Purification and properties of glutamine synthetase from Douglas fir roots

Glutamine synthetase (GS. EC 6.3.1.2) was purified to apparent electrophoretic homogeneity from roots of Pseudotsuga menziesii (Mirb) Franco by a three-step procedure involving diethylaminoethyl (DEAE)-Trisacryl chromatography, affinity chromatography on Matrex Gel Red A. and preparative polyacrylamide gel electrophoresis. The enzyme was purified 40-fold with a 16% recovery. The native enzyme had a molecular mass of 460 ± 5 kDa as estimated by gel filtration, interpolation of the Ferguson plots and non-denaturing gradient-PAGE. It was composed of two different subunits of 54 and 64 kDa. Affinity constants for glutamate (Glu), glutamine (Gln), ATP and ADP were 2.6, 10.5, 0.5 and 0.083 m M . respectively. The enzyme exhibited a negative cooperativity for ammonium (Hill number of 0.7) with two K_m values which were 11 and 75 μ M in the presence of ammonium concentrations lower and higher than 1.3 m M , respectively. Glycine and ADP appeared as potential inhibitors of the GS activity. The optimum pH values were 7.2 and 7.6 for the transferase and the biosynthetic assays, respectively. The enzyme lost 30% of its activity within 25 days of storage at 4°C. The optimum temperatures of activity were 40°C and 45°C for the transferase and bio-synthetic activities, respectively.     

A neural network based pattern recognition system for somatic embryos of Douglas fir

A pattern recognition system was developed to classify Douglas fir somatic embryos by employing an image analysis system and two neural network based classifiers. The contour of embryo images was segmented, digitalized and converted to numerical values after the discrete and fast Fourier transformation. These values, or Fourier features, along with some other shape factors, were used for embryo classification. The pattern recognition system used a hierarchical decision tree to classify Douglas fir embryos into three normal and one abnormal embryo classes. An accuracy of greater than 80% was achieved for normal embryos. This system provides an objective and efficient method of classifying embryos of Douglas fir. It will be a useful tool for kinetic studies and process optimization of conifer somatic embryogenesis.     

In vitro effect of formaldehyde on Douglas fir pollen

Formaldehyde (HCHO) concentrations of up to 479 mmol m^–3 have been reported in urban fogwater, yet little is known about the phytotoxicity of HCHO at such concentrations. We report that formaldehyde at target concentrations of 300, 600, 900 and 1200mmol m^–3 reduced germination of Douglas fir [ Pseudotsuga menziesii (Mirbel) Franco.] pollen in vitro . Formaldehyde concentrations and pH in media containing pollen decreased over 25 h. These effects were not seen with heat-killed pollen nor with media lacking pollen, which may suggest uptake or detoxification by live pollen. Stress was indicated by ion leakage from pollen, which increased within 20 h in all HCHO treatments. Reduced pollen viability was indicated by TTC staining of pollen, which decreased after HCHO treatment.     

Molecular analysis of bacterial communities associated with the roots of Douglas fir (Pseudotsuga menziesii) colonized by different ectomycorrhizal fungi

We studied the effect of ectomycorrhizal fungi on bacterial communities colonizing roots of Douglas fir (Pseudotsuga menziesii). Mycorrhizal tips were cleaned of soil and separated based on gross morphological characteristics. Sequencing of the internal transcribed spacers of the nuclear rRNA gene cluster indicated that the majority of the tips were colonized by fungi in the Russulaceae, with the genera Russula and Lactarius comprising 70% of the tips. Because coamplification of organellar 16S rRNA genes can interfere with bacterial community analysis of root tips, we developed and tested a new primer pair that permits amplification of bacterial 16S rRNA genes but discriminates more effectively against organellar sequences than commonly used bacterial primer sets. We then used terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of the 16S rRNA gene to examine differences in bacterial communities associated with the mycorrhizal tips. Cluster analysis of T-RFLP profiles indicated that there were different bacterial communities among the root tips; however, the communities did not seem to be affected by the taxonomic identity of the ectomycorrhizal fungi. Terminal restriction fragment profiling and sequencing of cloned partial 16S rRNA genes indicated that most bacteria on the ectomycorrhizal tips were related to the Alphaproteobacteria and the Bacteroidetes group.     

In vitro fertilization from co-cultured pollen tubes and female gametophytes of Douglas fir (Pseudotsuga menziesii)

 Our previous attempt on in vitro fertilization (IVF) in conifers resulted in pollen tube penetration of female gametophytes, but because of the rapid decline in egg viability, no further interaction occurred. In this report, we describe for the first time that IVF has been achieved in conifers. Using Douglas fir (Pseudotsuga menziesii), we describe a two-step process which involved induction of pollen tubes in culture followed by introduction of isolated female gametophytes at the tips of growing pollen tubes. Pollen tubes penetrated the introduced isolated female gametophytes at various places, but a number of tubes entered the egg cell through the neck cells similar to the in vivo condition. Under our current culture conditions, longevity of pollen tubes and eggs has been improved resulting in the release of sperms, fusion of gametes, and initial formation of the proembryo. Continued plasmolysis of the egg limited the number of successful gametic interactions. IVF has been accomplished in flowering plants in several ways, but the gametophyte-gametophyte IVF system described in this paper is unique. IVF offers a novel breeding technology that takes advantage of the sexual reproductive route. When coupled with hybridization and genetic transformation, IVF could result in the development of stable novel genotypes of economically superior trees. Received: 28 October 1997 / Accepted: 9 December 1997     

Uptake and translocation of manganese in seedlings of two varieties of Douglas fir (Pseudotsuga menziesii var. viridis and glauca)

Douglas fir (Pseudotsuga menziesii) variety glauca (DFG) but not the variety viridis (DFV) showed symptoms of manganese (Mn) toxicity in some field sites. We hypothesized that these two varieties differed in Mn metabolism. To test this hypothesis, biomass partitioning, Mn concentrations, subcellular localization and 54Mn-transport were investigated. Total Mn uptake was three-times higher in DFG than in DFV. DFV retained > 90% of 54Mn in roots, whereas > 60% was transported to the shoot in DFG. The epidermis was probably the most efficient Mn barrier since DFV contained lower Mn concentrations in cortical cells and vacuoles of roots than DFG. In both varieties, xylem loading was restricted and phloem transport was low. However, sieve cells still contained high Mn concentrations. DFV displayed higher biomass production and higher shoot : root ratios than DFG. Our results clearly show that both varieties of Douglas fir differ significantly in Mn-uptake and allocation patterns rendering DFG more vulnerable to Mn toxicity.     

Detection and identification of gibberellins in Douglas fir (Pseudotsuga menziesii) shoots

Extracts of Douglas fir ( Pseudotsuga menziesii [Mirb.] Franco) shoots were purified by reversed and normal phase HPLC; gibberellin (GA)-like compounds detected by radioimmunoassay with antibodies against GA₄ and the Tan-ginbozu dwarf rice micro-drop biossay were analyzed by GC-MS. Three major components were identified as GA₄, GA₇, and GA₉ while smaller amounts of GA¹, GA₃ and putative GA⁹-glucosyl ester were also present.     

Pseudotsuga menziesii chemical races of California and Oregon

Cortical oleoresin from more than 300 Douglas-fir trees sampled in over 30 localities throughout California and southern Oregon was analyzed for its monoterpene hydrocarbon composition. The results confirmed the existence of a distinct chemical race found in its pure unhybridized form throughout the Sierra Nevada as well as near Lompoc in the Purissima Hills of coastal California. Chemical features characterizing this race included near-absence of sabinene and presence of larger amounts of - and β-pinenes in the cortical essential oil. Elsewhere in California and southernmost Oregon, the Sierra Nevada race has extensively intergraded with the coastal Douglas-fir race from western Oregon, Washington, and British Columbia (var. menziesii), a race producing large amounts of sabinene and lesser amounts of - and β-pinenes. On the basis of chemical composition, the Sierra Nevada race was judged to be closer to the inland Douglas-fir races (var. glauca) than to the coastal race. Evolution of the Sierra Nevada race is explained by the emergence of the Great Basin arid regions during the PliocenePleistocene epochs which disjoined Western Douglas-fir populations from their eastern inland counterparts.     

Hydraulic control of stomatal conductance in Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] and alder [Alnus rubra (Bong)] seedlings

Experiments were conducted on 1-year-old Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] and 2- to 3-month-old alder [Alnus rubra (Bong)] seedlings growing in drying soils to determine the relative influence of root and leaf water status on stomatal conductance (g_c). The water status of shoots was manipulated independently of that of the roots using a pressure chamber that enclosed the root system. Pressurizing the chamber increases the turgor of cells in the shoot but not in the roots. Seedling shoots were enclosed in a whole-plant cuvette and transpiration and net photosynthesis rates measured continuously. In both species, stomatal closure in response to soil drying was progressively reversed with increasing pressurization. Responses occurred within minutes of pressurization and measurements almost immediately returned to pre-pressurization levels when the pressure was released. Even in wet soils there was a significant increase in g_c with pressurization. In Douglas fir, the stomatal response to pressurization was the same for seedlings grown in dry soils for up to 120 d as for those subjected to drought stress over 40 to 60 d. The stomatal conductance of both Douglas fir and alder seedlings was less sensitive to root chamber pressure at higher vapour pressure deficits (D), and stomatal closure in response to increasing D from 1.04 to 2.06 kPa was only partially reversed by pressurization. Our results are in contrast to those of other studies on herbaceous species, even though we followed the same experimental approach. They suggest that it is not always appropriate to invoke a ‘feedforward’ model of short-term stomatal response to soil drying, whereby chemical messengers from the roots bring about stomatal closure.     

Responses of the western spruce budworm to varying levels of nitrogen and terpenes

Summary An agar diet study using western spruce budworm populations from Idaho and New Mexico was carried out to determine the effects of varying concentrations of nitrogen, beta-pinene, and bornyl acetate on larval growth and survival. Increased availability of nitrogen resulted in increased larval growth rate and survival to the adult stage. Larval growth rates from the Idaho population were higher on the high-nitrogen diet than were growth rates from the New Mexico population. The high level of beta-pinene improved larval growth at the high-nitrogen concentration. Bornyl acetate significantly reduced larval growth at both the low and high levels of nitrogen although the effect was greatest with the high-nitrogen diet. High bornyl acetate concentrations also significantly reduced survival to the adult stage. At high-nitrogen levels, a high concentration of bornyl acetate reduced larval growth rates and adult survival to a level similar to that occurring at the low nitrogen and low bornyl acetate concentrations.     

Occurrence of vesicular-arbuscular mycorrhizae in Pseudotsuga menziesii and Tsuga heterophylla seedlings grown in Oregon Coast Range soils

 Vesicular-arbuscular mycorrhizae (VAM) were common in seedlings of Pseudotsuga menziesii and Tsuga heterophylla grown in a greenhouse soil bioassay in soils collected from the Oregon Coast Range. Although root samples were heavily colonized by ectomycorrhizal fungi (EM), VAM colonization was observed in the cortical cells of both secondary and feeder roots. Vesicles, arbuscules, and hyphae typical of VAM occurred in 48% of 61 P. menziesii and 25% of 57 T. heterophylla seedlings. The ecological significance of VAM presence in the Pinaceae, as well as interactions among VAM, EM, and the plant host, deserve future investigation. Accepted: 16 August 1995     

Multiple-host fungi are the most frequent and abundant ectomycorrhizal types in a mixed stand of Douglas fir (Pseudotsuga menziesii) and bishop pine (Pinus muricata)

The ectomycorrhizal fungal associations of Douglas fir ( Pseudotsuga menziesii D. Don) and bishop pine ( Pinus muricata D. Don) were investigated in a mixed forest stand. We identified fungi directly from field-collected ectomycorrhizal (ECM) root tips using PCR-based methods. Sixteen species of fungi were found, of which twelve associated with both hosts. Rhizopogon parksii Smith was specific to Douglas fir. Three other species colonized only one of the hosts, but were too infrequent to draw conclusions about specificity. Seventy-four percent of the biomass of ECM root tips sampled in the stand were colonized by members of the Thelephoraceae and Russulaceae. All 12 species of fungi that associated with both tree species did so within a 10×40 cm soil volume, suggesting that individual fungal genotypes linked the putatively competing tree hosts.     

Physiological effects of five months exposure to low concentrations of O3 and NH3 on Douglas fir (Pseudotsuga menziesii)

Three years old seedlings of Douglas fir (Pseudotsuga menziesii) were exposed lo filtered air, O₃ (day and night concentrations of 78 and 30 μgm^?3: respectively). NH₃ (54 μg m^?3) and to a mixture of NH₃+O₃ (day and night concentrations of 49 + 83 and 49 + 44 μg m^?3 respectively), for 5 months in fumigation chambers. Both gas exchange and chlorophyll fluorescence were measured on shoots which had sprouted at the beginning of the exposure period. After 4. 8, 10 and 20 weeks of exposure, light response curves of electron transport rate (J) were determined, in which J was deduced from chlorophyll fluorescence. Net CO₂ assimiialion was measured at maximum light intensity of 560) μmol m^?2 S^?1 (P_n.560). After 8 and 10 weeks of exposure also light response curves of CO₂ assimilation were assessed. Shoots exposed to O₃ showed a reduction in net CO₂ assimilation as compared to the control shoots during the entire exposure period. The reduction was related lo a lower chlorophyll content and a lower electron transport rate, whereas no effect on quantum yield efficiency (qy) was observed. In contrast, shoots exposed to NH₃ showed a positive effect on photosynthesis. Shoots exposed to NH₃. + O₃ showed a rapid increase in P_n.560, in the period between 4 and 8 weeks to a level equal of that of the NH₃-treatment. After this period a decline in P_n.560 was observed. After 10 weeks of exposure shoots exposed to O₃ showed an increased transpiration rate in the dark as compared to the control shoots. In addition, water use efficiency (WUE) declined as a result of an increase in leaf conductance. Both observations indicate that the stomatal apparatus was affected by O₃. A high transpiration rate in the dark was also found for shoots esposed to NHX. However, shoots exposed to NH₃+ O₃ showed neither an effect on WUE, nor an effect on transpiration rate in the dark. The possibility that NH₃ delayed the O₃ induced effects on photosynthesis and stomatal conductance is discussed.     

Genetic maladaptation of coastal Douglas-fir seedlings to future climates

Climates are expected to warm considerably over the next century, resulting in expectations that plant populations will not be adapted to future climates. We estimated the risk of maladaptation of current populations of coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) to future climates as the proportion of nonoverlap between two normal distributions where the means and genetic variances of current and future populations are determined from genecological models derived from seedling common garden studies. The risk of maladaptation was large for most traits when compared with the risk associated with current transfers within seed zones, particularly for the more drastic climate change scenario. For example, the proportion of nonoverlap for a composite trait representing bud set, emergence, growth, and root : shoot ratio was as high as 0.90. We recommend augmenting within-population variation by mixing local populations with some proportion of populations from lower elevations and further south. Populations expected to be adapted to climates a century from now come from locations as far down in elevation as 450–1130 m and as far south in latitude as 1.8–4.9°.     

Aphid-induced reduction of shoot and root growth in Douglas-fir seedlings

1. The short‐ and long‐term effects of photosynthate removal by sap‐sucking herbivores on plant growth were examined by experimentally manipulating densities of an aphid Cinara pseudotsugae (Wilson) on 2‐year‐old Douglas‐fir seedlings Pseudotsuga menziesii (Mirb.) Franco under greenhouse conditions. An 18‐week test was conducted to determine short‐term effects. Effects of long‐term aphid feeding were examined by exposing seedlings to aphid feeding for two consecutive growing seasons. A third experiment evaluated the extent of recovery during 1 year following a single season of aphid feeding. At least 35 seedlings were used in each test. 2. Volume and dry weight of both shoots and roots decreased significantly with increasing aphid feeding in all three experiments. 3. The most significant and severe aphid effect was reduced root tissue density, suggesting carbohydrate depletion due to translocation from roots to shoots. 4. There was no sign of recovery, of either root or shoot growth, during the year following one season of feeding. 5. The results of this study indicate that short‐term feeding by aphids can affect plant growth and structure for a relatively long time.