

Biomass↗

Enhanced enzymatic hydrolysis of steam-exploded Douglas fir wood by alkali-oxygen post-treatment.

Good enzymatic hydrolysis of steam-exploded Douglas fir wood (SEDW) cannot be achieved owing to the very high lignin content ( >40%) that remains associated with this substrate. Thus, in this study, we investigated the use of alkali-oxygen treatment as a posttreatment to delignify SEDW and also considered the enzymatic hydrolyzability of the delignified SEDW. The results showed that under optimized conditions of 15% NaOH, 5% consistency, 110 degrees C, and 3 h, approx 84% of the lignin in SEDW could be removed. The resulting delignified SEDW had good hydrolyzability, and cellulose-to-glucose conversion yields of over 90 and 100% could be achieved within 48 h with 20 and 40 filter paper units/g of cellulose enzyme loadings, respectively. It was also indicated that severe conditions, such as high NaOH concentration and high temperature, should not be utilized in oxygen delignification of SEDW in order to avoid extensive condensation of lignin and significant degradation of cellulose.

Biotechnology↗