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Differences in stem respiration responses to temperature between balsam fir trees in thinned and unthinned stands.

The change in stem respiration rates with change of temperature was significantly lower in a recently thinned balsam fir (Abies balsamea (L.) Mill.) stand than in an unthinned stand. A comparison of respiration measurements made in the morning with those made in the afternoon indicated that there was more variation among afternoon readings and greater responses to changes in temperature among morning readings. Lower respiration rates in the afternoon were more common in the thinned stand than in the unthinned stand.

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Evaluation of transpiration in a Douglas-fir stand by means of sap flow measurements.

Transpiration of a Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) stand was evaluated by sap flow measurements during a 4-month period. Between-tree variation in sap flow depended on crown class. On a sunny day, total transpiration was 1.6, 8.0 and 22.0 liters day(-1) for suppressed, codominant and dominant trees, respectively. Transpiration estimated by sap flow fell below potential evapotranspiration when available soil water decreased below 30% of its maximum value. Sap flow measurements gave transpiration values similar to those obtained by the water balance method.

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Seasonal changes in levels of cytokinin-like compounds from Douglas-fir xylem extrudate.

High-performance liquid chromatography, immunochromatography, and radioimmunoassay were used to identify cytokinin-like bases and glycosides in xylem sap of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco). Isopentenyladenosine-type (isopentenyladenine and isopentenyladenosine) and zeatin-riboside type (zeatin, zeatin riboside, and dihydrozeatin riboside) cytokinins were detected during springtime. A glucosyl conjugate of zeatin riboside was also present in small amounts. Levels of cytokinin-like compounds varied throughout the spring but were generally highest in late April to early May.

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Root growth in Sitka spruce and Douglas-fir transplants: dependence on the shoot and stored carbohydrates.

When dormant Sitka spruce (Picea sitchensis (Bong.) Carr.) transplants were lifted from the nursery in March and transferred to favorable growing conditions in a controlled environment chamber, new roots emerged and elongated even when the plants were bark-ringed so that the roots could not receive metabolites from the shoot by phloem translocation. Carbohydrate analysis showed that starch reserves in the roots of the bark-ringed plants were depleted, indicating that these reserves provided the energy source for new root growth. The shoot also had a role in new root growth as excised root systems showed greatly reduced root growth. This response is compared to that of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) in which bark-ringing prevented root growth indicating that current photosynthate is needed for new root growth. The findings are discussed in relation to the problems of establishing these species in forest plantations.

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The influence of glutamine on growth and viability of cell suspension cultures of Douglas-fir after exposure to polyethylene glycol.

The response of cell cultures of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) to osmotic stress was studied by measuring cell growth and viability after exposure to polyethylene glycol (PEG) (M(r) 6000-8000). Growth of cells inoculated in a medium containing 10% PEG was slightly inhibited, whereas growth in a medium containing 15% PEG was severely inhibited. Cells grown for 6 days in nutrient medium and then subcultured in a medium containing 15% PEG to induce water stress showed high viabilities, whereas cells grown for longer than 6 days before exposure to PEG showed decreased viabilities after subculture. Cells grown in medium containing 30 mM glutamine were significantly more resistant to PEG-induced water stress, as measured by viability, than cells grown in medium without glutamine.

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Evidence of cytokinin bases, ribosides and glucosides in roots of Douglas-fir, Pseudotsuga menziesii.

Concentrations of cytokinin-like compounds were measured in root tips of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) collected in spring. Cytokinin bases and ribosides were estimated by radioimmunoassay after purification by immunoaffinity (zeatin riboside and isopentenyladenosine antibodies coupled to cellulose gel) and high performance liquid chromatography. Cytokinin glucosides were similarly analyzed in root extracts treated with beta-glucosidase and octadecyl-silica column chromatography in conjunction with immunoaffinity purification. The identity of predominant cytokinins obtained after enzymatic degradation was confirmed by gas chromatography-mass spectrometry. Each of the observed cytokinin bases and ribosides seemed to have a corresponding hexose conjugate form. Total quantity of cytokinin glucosides was twice that of cytokinin bases and ribosides, and the major compound was isopentenyladenosine hexose conjugate.

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Effects of aluminum on growth and nutrient status of Douglas-fir seedlings grown in culture solution.

Effects of Al on growth, nutrient uptake and proton efflux were studied in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings grown for about nine months in culture solutions with a pH between 3.4 and 3.6 and with both calcium and magnesium (Ca + Mg) at a concentration of 0.1, 0.5 or 2.5 mM. In the absence of Al, plant dry matter production and root development increased with increasing concentrations of (Ca + Mg) in the culture solution. At the low and intermediate (Ca + Mg) concentrations, optimal root and shoot development were observed at an Al concentration of 4 mg l(-1). At the highest (Ca + Mg) concentration, Al up to 4 mg l(-1) did not affect growth, but at higher concentrations, it significantly reduced both shoot and root growth. As the concentration of (Ca + Mg) in the nutrient solution increased, the concentrations of Ca and Mg increased in shoots and roots also. The concentrations of Ca and Mg in the roots were unaffected by the presence of Al, whereas in the shoots they were either unaffected, or increased, by Al. Concentrations of Al in, or on, roots, or in shoots, did not change in response to changing concentrations of Ca + Mg in the nutrient medium. In general, concentrations of P and K in shoots and roots were higher in seedlings grown in nutrient solutions containing Al. Stimulation of growth by moderate concentrations of Al, which was observed at suboptimal (Ca + Mg) concentrations, was associated with a low NH(4) preference and a low root proton efflux. The Al-induced increase in internal K concentration and reduction in NH(4) preference may be explained by a lower efflux of K and NO(3), respectively.

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Selection and physiology of cell cultures of Douglas-fir grown under conditions of water stress.

Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) cell cultures sampled 3, 6, or 9 days after subculture in nutrient medium were able to survive subsequent subculture in a medium containing 15% polyethylene glycol (PEG) (M(r) 6000-8000) (-1.21 MPa), whereas cell sampled 12 or 16 days after subculture in nutrient medium became senescent when transferred to a medium containing 15% PEG. Cells sampled after subculture for 3, 6, or 9 days in nutrient medium had lower fresh weight/dry weight ratios, lower osmotic potentials, smaller cell diameters, and higher turgor pressures than cells sampled after 12 or 16 days subculture in nutrient medium. Cells surviving subculture to a medium containing 15% PEG did not increase in dry weight for 5 weeks even though the medium was exchanged every 7 days. After 5 weeks, however, dry weight growth resumed and reached 75% of the level attained by control cells grown on PEG-free medium. Long-term growth on a medium containing 15% PEG (PEG-selected cells) could only be sustained if the medium was supplemented with 30 mM glutamine. The PEG-selected cells grew in small clusters, were isodiametric, and had chlorophyll contents 50% higher than unselected cells. The PEG-selected cells also showed lowered cellular osmotic potentials, presumably due to osmoregulation. Turgor pressures of PEG-selected cells were greater than or equal to those of unselected cells.

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New root growth of Douglas-fir seedlings at low carbon dioxide concentration.

New root growth of conifer nursery seedlings is dependent on light, but whether this is necessary only for photosynthesis, or also has some other root growth promoting effect is unknown. This question was investigated using one-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings grown at two nurseries from the same seed lot and taken from cold storage in March and April. New root production was examined in two 10-day root growth capacity (RGC) experiments. Seedlings were subjected to one of four treatments: (1) control, (2) exclusion of light, (3) low CO(2) concentration, and (4) girdling to separate the phloem connection between shoot and root. Shoots were enclosed in Plexiglas cuvettes and supplied with scrubbed air to reduce the CO(2) concentration to 11 to 22 microl l(-1) in the light. The closed system used in Experiment 1 was opened to the atmosphere briefly each day to restore O(2) to ambient, but this was unnecessary for the open system used in Experiment 2. In Experiment 1, new root production was affected by treatments in the order: control > low CO(2) concentration > dark > girdling. Greater new root production in the low CO(2) treatment compared with the dark treatment was attributed to brief increases in CO(2) to ambient concentrations when O(2) was restored each day. In Experiment 2, new root production at the low CO(2) concentration in the light was essentially the same as in darkness, but only 17% of the control value. Thus light appeared to play no part in new root production other than permitting photosynthesis. Limited production of new roots occurred in the absence of photosynthesis, which was further reduced by girdling, presumably because, after girdling, only root system reserves could be used.

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Cardiopulmonary responses after spontaneous inhalation of Douglas fir smoke in goats.

In the majority of clinical cases, smoke inhalation results in a self-limited lung injury mostly confined to the airways. In this study, an animal model of inhalation injury was developed that reflected similar pathophysiology. Cardiopulmonary parameters were studied in awake, instrumented goats following spontaneous inhalation of characterized Douglas fir smoke. Peak carboxyhemoglobin levels averaged 37% during a mean exposure time of 33 minutes. All animals survived the 24-hour study period, and showed only transient abnormalities in lung fluid balance and gas exchange, with no change in lung mechanics or plasma eicosanoid (TxB2 and 6-keto-PGF1 alpha) levels. However, extravascular lung water at 24 hours was increased 33%, suggesting the presence of some airway edema and retained secretions. We feel this model fairly represents the majority of clinical smoke inhalation cases. This model is compared to other large animal inhalation injury models producing more severe lung injury.

6-Ketoprostaglandin F1 alpha↗

Seed parasitism redirects ovule development in Douglas fir.

Many parasitic species of insects complete their entire development in seeds. They feed off storage reserves within the ovule. These reserves only normally accumulate in fertilized ovules. Consequently, female insects that oviposit their eggs directly into the plant ovule need to be able to select correctly, as unfertilized ovules of conifers normally become so-called empty seed. We provide clear evidence that in conifers, seed-parasitizing insects do not need to discriminate between fertilized and unfertilized plant ovules when ovipositing their eggs. A host-specific insect, the chalcid Megastigmus spermotrophus Wachtl (Hymenoptera: Torymidae), lays its eggs in ovules of Douglas fir (Pseudotsuga menziesii (Mirbel) Franco) before fertilization has taken place in the plant. Oviposition not only prevents the expected degeneration and death of unfertilized ovules, but it induces energy reserve accumulation. Ovules that would otherwise develop as empty seed are redirected in their development by the insect to provide food for the developing larvae. Instead of the insect exploiting normal events during seed development, the insect manipulates seed development for its own reproductive advantage.

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Regulation of Oleoresinosis in Grand Fir (Abies grandis) (Coordinate Induction of Monoterpene and Diterpene Cyclases and Two Cytochrome P450-Dependent Diterpenoid Hydroxylases by Stem Wounding).

Oleoresin (pitch) is a defensive secretion composed of monoterpene olefins (turpentine) and diterpene resin acids (rosin) that is produced in grand fir (Abies grandis Lindl.) stems in response to wounding. Monoterpene and diterpene biosynthesis are coordinately induced in wounded stems as determined by monitoring the activity of monoterpene and diterpene cyclases, as well as two cytochrome P450-dependent diterpenoid hydroxylases involved in the formation of ([mdash])-abietic acid, the principal resin acid of this species. The activity of these enzymes reaches maximum levels that are 5- to 100-fold higher than those of nowwounded control stems 10 d after wounding and this is followed by a synchronous decline. The increase in biosynthetic activity is consequently followed by the accumulation of a viscous mass of resin acids, with the loss of the volatile monoterpenes, at the site of injury. The observed coordinate induction of monoterpene olefin and abietic acid bio-synthesis and the results of oleoresin analysis are consistent with the role of the volatile monoterpenes as a solvent for the mobilization and deposition of resin acids at the wound site to seal the injury with a rosin barrier after the evaporation of the turpentine. The last step of resin acid biosynthesis is catalyzed by an operationally soluble aldehyde dehydrogenase that is not inducible by wounding but seemingly is expressed constitutively at a high level. In vivo [14C]acetate feeding and resin analysis indicate that this enzyme is not efficiently coupled to the earlier steps of the pathway.

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Regulation of oleoresinosis in grand fir (Abies grandis). Differential transcriptional control of monoterpene, sesquiterpene, and diterpene synthase genes in response to wounding

Grand fir (Abies grandis Lindl.) has been developed as a model system for the study of wound-induced oleoresinosis in conifers as a response to insect attack. Oleoresin is a roughly equal mixture of turpentine (85% monoterpenes [C10] and 15% sesquiterpenes [C15]) and rosin (diterpene [C20] resin acids) that acts to seal wounds and is toxic to both invading insects and their pathogenic fungal symbionts. The dynamic regulation of wound-induced oleoresin formation was studied over 29 d at the enzyme level by in vitro assay of the three classes of synthases directly responsible for the formation of monoterpenes, sesquiterpenes, and diterpenes from the corresponding C10, C15, and C20 prenyl diphosphate precursors, and at the gene level by RNA-blot hybridization using terpene synthase class-directed DNA probes. In overall appearance, the shapes of the time-course curves for all classes of synthase activities are similar, suggesting coordinate formation of all of the terpenoid types. However, closer inspection indicates that the monoterpene synthases arise earlier, as shown by an abbreviated time course over 6 to 48 h. RNA-blot analyses indicated that the genes for all three classes of enzymes are transcriptionally activated in response to wounding, with the monoterpene synthases up-regulated first (transcripts detectable 2 h after wounding), in agreement with the results of cell-free assays of monoterpene synthase activity, followed by the coordinately regulated sesquiterpene synthases and diterpene synthases (transcription beginning on d 3-4). The differential timing in the production of oleoresin components of this defense response is consistent with the immediate formation of monoterpenes to act as insect toxins and their later generation at solvent levels for the mobilization of resin acids responsible for wound sealing.

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Influence of light intensity at different temperatures on rate of respiration of douglas-fir seedlings.

The rate of photorespiration of Douglas-fir seedlings was measured under different light intensities by: (1) extrapolating the curve for CO(2) uptake in relation to atmospheric CO(2) content to zero CO(2) content, and (2) measuring CO(2) evolution of the plants into a CO(2)-free airstream. Different results, obtained from these techniques, were believed to be caused by a severe restriction of the photosynthetic activity when the latter was used. With the first method, CO(2) evolution was lower than the dark respiration rate at low light intensity. For all temperatures studied (6 degrees , 20 degrees , 28 degrees ) a further increase in light intensity raised the CO(2) evolution above dark respiration before it leveled off. The rate of CO(2) evolution was stimulated by increase in temperature at all light intensities. With the CO(2)-free air method, CO(2) evolution in the light was less than dark respiration at all light intensities.

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Dynamics of photoassimilated carbon in douglas fir seedlings.

The relations between CO(2) uptake, translocation, and carbon accumulation in several vegetative components of Douglas fir seedlings (Pseudotsuga menziesii [Mirb.] Franco) have been quantified using (14)CO(2). Seedlings were exposed to a constant specific radioactivity of (14)CO(2) and a repeating daily pattern of temperature and light for 4 consecutive days. Results of (14)C analysis, which indicated a transitory pattern of photoassimilated carbon movement, were extrapolated to a "steady rate" using a compartment analysis. Accumulation rates of photoassimilated carbon, relative to tissue carbon, were new needles, 0.94%/day, old needles, 1.14%/day, new shoots 0.38%/day, stem, 0.16%/day, and roots, 0.50%/day. Therefore, the source of carbon, the needles, is also the strongest sink.

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Determination of Indole-3-acetic Acid in Douglas Fir Using a Deuterated Analog and Selected Ion Monitoring: Comparison of Microquantities in Seedling and Adult Tree.

Indole-3-acetic acid (IAA) content in shoot tips of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) trees and seedlings was determined by combined gas chromatography-mass spectrometry using a deuterated analog (d(2)-IAA) as an internal standard and the technique of selected ion monitoring. Ratios of the peak heights of the deuterated analog internal standard to endogenous IAA revealed a slightly higher content of IAA in seedlings compared with the shoot tips collected in June. The relatively high level of IAA (2.9 micrograms per gram fresh weight) in seedlings is discussed in relation to in vitro propagation of this species.

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Proanthocyanidins and potential precursors in needles of douglas fir and in cell suspension cultures derived from seedling shoot tissues.

Proanthocyanidins and their potential precursors have been analyzed by paper chromatography and C(18) reversed phase columns with high performance liquid chromatography. Total proanthocyanidins on a dry weight basis in cell suspension cultures derived from seedlings of Douglas fir (Pseudotsuga menziesii) were equal to or greater than those found in mature needles of randomly selected outdoor-grown trees. The major monomer and dimer were catechin and epicatechin-catechin, respectively. Although only procyanidins were detectable in cell suspension cultures, mature needles of outdoor-grown trees contained prodelphinidins as well. Immature needles (flush growth) of the same trees contained only trace amounts of prodelphinidins. Eriodictyol-7-glucoside and dihydroquercetin-3'-glucoside were present in all tissues examined. The amount of eriodictyol-7-glucoside was strongly correlated with total proanthocyanidins in immature needles of flush growth (r = 0.89, p = 0.001). The most complex pattern of flavonoids was found in flush growth needles, which contained in addition to the above, naringenin-7-glucoside and five to six flavone glycosides. Chlorogenic acid was detected only in seedlings and in flush growth needles.

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Incorporation of [C]Phenylalanine into Flavan-3-ols and Procyanidins in Cell Suspension Cultures of Douglas Fir.

L-[(14)C]Phenylalanine, fed to cell suspension cultures of Douglas fir, (Pseudotsuga menziesii Franco) was incorporated simultaneously, but at different rates, into (+)-catechin, (-)-epicatechin, and procyanidins of increasing molecular weight. Asymmetric labeling of dimers and polymers was demonstrated, with more label appearing in the upper than in the lower or terminal unit. In addition, the total pool of free monomers was 10 to 30 times more highly labeled than was this lower, terminal unit of dimers and higher oligomers. Since the dimer, epicatechin-catechin, contained more label than catechin-catechin, it is concluded that the carbocation with the 2,3-cis stereochemistry of (-)-epicatechin was formed more rapidly than was that of the 2,3-trans type of (+)-catechin.

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