Modeling the production of sugar and byproducts from acid bisulfite pretreatment and enzymatic hydrolysis of Douglas-fir.

The aim of this work was to investigate the kinetics of multiple chemicals in acid bisulfite pretreatment and the relationship between total sugar yields and pretreatment factors (temperature and time). The results showed Saeman model accurately fitted the pretreatment process. According to this kinetic model, a maximum hemicellulose hydrolysis yield was achieved at a treatment time of 75min with a temperature of 145°C. Meantime, the concentrations of acetic acid, hydroxymethylfurfural (HMF), and furfural were 1.54, 0.60, and 1.15gL-1, respectively. Also, a Lorentzian function described the relationship between total sugar yield and pretreatment factors: temperature and time. The regression parameters from this mathematical fitting have accurately reflected the maximum total sugar yield and the optimal treatment conditions were determined to be 145°C and 110min.

Local adaptation in migrated interior Douglas-fir seedlings is mediated by ectomycorrhizas and other soil factors.

Separating edaphic impacts on tree distributions from those of climate and geography is notoriously difficult. Aboveground and belowground factors play important roles, and determining their relative contribution to tree success will greatly assist in refining predictive models and forestry strategies in a changing climate. In a common glasshouse, seedlings of interior Douglas-fir (Pseudotsuga menziesii var. glauca) from multiple populations were grown in multiple forest soils. Fungicide was applied to half of the seedlings to separate soil fungal and nonfungal impacts on seedling performance. Soils of varying geographic and climatic distance from seed origin were compared, using a transfer function approach. Seedling height and biomass were optimized following seed transfer into drier soils, whereas survival was optimized when elevation transfer was minimised. Fungicide application reduced ectomycorrhizal root colonization by c. 50%, with treated seedlings exhibiting greater survival but reduced biomass. Local adaptation of Douglas-fir populations to soils was mediated by soil fungi to some extent in 56% of soil origin by response variable combinations. Mediation by edaphic factors in general occurred in 81% of combinations. Soil biota, hitherto unaccounted for in climate models, interacts with biogeography to influence plant ranges in a changing climate.

Conifer somatic embryogenesis: improvements by supplementation of medium with oxidation-reduction agents.

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue (ET) and continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment has been shown to decrease this recalcitrance. Glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid and dehydroascorbate analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiological concentrations. Major differences in stage-specific oxidation-reduction (redox) agents were observed. A simple bioassay was used to evaluate potential growth-promotion of natural and inorganic redox agents added to early-stage somatic embryo growth medium. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase initiation of loblolly pine. Low-cost reducing agents sodium dithionite and sodium thiosulfate increased ET initiation for loblolly pine and Douglas fir (Mirb) Franco. Germination medium supplementation with GSSG increased somatic embryo germination. Early-stage somatic embryos grown on medium with or without sodium thiosulfate did not differ in GSH or GSSG content, suggesting that sodium thiosulfate-mediated growth stimulation does not involve GSH or GSSG. We have developed information demonstrating that alteration of the redox environment in vitro can improve ET initiation, early-stage embryo development and somatic embryo germination in loblolly pine.

Phytoremediation of cadmium-contaminated soils by young Douglas fir trees: effects of cadmium exposure on cell wall composition.

Douglas fir trees grown on an artificially Cd-contaminated soil, can tolerate this trace element (up to 68 mg/kg in soil) during several months. Most of the absorbed Cd is retained in roots (25 mg/kg DM), but transfer to aerial part is also effective. Showing the highest content, up to 6 mg/kg DM, among all the aboveground parts, barks seem to be a preferred storage compartment. However, the transfer factor is quite low, about 0.3. Another objective of this study was to compare the cell wall components of trees exposed to increasing Cd amounts in soil. A decrease in lignin and an increase in pectin contents were observed in response to increasing soil cadmium concentration. A concurrent reduction in methyl-esterification of pectin suggests than the structure of this major binding site could therefore be modified as a reaction to cadmium contamination. Future prospects will focus on the modulation of pectin composition in response to Cd exposure.

Alkaline hydrogen peroxide pretreatment of softwood: hemicellulose degradation pathways.

This study investigated softwood hemicelluloses degradation pathways during alkaline hydrogen peroxide (AHP) pretreatment of Douglas fir. It was found that glucomannan is much more susceptible to alkaline pretreatment than xylan. Organic acids, including lactic, succinic, glycolic and formic acid are the predominant products from glucomannan degradation. At low treatment temperature (90°C), a small amount of formic acid is produced from glucomannan, whereas glucomannan degradation to lactic acid and succinic acid becomes the main reactions at 140°C and 180°C. The addition of H2O2 during alkaline pretreatment of D. fir led to a significant removal of lignin, which subsequently facilitated glucomannan solubilization. However, H2O2 has little direct effect on the glucomannan degradation reaction. The main degradation pathways involved in glucomannan conversion to organics acids are elucidated. The results from this study demonstrate the potential to optimize pretreatment conditions to maximize the value of biomass hemicellulose.

Metal stress and decreased tree growth in response to biosolids application in greenhouse seedlings and in situ Douglas-fir stands.

To assess physiological impacts of biosolids on trees, metal contaminants and phytochelatins were measured in Douglas-fir stands amended with biosolids in 1982. A subsequent greenhouse study compared these same soils to soils amended with fresh wastewater treatment plant biosolids. Biosolids-amended field soils had significantly higher organic matter, lower pH, and elevated metals even after 25 years. In the field study, no beneficial growth effects were detected in biosolids-amended stands and in the greenhouse study both fresh and historic biosolids amendments resulted in lower seedling growth rates. Phytochelatins - bioindicators of intracellular metal stress - were elevated in foliage of biosolids-amended stands, and significantly higher in roots of seedlings grown with fresh biosolids. These results demonstrate that biosolids amendments have short- and long-term negative effects that may counteract the expected tree growth benefits.

Enzymatic saccharification of woody biomass micro/nanofibrillated by continuous extrusion process II: effect of hot-compressed water treatment.

An extrusion process involving a twin-screw extruder was used for the micro/nanofibrillation of Douglas fir and Eucalyptus treated with hot-compressed water (HCW). Partial removal of hemicellulose and lignin by HCW treatment effectively improved the fibrillation by extrusion. Only HCW treatment produced glucose less than 5 weight percent (wt.%) in Douglas fir in a temperature range of 140-180 degrees C by enzymatic hydrolysis. Glucose production yields of 18 and 26 wt.% were obtained by HCW treatment at 170 and 180 degrees C, respectively, in Eucalyptus. Use of extrusion after HCW treatment drastically improved monosaccharide production yield in both woods. In the case of Douglas fir, the obtained values were 5 times higher than those obtained by HCW treatment alone. Total monosaccharide production yields were higher in Eucalyptus than in Douglas fir. The extruded production had a fine fibrous morphology on a sub-micro/nanoscopic scale. This result shows the great potential of the extrusion process after HCW treatment as a cost-effective pretreatment for enzymatic saccharification of woody biomass.

Coupling tree-ring delta13C and delta15N to test the effect of fertilization on mature Douglas-fir (Pseudotsuga menziesii var. glauca) stands across the Interior northwest, USA.

Nitrogen (N) fertilization causes long-term increases in biomass production in many N-limited forests around the world, but the mechanistic basis underlying the increase is often unclear. One possibility, especially in summer-dry climates, is that N fertilization increases the efficiency with which a finite water supply is consumed to support photosynthesis. This increase is achieved by a reduction in the canopy-integrated concentration of internal CO(2) and thus discrimination against (13)C. We used stable isotopes of carbon (delta(13)C) in tree rings to experimentally test the physiological impact of N fertilization on mature Douglas-fir (Pseudotsuga menziesii Franco var. glauca) stands across the geographic extent of the Intermountain West, USA. The concentration and the stable isotopes of N (delta(15)N) in tree rings were also used to assess the presence and activity of fertilizer N. We hypothesized that N fertilization would (i) increase delta(15)N and N concentration of stemwood relative to non-fertilized stands and (ii) increase stemwood delta(13)C as photosynthetic gas exchange responded to the additional N. This experiment included two rates of urea addition, 178 kg ha(-1) (low) and 357 kg ha(-1) (high), which were applied twice over a 6-year interval bracketed by the 18 years of wood production measured in this study. Foliar N concentrations measured the year after each fertilization treatment suggest that the fertilizer N had been assimilated by the trees (P < 0.001). The N fertilization significantly enriched stemwood delta(15)N by 1.3 per thousand at the low fertilization rate and by 2.4 per thousand at the high rate (P < 0.001) despite variation in soil N between sites. However, we found no significant effect of the N fertilizer on delta(13)C of the annual rings (P = 0.76). These data lead us to suggest that alternative mechanisms underlie the growth response to fertilizer, i.e., increase in canopy area and shifts in biomass allocation.

Phytohormones and their metabolites during long shoot development in Douglas-fir following cone induction by gibberellin injection.

Changes in plant hormones and metabolites in long-shoot stems of interior Douglas-fir (Pseudotsuga menziesii var. glauca (Beissn.) Franco) during cone induction by gibberellic acid (GA) treatment were analyzed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode. A mixture of GA(4) and GA(7), including small amounts of GA(3) and GA(1), was stem-injected into each tree in amounts of 0, 4, 40 or 400 mg. One week after injection, concentrations of GA(4), GA(7) and GA(3) were elevated in all GA-treated samples. The ratio of GA(4) to GA(7) decreased significantly at Week 3. Absolute concentrations of all gibberellins declined sharply at Week 3 after GA application. After 5 weeks, GA(1) and GA(4) were below detection limits in all samples, and GA(7) and GA(3) were found only in the samples from trees treated with 40 or 400 mg of GA. Endogenous indole-3-acetic acid (IAA) concentrations increased following GA injection, and peaked at Week 2 or Week 3 in the trees treated with 40 or 400 mg GA, respectively. Injection of 400 mg of GA brought about a twofold increase in IAA concentration compared with control values. Injection of 40 and 400 mg of GA caused significant increases in stem dry mass in Week 5. Seed orchard data revealed that injection of either 40 or 400 mg GA enhanced female cone formation, whereas male cone formation was enhanced only by 400 mg GA. Slight decreases in concentrations of abscisic acid (ABA) and isopentenyl adenosine were observed after GA application. No significant changes were detected in the concentrations of ABA metabolites except for a slight decrease in the concentration of 7'-hydroxy ABA. The concentration of ABA declined during the growing season and the concentration of ABA glucose ester increased correspondingly.

Fire resistance of Douglas fir (Pseudotsuga menzieesi) treated with borates and natural extractives.

The objective of this study was to determine fire resistance of Douglas fir (Pseudotsuga menzieesi (Mirb.) Franco) specimens treated with borate supplemented aqueous solutions of brutia pine bark powder, acorn powder, sumach leaf powder, and gall-nut powder. Boric acid (BA) and borax (BX) were used as borates which are the most commonly used fire retardants in wood preservation industry. Natural extractives (brutia pine bark powder, sumach leaf powder, acorn powder, and gall-nut powder) were also used which have toxic efficiency against insects and fungi due to their tannin contents. A commercial treatment compound Tanalith-CBC (copper-borate-chromate), which is an impregnation chemical, is used for comparison. The fire test method was performed in three stages: flame stage, without flame stage, and glowing stage. Results indicated that the lowest temperature for flame stage, without flame stage, and glowing stage were obtained for specimens treated with BA and BX mixture (7:3; weight:weight). The lowest mass loss was found for the specimens treated with a mixture of BA and BX. Natural extractives did not improve fire resistance of the samples. However, boric acid and borax had excellent fire retardant effectiveness over untreated and treated samples with natural extractives.

Hormonal control of second flushing in Douglas-fir shoots.

Spring-flushing, over-wintered buds of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) produce new buds that may follow various developmental pathways. These include second flushing in early summer or dormancy before flushing during the following spring. Second flushing usually entails an initial release of apical dominance as some of the current-season upper lateral buds grow out. Four hypotheses concerning control of current bud outgrowth in spring-flushing shoots were tested: (1) apically derived auxin in the terminal spring-flushing shoot suppresses lateral bud outgrowth (second flushing); (2) cytokinin (0.5 mM benzyladenine) spray treatments given midway through the spring flush period induce bud formation; (3) similar cytokinin spray treatments induce the outgrowth of existing current lateral buds; and (4) defoliation of the terminal spring-flushing shoot promotes second flushing. Hypothesis 1 was supported by data demonstrating that decapitation-released apical dominance was completely restored by treatment with exogenous auxin (22.5 or 45 mM naphthalene acetic acid) (Thimann-Skoog test). Hypothesis 2 was marginally supported by a small, but significant increase in bud number; and Hypothesis 3 was strongly supported by a large increase in the number of outgrowing buds following cytokinin applications. Defoliation produced similar results to cytokinin application. We conclude that auxin and cytokinin play important repressive and promotive roles, respectively, in the control of second flushing in the terminal spring-flushing Douglas-fir shoot.

Growth response of Douglas-fir seedlings to nitrogen fertilization: importance of Rubisco activation state and respiration rates.

High foliar nitrogen concentration ([N]) is associated with high rates of photosynthesis and thus high tree productivity; however, at excessive [N], tree productivity is reduced. Reports of excessive [N] in the Douglas-fir forests of the Oregon Coast Range prompted this investigation of growth and needle physiological responses to increasing foliar N concentrations in 1-year-old Douglas-fir seedlings. After 1 year of N fertilization, total seedling biomass increased with each successive increase in N fertilizer concentration, except in the highest N fertilization treatment. Of the many physiological responses that were analyzed, only photosynthetic capacity (i.e., Vcmax), respiration rates and leaf specific conductance (KL) differed significantly between N treatments. Photosynthetic capacity showed a curvilinear relationship with foliar [N], reaching an apparent maximum rate when needle N concentrations exceeded about 12 mg g(-1). In vitro measurements of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity suggested that photosynthetic capacity was best related to activated, not total, Rubisco content. Rubisco activation state declined as foliar [N] increased, and based on its significant correlation (r2= 0.63) with foliar Mn:Mg ratios, it may be related to Mn inactivation of Rubisco. Respiration rates increased linearly as foliar N concentration increased (r2= 0.84). The value of K(L) also increased as foliar [N] increased, reaching a maximum when foliar [N] exceeded about 10 mg g(-1). Changes in K(L) were unrelated to changes in leaf area or sapwood area because leaf area to sapwood area ratios remained constant. Cumulative effects of the observed physiological responses to N fertilization were analyzed by modeling annual net CO2 assimilation (Anet) based on treatment specific values of Vcmax, dark respiration (Rdark) and KL. Estimates of Anet were highly correlated with measured total seedling biomass (r2= 0.992), suggesting that long-term, cumulative effects of maximum Rubisco carboxylation, Rdark and KL responses to N fertilization may limit seedling production when foliar N exceeds about 13 mg g(-1) or is reduced to less than about 11 mg g(-1).

Changes in anatomy and terpene chemistry in roots of Douglas-fir seedlings following treatment with methyl jasmonate.

Replicated trials were conducted on two full-sibling families of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings. In response to the application of a 0.01% solution of methyl jasmonate (MeJA) to the soil of potted seedlings, numerous anatomical and chemical changes were observed in the roots, stem and foliage. These changes were, for the most part, similar for both sib groups. Methyl jasmonate induced traumatic resin duct formation in roots and stems. Chemical differences between MeJA-treated and control seedlings were mainly limited to the roots and stem, though some changes also occurred in the foliage. A total of 35 terpenoids were observed in the P. menziesii seedlings. In response to MeJA treatment, several of the 22 detected monoterpenoids (linalool, bornyl acetate, camphene, myrcene, alpha- and beta-pinene, tricyclene and beta-phellandrene) increased significantly in roots and stems, whereas (E)-beta-ocimene decreased significantly in the foliage. Four of the five detected sesquiterpenoids (alpha-humulene, germacrene D, longifolene and (E)-caryophyllene) increased significantly following MeJA application, mainly in the root and stem. Four of the eight detected diterpenoids (abietate, levopimarate, palustrate and sandaracopimarate) increased in response to MeJA treatment, but only in root and stem tissue. This study provides the first description of the effects of MeJA applied to roots through the soil on the anatomy and terpene chemistry of a gymnosperm. This comprehensive inventory of terpenoids in P. menziesii, with and without MeJA treatment, may facilitate identification of terpenoid-related resistance traits. Potential practical applications of MeJA treatment of conifer roots as a pest management strategy are discussed.

Methyl jasmonate-induced ethylene production is responsible for conifer phloem defense responses and reprogramming of stem cambial zone for traumatic resin duct formation.

Conifer stem pest resistance includes constitutive defenses that discourage invasion and inducible defenses, including phenolic and terpenoid resin synthesis. Recently, methyl jasmonate (MJ) was shown to induce conifer resin and phenolic defenses; however, it is not known if MJ is the direct effector or if there is a downstream signal. Exogenous applications of MJ, methyl salicylate, and ethylene were used to assess inducible defense signaling mechanisms in conifer stems. MJ and ethylene but not methyl salicylate caused enhanced phenolic synthesis in polyphenolic parenchyma cells, early sclereid lignification, and reprogramming of the cambial zone to form traumatic resin ducts in Pseudotsuga menziesii and Sequoiadendron giganteum. Similar responses in internodes above and below treated internodes indicate transport of a signal giving a systemic response. Studies focusing on P. menziesii showed MJ induced ethylene production earlier and 77-fold higher than wounding. Ethylene production was also induced in internodes above the MJ-treated internode. Pretreatment of P. menziesii stems with the ethylene response inhibitor 1-methylcyclopropene inhibited MJ and wound responses. Wounding increased 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase protein, but MJ treatment produced a higher and more rapid ACC oxidase increase. ACC oxidase was most abundant in ray parenchyma cells, followed by cambial zone cells and resin duct epithelia. The data show these MJ-induced defense responses are mediated by ethylene. The cambial zone xylem mother cells are reprogrammed to differentiate into resin-secreting epithelial cells by an MJ-induced ethylene burst, whereas polyphenolic parenchyma cells are activated to increase polyphenol production. The results also indicate a central role of ray parenchyma in ethylene-induced defense.

Brassinolide improves embryogenic tissue initiation in conifers and rice.

Somatic embryogenesis (SE), the most promising technology for the large-scale production of high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and the uniformity of future U.S. forests. To be successful for commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine ( Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation percentages of loblolly pine, Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco], and Norway spruce ( Picea abies L., Karst.) were improved through the use of brassinolide. Brassinosteroids, which include brassinolide, are a relatively new group of natural plant growth regulators that are found in many plant species. They have been shown to have diverse, tissue-specific, and species-specific effects, including the stimulation of cell elongation and ethylene production and increasing resistance to abiotic stress. In our media, brassinolide was effective at concentrations ranging from 0.005-0.25 micro M. Using control medium (no brassinolide) and brassinolide-supplemented (0.1 micro M) medium, we achieved improved initiation percentages in loblolly pine, Douglas-fir, Norway spruce, and rice-15.0% to 30.1%, 16.1% to 36.3%, 34.6% to 47.4%, and 10%, respectively. Brassinolide increased the weight of loblolly pine embryogenic tissue by 66% and stimulated initiation in the more recalcitrant families of loblolly pine and Douglas-fir, thus compensating somewhat for genotypic differences in initiation. Initiation percentages in loblolly pine were improved through the combination of modified 1/2-P6 salts, 50 mg/l activated carbon (AC), adjusted levels of Cu and Zn (to compensate for adsorption by AC), 1.5% maltose, 2% myo-inositol (to raise the osmotic level, partially simulating the megagametophyte environment), 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l alpha-naphthaleneacetic acid, 0.63 mg/l 6-benzylaminopurine, 0.61 mg/l kinetin, 3.4 mg/l silver nitrate, 10 micro M cGMP, 0.1 micro M brassinolide, and 2 g/l Gelrite. Across 12 open-pollinated families of loblolly pine, initiation percentages ranged from 2.5% to 50.7%, averaging 22.5%.

Methyl jasmonate induces changes mimicking anatomical defenses in diverse members of the Pinaceae.

Conifers have defenses such as the production of phenolic compounds and resins that can be induced by bark beetles and other invading organisms, but the signaling agents involved are unknown. The anatomical effects of methyl jasmonate (MJ), a potent inducer of certain plant defenses, were compared with wounding of the bark of 12-15-year-old trees of five conifer species. Wounding in all species resulted in tissue necrosis and wound periderm development immediately around the wound site. One cm from the wound, swelling of phloem polyphenolic parenchyma cells and phenolic accumulation were observed in Pseudotsuga menziesii (Mirb.) Franco, Picea pungens Engelman, Larix occidentalis Nutt. and Pinus monticola Douglas ex D. Don, but not in Taxus brevifolia Nutt. Traumatic resin ducts were formed in response to wounding in three species of Pinaceae, but not in P. monticola, which formed irregular clusters of cells rather than ducts. Taxus brevifolia did not form resin ducts in response to either wounding or MJ treatment. In the Pinaceae species studied, surface application of 100 mM MJ caused similar anatomical changes to those observed in response to wounding, including phenolic accumulation, cell swelling and traumatic resin duct formation, but it did not induce a wound periderm. Traumatic resin ducts differed in size among the study species, ranging from small in L. occidentalis to very large in P. menziesii. In P. menziesii, P. pungens and L. occidentalis, traumatic resin ducts were more abundant after MJ treatment than after wounding. We conclude that the octadecanoid pathway is likely involved in defense responses in stems of the Pinaceae, but not necessarily in other taxa.