Differences in carbon isotope leaf-to-phloem fractionation and mixing patterns along a vertical gradient in mature European beech and Douglas fir.

While photosynthetic isotope discrimination is well understood, the postphotosynthetic and transport-related fractionation mechanisms that influence phloem and subsequently tree ring δ13 C are less investigated and may vary among species. We studied the seasonal and diel courses of leaf-to-phloem δ13 C differences of water-soluble organic matter (WSOM) in vertical crown gradients and followed the assimilate transport via the branches to the trunk phloem at breast height in European beech (Fagus sylvatica) and Douglas fir (Pseudotsuga menziesii). δ13 C of individual sugars and cyclitols from a subsample was determined by compound-specific isotope analysis. In beech, leaf-to-phloem δ13 C differences in WSOM increased with height and were partly caused by biochemical isotope fractionation between leaf compounds. 13 C-Enrichment of phloem sugars relative to leaf sucrose implies an additional isotope fractionation mechanism related to leaf assimilate export. In Douglas fir, leaf-to-phloem δ13 C differences were much smaller and isotopically invariant pinitol strongly influenced leaf and phloem WSOM. Trunk phloem WSOM at breast height reflected canopy-integrated δ13 C in beech but not in Douglas fir. Our results demonstrate that leaf-to-phloem isotope fractionation and δ13 C mixing patterns along vertical gradients can differ between tree species. These effects have to be considered for functional interpretations of trunk phloem and tree ring δ13 C.

Increased water deficit decreases Douglas fir growth throughout western US forests.

Changes in tree growth rates can affect tree mortality and forest feedbacks to the global carbon cycle. As air temperature increases, evaporative demand also increases, increasing effective drought in forest ecosystems. Using a spatially comprehensive network of Douglas fir (Pseudotsuga menziesii) chronologies from 122 locations that represent distinct climate environments in the western United States, we show that increased temperature decreases growth via vapor pressure deficit (VPD) across all latitudes. Using an ensemble of global circulation models, we project an increase in both the mean VPD associated with the lowest growth extremes and the probability of exceeding these VPD values. As temperature continues to increase in future decades, we can expect deficit-related stress to increase and consequently Douglas fir growth to decrease throughout its US range.

Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii [Mirb.].

BACKGROUND: To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g- 1 f.w.). Moreover, we observed similar differences in yield between 2ry and 3ry lines of SD4 (2400 vs 3921 g- 1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2ry vs 1ry and 2ry vs 3ry lines at histo-cytological (using LC-MS/MS) and proteomic levels. RESULTS: Repetitive somatic embryogenesis dramatically improved the proliferating lines' cellular organization (genotype SD4's most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2ry and (even more) 3ry lines. Among 2300-2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on "Proteolysis" and "Catabolic process" Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same "Plant development" and "Defense response" biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3ry lines. CONCLUSIONS: This first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines' embryogenic status.

Transfer of 13 C between paired Douglas-fir seedlings reveals plant kinship effects and uptake of exudates by ectomycorrhizas.

Processes governing the fixation, partitioning, and mineralization of carbon in soils are under increasing scrutiny as we develop a more comprehensive understanding of global carbon cycling. Here we examined fixation by Douglas-fir seedlings and transfer to associated ectomycorrhizal fungi, soil microbes, and full-sibling or nonsibling neighbouring seedlings. Stable isotope probing with 99% 13 C-CO2 was applied to trace 13 C-labelled photosynthate throughout plants, fungi, and soil microbes in an experiment designed to assess the effect of relatedness on 13 C transfer between plant pairs. The fixation and transfer of the 13 C label to plant, fungal, and soil microbial tissue was examined in biomass and phospholipid fatty acids. After a 6 d chase period, c. 26.8% of the 13 C remaining in the system was translocated below ground. Enrichment was proportionally greatest in ectomycorrhizal biomass. The presence of mesh barriers (0.5 or 35 µm) between seedlings did not restrict 13 C transfer. Fungi were the primary recipients of 13 C-labelled photosynthate throughout the system, representing 60-70% of total 13 C-enriched phospholipids. Full-sibling pairs exhibited significantly greater 13 C transfer to recipient roots in two of four Douglas-fir families, representing three- and fourfold increases (+ c. 4 µg excess 13 C) compared with nonsibling pairs. The existence of a root/mycorrhizal exudation-hyphal uptake pathway was supported.

Douglas fir (pseudotsuga menziesii) plantlets responses to as, PB, and sb-contaminated soils from former mines.

Phytoremediation of metalloids by conifers is not widely studied although they may be relevant for several contaminated sites, especially those located in cold areas and sometimes under dry climates. Here, seeds of Douglas fir were sown in greenhouse on three soils collected in two French former mines: a gold mine (soils L1 and L2) and a lead and silver mine (soil P). These soils are highly contaminated by Pb, As, and Sb at different concentrations. Plants were harvested after ten weeks. Growth parameters, primary metabolite content, and shoot and root ionomes were determined. Douglas firs grown on the soils L1 and P had a lower biomass than controls and a higher oxidation status whereas those grown on the soil L2 exhibited a more developed root system and only slight modifications of carbon and nitrogen nutrition. Based on trace element (TE) concentrations in shoots and roots and their translocation factor (TF), Douglas fir could be a relevant candidate for As phytoextraction (0.8 g. kg(-1) dry weight in shoots and a TF of 1.1) and may be used to phytostabilize Pb and Sb (8.8 g and 127 mg. kg(-1) in roots for Pb and Sb, respectively, and TF lower than 0.1).

In vitro protein profiles in the early and late stages of Douglas-fir xylogenesis.

The process of wood formation is of great interest to control and manipulate wood quality for economically important gymnosperms. A Douglas-fir tissue culture system was developed that could be induced to differentiate into tracheary elements (fibers) making it possible to monitor xylogenesis in vitro by a proteomics approach. Two proteomes were analyzed and compared, one from an early and one from a late stage of the fiber differentiation process. After 18 weeks in a differentiation-inducing medium, 80% of the callus cells were elongated while 20% showed advanced spiral thickening indicating full wood fiber differentiation. Based on 2D electrophoresis, MS, and data analyses (data are available via ProteomeXchange with identifier PXD001484.), it was shown that in nondifferentiated callus (representing an early stage of development), proteins related to protein metabolism, cellular energy, and primary cell wall metabolism were abundant. By comparison, in cells actively differentiating wood fibers (representing a late stage of development), proteins involved in cell wall polysaccharide biosynthesis predominated together with housekeeping and stress-associated proteins.

Tree-ring stable isotopes record the impact of a foliar fungal pathogen on CO(2) assimilation and growth in Douglas-fir.

Swiss needle cast (SNC) is a fungal disease of Douglas-fir (Pseudotsuga menziesii) that has recently become prevalent in coastal areas of the Pacific Northwest. We used growth measurements and stable isotopes of carbon and oxygen in tree-rings of Douglas-fir and a non-susceptible reference species (western hemlock, Tsuga heterophylla) to evaluate their use as proxies for variation in past SNC infection, particularly in relation to potential explanatory climate factors. We sampled trees from an Oregon site where a fungicide trial took place from 1996 to 2000, which enabled the comparison of stable isotope values between trees with and without disease. Carbon stable isotope discrimination (Δ(13)C) of treated Douglas-fir tree-rings was greater than that of untreated Douglas-fir tree-rings during the fungicide treatment period. Both annual growth and tree-ring Δ(13)C increased with treatment such that treated Douglas-fir had values similar to co-occurring western hemlock during the treatment period. There was no difference in the tree-ring oxygen stable isotope ratio between treated and untreated Douglas-fir. Tree-ring Δ(13)C of diseased Douglas-fir was negatively correlated with relative humidity during the two previous summers, consistent with increased leaf colonization by SNC under high humidity conditions that leads to greater disease severity in following years.

Soil microbe active community composition and capability of responding to litter addition after 12 years of no inputs.

One explanation given for the high microbial diversity found in soils is that they contain a large inactive biomass that is able to persist in soils for long periods of time. This persistent microbial fraction may help to buffer the functionality of the soil community during times of low nutrients by providing a reservoir of specialized functions that can be reactivated when conditions improve. A study was designed to test the hypothesis: in soils lacking fresh root or detrital inputs, microbial community composition may persist relatively unchanged. Upon addition of new inputs, this community will be stimulated to grow and break down litter similarly to control soils. Soils from two of the Detrital Input and Removal Treatments (DIRT) at the H. J. Andrews Experimental Forest, the no-input and control treatment plots, were used in a microcosm experiment where Douglas-fir needles were added to soils. After 3 and 151 days of incubation, soil microbial DNA and RNA was extracted and characterized using quantitative PCR (qPCR) and 454 pyrosequencing. The abundance of 16S and 28S gene copies and RNA copies did not vary with soil type or amendment; however, treatment differences were observed in the abundance of archaeal ammonia-oxidizing amoA gene abundance. Analysis of ∼110,000 bacterial sequences showed a significant change in the active (RNA-based) community between day 3 and day 151, but microbial composition was similar between soil types. These results show that even after 12 years of plant litter exclusion, the legacy of community composition was well buffered against a dramatic disturbance.

Decomposition and nitrogen dynamics of (15)N-labeled leaf, root, and twig litter in temperate coniferous forests.

Litter nutrient dynamics contribute significantly to biogeochemical cycling in forest ecosystems. We examined how site environment and initial substrate quality influence decomposition and nitrogen (N) dynamics of multiple litter types. A 2.5-year decomposition study was installed in the Oregon Coast Range and West Cascades using (15)N-labeled litter from Acer macrophyllum, Picea sitchensis, and Pseudotsuga menziesii. Mass loss for leaf litter was similar between the two sites, while root and twig litter exhibited greater mass loss in the Coast Range. Mass loss was greatest from leaves and roots, and species differences in mass loss were more prominent in the Coast Range. All litter types and species mineralized N early in the decomposition process; only A. macrophyllum leaves exhibited a net N immobilization phase. There were no site differences with respect to litter N dynamics despite differences in site N availability, and litter N mineralization patterns were species-specific. For multiple litter × species combinations, the difference between gross and net N mineralization was significant, and gross mineralization was 7-20 % greater than net mineralization. The mineralization results suggest that initial litter chemistry may be an important driver of litter N dynamics. Our study demonstrates that greater amounts of N are cycling through these systems than may be quantified by only measuring net mineralization and challenges current leaf-based biogeochemical theory regarding patterns of N immobilization and mineralization.

Changes to the N cycle following bark beetle outbreaks in two contrasting conifer forest types.

Outbreaks of Dendroctonus beetles are causing extensive mortality in conifer forests throughout North America. However, nitrogen (N) cycling impacts among forest types are not well known. We quantified beetle-induced changes in forest structure, soil temperature, and N cycling in Douglas-fir (Pseudotsuga menziesii) forests of Greater Yellowstone (WY, USA), and compared them to published lodgepole pine (Pinus contorta var. latifolia) data. Five undisturbed stands were compared to five beetle-killed stands (4-5 years post-outbreak). We hypothesized greater N cycling responses in Douglas-fir due to higher overall N stocks. Undisturbed Douglas-fir stands had greater litter N pools, soil N, and net N mineralization than lodgepole pine. Several responses to disturbance were similar between forest types, including a pulse of N-enriched litter, doubling of soil N availability, 30-50 % increase in understory cover, and 20 % increase in foliar N concentration of unattacked trees. However, the response of some ecosystem properties notably varied by host forest type. Soil temperature was unaffected in Douglas-fir, but lowered in lodgepole pine. Fresh foliar %N was uncorrelated with net N mineralization in Douglas-fir, but positively correlated in lodgepole pine. Though soil ammonium and nitrate, net N mineralization, and net nitrification all doubled, they remained low in both forest types (<6 µg N g soil(-1) NH(4) (+)or NO(3) (-); <25 µg N g soil(-1) year(-1) net N mineralization; <8 µg N g soil(-1) year(-1) net nitrification). Results suggest that beetle disturbance affected litter and soil N cycling similarly in each forest type, despite substantial differences in pre-disturbance biogeochemistry. In contrast, soil temperature and soil N-foliar N linkages differed between host forest types. This result suggests that disturbance type may be a better predictor of litter and soil N responses than forest type due to similar disturbance mechanisms and disturbance legacies across both host-beetle systems.

Water stress, shoot growth and storage of non-structural carbohydrates along a tree height gradient in a tall conifer.

We analysed concentrations of starch, sucrose, glucose and fructose in upper branch wood, foliage and trunk sapwood of Douglas-fir trees in height classes ranging from ~2 to ~57 m. Mean concentrations of non-structural carbohydrates (NSC) for all tissues were highest in the tallest height class and lowest in the lowest height class, and height-related trends in NSC were most pronounced in branches. Throughout a 17-month sampling period, mean values of branch NSC from the 57 m trees ranged between 30 and 377% greater than the 2 m trees. Branch NSC was inversely correlated with midday shoot water potential (Ψ(l)), shoot osmotic potential at full turgor (Ψ) and shoot extension. Temporal fluctuation in branch NSC was inversely correlated with height, and positively correlated with midday Ψ(l) , Ψ and shoot extension. The positive correlation between height and storage of NSC, and the negative correlation between NSC storage and shoot extension provide evidence that size-related growth decline in trees is not strongly associated with constraints on photosynthesis. The negative correlation between height and fluctuation in NSC suggests that mobilization of photosynthate in taller trees is constrained by some factor such as reductions in turgor-driven cell expansion or constraints on phloem transport.

Climate-related trends in sapwood biophysical properties in two conifers: avoidance of hydraulic dysfunction through coordinated adjustments in xylem efficiency, safety and capacitance.

In the Pacific north-west, the Cascade Mountain Range blocks much of the precipitation and maritime influence of the Pacific Ocean, resulting in distinct climates east and west of the mountains. The current study aimed to investigate relationships between water storage and transport properties in populations of Douglas-fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa) adapted to both climates. Sapwood thickness, capacitance, vulnerability to embolism, and axial and radial conductivity were measured on samples collected from trunks of mature trees. The sapwood of ponderosa pine was three to four times thicker than Douglas-fir. Radial conductivity was higher in west-side populations of both species, but axial conductivity was higher in the east-side populations and in Douglas-fir. Eastern populations of both species had sapwood that was more vulnerable to embolism than west-side populations. Sapwood capacitance was similar between species, but was about twice as great in east-side populations (580 kg m⁻³ MPa⁻¹) as in west-side populations (274 kg m⁻³ MPa⁻¹). Capacitance was positively correlated with both mean embolism pressure and axial conductivity across species and populations, suggesting that coordinated adjustments in xylem efficiency, safety and water storage capacity may serve to avoid embolism along a gradient of increasing aridity.

Estimation of canopy average mesophyll conductance using δ(13) C of phloem contents.

Conductance to CO(2) inside leaves, known as mesophyll conductance (g(m)), imposes large limitations on photosynthesis. Because g(m) is difficult to quantify, it is often neglected in calculations of (13)C photosynthetic discrimination. The 'soluble sugar method' estimates g(m) via differences between observed photosynthetic discrimination, calculated from the δ(13)C of soluble sugars, and discrimination when g(m) is infinite. We expand upon this approach and calculate a photosynthesis-weighted average for canopy mesophyll conductance ((c) g(m)) using δ(13)C of stem phloem contents. We measured gas exchange at three canopy positions and collected stem phloem contents in mature trees of three conifer species (Pseudotsuga menziesii, Thuja plicata and Larix occidentalis). We generated species-specific and seasonally variable estimates of (c)g(m) . We found that (c)g(m) was significantly different among species (0.41, 0.22 and 0.09 mol m(-2) s(-1) for Larix, Pseudotsuga and Thuja, respectively), but was similar throughout the season. Ignoring respiratory and photorespiratory fractionations ((c)Δ(ef)) resulted in ≈30% underestimation of (c)g(m) in Larix and Pseudotsuga, but was innocuous in Thuja. Substantial errors (~1-4‰) in photosynthetic discrimination calculations were introduced by neglecting (c)g(m) and (c)Δ(ef) . Our method is easy to apply and cost-effective, captures species variation and would have captured seasonal variation had it existed. The method provides an average canopy value, which makes it suitable for parameterization of canopy-scale models of photosynthesis, even in tall trees.

pH affects ammonium, nitrate and proton fluxes in the apical region of conifer and soybean roots.

The effect of pH on nitrate and ammonium uptake in the high-affinity transport system and low-affinity transport system ranges was compared in two conifers and one crop species. Many conifers grow on acidic soils, thus their preference for ammonium vs nitrate uptake can differ from that of crop plants, and the effect of pH on nitrogen (N) uptake may differ. Proton, ammonium and nitrate net fluxes were measured at seedling root tips and 5, 10, 20 and 30 mm from the tips using a non-invasive microelectrode ion flux measurement system in solutions of 50 or 1500 microM NH(4)NO(3) at pH 4 and 7. In Glycine max and Pinus contorta, efflux of protons was observed at pH 7 while pH 4 resulted in net proton uptake in some root regions. Pseudotsuga menziesii roots consistently showed proton efflux behind the root tip, and thus appear better adapted to maintain proton efflux in acid soils. P. menziesii's ability to maintain ammonium uptake at low pH may relate to its ability to maintain proton efflux. In all three species, net nitrate uptake was greatest at neutral pH. Net ammonium uptake in G. max and net nitrate uptake in P. menziesii were greatly reduced at pH 4, particularly at high N concentration, thus N concentration should be considered when determining optimum pH for N uptake. In P. menziesii and G. max, net N uptake was greater in 1500 than 50 microM NH(4)NO(3) solution, but flux profiles of all ions varied among species.

Distinct developmental mechanisms reflect the independent origins of leaves in vascular plants.

Vascular plants diverged more than 400 million years ago into two lineages, the lycophytes and the euphyllophytes . Leaf-like organs evolved independently in these two groups . Microphylls in lycophytes are hypothesized to have originated as lateral outgrowths of tissue that later became vascularized (the enation theory) or through the sterilization of sporangia (the sterilization hypothesis) . Megaphylls in euphyllophytes are thought to represent modified lateral branches . The fossil record also indicates that the seed plant megaphyll evolved uniquely in the ancestor of seed plants, independent of megaphylls in ferns, because seed plants evolved from leafless progymnosperm ancestors . Surprisingly, a recent study of KNOX and ARP gene expression in a lycophyte was reported to indicate recruitment of a similar mechanism for determinacy in both types of leaves . We examined the expression of Class III HD-Zip genes in the lycophyte Selaginella kraussiana and in two gymnosperms, Ginkgo and Pseudotsuga. Our data indicate that mechanisms promoting leaf initiation, vascularization, and polarity are quite different in lycophytes and seed plants, consistent with the hypotheses that megaphylls originated as lateral branches whereas microphylls originated as tissue outgrowths.

Forest fuel reduction alters fire severity and long-term carbon storage in three Pacific Northwest ecosystems.

Two forest management objectives being debated in the context of federally managed landscapes in the U.S. Pacific Northwest involve a perceived trade-off between fire restoration and carbon sequestration. The former strategy would reduce fuel (and therefore C) that has accumulated through a century of fire suppression and exclusion which has led to extreme fire risk in some areas. The latter strategy would manage forests for enhanced C sequestration as a method of reducing atmospheric CO2 and associated threats from global climate change. We explored the trade-off between these two strategies by employing a forest ecosystem simulation model, STANDCARB, to examine the effects of fuel reduction on fire severity and the resulting long-term C dynamics among three Pacific Northwest ecosystems: the east Cascades ponderosa pine forests, the west Cascades western hemlock-Douglas-fir forests, and the Coast Range western hemlock-Sitka spruce forests. Our simulations indicate that fuel reduction treatments in these ecosystems consistently reduced fire severity. However, reducing the fraction by which C is lost in a wildfire requires the removal of a much greater amount of C, since most of the C stored in forest biomass (stem wood, branches, coarse woody debris) remains unconsumed even by high-severity wildfires. For this reason, all of the fuel reduction treatments simulated for the west Cascades and Coast Range ecosystems as well as most of the treatments simulated for the east Cascades resulted in a reduced mean stand C storage. One suggested method of compensating for such losses in C storage is to utilize C harvested in fuel reduction treatments as biofuels. Our analysis indicates that this will not be an effective strategy in the west Cascades and Coast Range over the next 100 years. We suggest that forest management plans aimed solely at ameliorating increases in atmospheric CO2 should forgo fuel reduction treatments in these ecosystems, with the possible exception of some east Cascades ponderosa pine stands with uncharacteristic levels of understory fuel accumulation. Balancing a demand for maximal landscape C storage with the demand for reduced wildfire severity will likely require treatments to be applied strategically throughout the landscape rather than indiscriminately treating all stands.

Dynamic changes in concentrations of auxin, cytokinin, ABA and selected metabolites in multiple genotypes of Douglas-fir (Pseudotsuga menziesii) during a growing season.

Changes in concentrations of several endogenous phytohormones and metabolites were analyzed in the long shoots of nine genotypes of coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. menziesii) at five developmental stages: (1) closed buds, (2) flushing buds, (3) rapidly elongating shoots, (4) growing shoots and (5) near full-length shoots during one growing season. When averaged across genotypes, indole-3-acetic acid (IAA) concentration was high at stages 1 and 3. The only pattern that correlated with cone productivity was the one that was unique to IAA, in which high concentrations at stages 3 and 4 were found in all genotypes with high female cone productivity. Concentrations of isopentenyl adenosine (iPA) decreased and zeatin riboside (ZR) concentrations increased as the buds initiated and differentiated; ZR was 30 and 28 ng g(-1) dry weight (DW) at stages 1 and 4, respectively, before increasing to 166 ng g(-1) DW at stage 5. Isopentenyl adenosine peaked at 92 ng g(-1) DW at stage 2 and declined to low concentrations at stages 4 and 5. Zeatin-O-glucoside was 30 ng g(-1) DW at stage 1, declined at stages 2 and 3 and increased at stages 4 and 5. High abscisic acid (ABA) concentrations were positively correlated with rapid shoot elongation (stages 1 and 2), but as growth slowed and terminated, ABA concentrations decreased. Abscisic acid was 7 microg g(-1) DW at stage 1, increased to 13 microg g(-1) DW at stage 2 and then declined. The glucosyl ester (GE) of ABA decreased rapidly in early summer, and increased inversely with an increase in ABA. Between stages 1 and 2, ABA-GE decreased from 10 to 0.2 microg g(-1) DW and then increased. Of the ABA catabolites studied, 7'-hydroxy-ABA was about 2 microg g(-1) DW at stage 1, declined at stages 2 and 3 and increased at stages 4 and 5; phaseic acid concentrations were low at all stages, whereas dihydrophaseic acid was detected only at stages 4 and 5.

Temporal variation of nonstructural carbohydrates in montane conifers: similarities and differences among developmental stages, species and environmental conditions.

Nonstructural carbohydrates (NSCs) are commonly used to assess the balance of carbon sources and sinks in plants. A notable application of this approach has been tests of hypotheses on carbon limitations of trees at their upper altitudinal limits, near the alpine. How NSCs vary in time is not well known in conifers during their critical seedling stage, despite the importance of knowing the temporal variations of NSCs to use snapshot measurements of NSCs to assess carbon balance. We measured NSCs in needles, separately as soluble sugars and starch; (1) over diurnal periods in seedlings of Pseudotsuga menziesii (Mirb.) Franco (a timberline species that does not occur up to treeline), (2) throughout the growth season in the seedlings of P. menziesii and Abies lasiocarpa (Hook.) Nutt. (a species that does occur up to treeline) growing along an elevation gradient in the timberline ecotone and furthermore (3) compared seedlings and co-occurring adults to assess variation with developmental stage. We also compared NSCs in seedlings grown under field or laboratory conditions to separate environmental from intrinsic factors affecting NSCs during early emergence. Diurnal variations in NSCs were minimal, especially when compared to seasonal variation, and were detectable mainly in relatively small midday maxima of soluble sugar concentrations. Seasonal patterns of NSCs were generally (and surprisingly) similar among field and laboratory seedlings and adults. Seasonal patterns of NSCs were dominated by progressive increases in soluble sugars until winter, and by early-season peaks in starch. Nonetheless, notable differences were detectable among ages, species and environmental conditions in (1) the timing and extent of the early-season maxima of starch and (2) the extent of the late-season maxima of soluble sugars. These differences in NSCs likely correspond with ecophysiologically relevant differences in carbon balance that could affect growth and survival of trees growing in the timberline ecotone.

Ultrastructural studies of Phellinus sulphurascens infection of Douglas-fir roots and immunolocalization of host pathogenesis-related proteins.

Interactions between roots of Douglas-fir (DF; Pseudotsuga menziesii) seedlings and the laminated root rot fungus Phellinus sulphurascens were investigated using scanning and transmission electron microscopy and immunogold labelling techniques. Scanning electron micrographs revealed that P. sulphurascens hyphae colonize root surfaces and initiate the penetration of root epidermal tissues by developing appressoria within 2 d postinoculation (dpi). During early colonization, intra- and intercellular fungal hyphae were detected. They efficiently disintegrate cellular components of the host including cell walls and membranes. P. sulphurascens hyphae penetrate host cell walls by forming narrow hyphal tips and a variety of haustoria-like structures which may play important roles in pathogenic interactions. Ovomucoid-WGA (wheat germ agglutinin) conjugated gold particles (10 nm) confirmed the occurrence and location of P. sulphurascens hyphae, while four specific host pathogenesis-related (PR) protein antibodies conjugated with protein A-gold complex (20 nm) showed the localization and abundance of these PR proteins in infected root tissues. A thaumatin-like protein and an endochitinase-like protein were both strongly evident and localized in host cell membranes. A DF-PR10 protein was localized in the cell walls and cytoplasm of host cells while an antimicrobial peptide occurred in host cell walls. A close association of some PR proteins with P. sulphurascens hyphae suggests their potential antifungal activities in DF roots.

Incorporating diffuse photosynthetically active radiation in a single-leaf model of canopy photosynthesis for a 56-year-old Douglas-fir forest.

A simple top-down model of canopy photosynthesis (P) was developed and tested in this study. The model (referred to as the Q(e)-MM model) is P = alphaQ (e) P (max)/(alphaQ ( e ) + P (max)), alpha and P (max) are quantum-use efficiency and potential P, respectively. Q (e) is given by Q (d) (0) + kQ (b) (0), where Q (d) (0) and Q (b) (0) are the diffuse and direct photosynthetically active radiation (PAR) incident on the canopy, respectively. Q (e) can be considered to be the effective incident PAR contributing to P and k is a measure of the contribution of Q (b) (0) to Q (e). When k = 1, the Q(e)-MM model becomes the regular Michaelis-Menten type model of P (referred to as the MM model). A major objective of this study was to determine how well the Q(e)-MM model could estimate P of a 56-year-old coastal Douglas-fir stand. To this end, we parameterized the Q(e)-MM model using five and half years of eddy-covariance measurements of CO(2) flux above the Douglas-fir stand. The Q(e)-MM model, with the incorporation of a function of air temperature, accounted for 74% of the variance in over 34,000 half-hourly P measurements. P estimated using the Q(e)-MM model had no systematic errors with respect to Q (d) (0). Although the Q(e)-MM model has only one more parameter than the MM model, it accounted for 30% more variance in P than the latter when total incident PAR exceeded 900 micromol m(-2) s(-1). On average, k was found to be 0.22. We show that this small value of k reflects the significant effect of the scattering of the solar beam and the fraction of light-limited sunlit leaves. We also show that the success of the Q(e)-MM model was due to the fact that a large fraction of the sunlit leaves were light-limited as a result of their orientation to the solar beam.