Tissue and cellular localization of condensed tannins in poplar roots and potential association with nitrogen uptake.

Condensed tannins are common in vegetative tissues of woody plants, including in roots. In hybrid poplar (Populus tremula x alba; also known as P. x canescens) CT assays indicated they were most concentrated in younger white roots and at the root tip. Furthermore, CT-specific staining of embedded tissue sections demonstrated accumulation in root cap cells and adjacent epidermal cells, as well as a more sporadic presence in cortex cells. In older, brown roots as well as roots with secondary growth (cork zone), CT concentration was significantly lower. The insoluble fraction of CTs was greatest in the cork zone. To determine if CT accumulation correlates with nutrient uptake in poplar roots, a microelectrode ion flux measurement (MIFE™) system was used to measure flux along the root axis. Greatest NH4 + uptake was measured near the root tip, but NO3- and Ca2+ did not vary along the root length. In agreement with earlier work, providing poplars with ample nitrogen led to higher accumulation of CTs across root zones. To test the functional importance of CTs in roots directly, CT-modified transgenic plants could be important tools.

Histological, chemical and gene expression differences between western redcedar seedlings resistant and susceptible to cedar leaf blight.

Introduction: Western redcedar (Thuja plicata) is an important species in the Cupressaceae both at economic and cultural levels in the Pacific Northwest of North America. In adult trees, the species produces one of the most weathering-resistant heartwoods among conifers, making it one of the preferred species for outdoor applications. However, young T. plicata plants are susceptible to infection with cedar leaf blight (Didymascella thujina), an important foliar pathogen that can be devastating in nurseries and small-spaced plantations. Despite that, variability in the resistance against D. thujina in T. plicata has been documented, and such variability can be used to breed T. plicata for resistance against the pathogen. Objective: This investigation aimed to discern the phenotypic and gene expression differences between resistant and susceptible T. plicata seedlings to shed light on the potential constitutive resistance mechanisms against cedar leaf blight in western redcedar. Methods: The study consisted of two parts. First, the histological differences between four resistant and four susceptible families that were never infected with the pathogen were investigated. And second, the differences between one resistant and one susceptible family that were infected and not infected with the pathogen were analyzed at the chemical (C, N, mineral nutrients, lignin, fiber, starch, and terpenes) and gene expression (RNA-Seq) levels. Results: The histological part showed that T. plicata seedlings resistant to D. thujina had constitutively thicker cuticles and lower stomatal densities than susceptible plants. The chemical analyses revealed that, regardless of their infection status, resistant plants had higher foliar concentrations of sabinene and α-thujene, and higher levels of expression of transcripts that code for leucine-rich repeat receptor-like protein kinases and for bark storage proteins. Conclusion: The data collected in this study shows that constitutive differences at the phenotypic (histological and chemical) and gene expression level exist between T. plicata seedlings susceptible and resistant to D. thujina. Such differences have potential use for marker-assisted selection and breeding for resistance against cedar leaf blight in western redcedar in the future.

Sporocarp nutrition of ectomycorrhizal fungi indicates an important role for endemic species in a high productivity temperate rainforest.

Endemic species of ectomycorrhizal fungi (EMF) are found throughout many biomes, but it is unclear whether their localized distribution is dictated by habitat filtering or geographical barriers to dispersal. We examined community composition (via long-read metabarcoding) and differences in sporocarp nutrition between endemic and cosmopolitan EMF species across perhumid temperate rainforests of British Columbia, characterized by soils with high nitrogen (N) supply alongside low phosphorus (P) and cation availability. Endemic EMF species, representing almost half of the community, had significantly greater sporocarp N (24% higher), potassium (+16%), and magnesium (+17%) concentrations than cosmopolitan species. Sporocarp P concentrations were comparatively low and did not differ by fungal range. However, sporocarp N% and P% were well correlated, supporting evidence for linkages in N and P acquisition. Endemics were more likely to occur on Tsuga heterophylla (a disjunct host genus) than Picea sitchensis (a circumpolar genus). The Inocybaceae and Thelephoraceae families had high proportions of endemic taxa, while species in Cortinariaceae were largely cosmopolitan, indicating some niche conservatism among genera. We conclude that superior adaptive traits in relation to perhumid soils were skewed toward the endemic community, underscoring the potentially important contribution of these localized fungi to rainforest nutrition and productivity.

Fatal ingestion of Taxus baccata: English yew.

Poisoning from consumption of foraged alternative medicine products is an uncommon yet recognized occurrence. Here, presented is the case of a 40-year-old woman who was witnessed to collapse with labored breathing and subsequently died despite emergency medical personnel attendance and resuscitation efforts. Autopsy revealed the presence of plant matter that was visually identified as leaves from Taxus baccata - the English Yew. Isolation of alkaloids from the plant material and subsequent identification of the same alkaloids in the decedent's blood by liquid chromatography-tandem mass spectrometry indicated a toxicological cause of death. This case illustrates a collaborative team approach among subject matter experts to unexpectedly discover and then confirm the sudden death of this woman from T. baccata toxicity.

Condensed tannins as antioxidants that protect poplar against oxidative stress from drought and UV-B.

Condensed tannins (CTs, proanthocyanidins) are widespread polymeric flavan-3-ols known for their ability to bind proteins. In poplar (Populus spp.), leaf condensed tannins are induced by both biotic and abiotic stresses, suggesting diverse biological functions. Here we demonstrate the ability of CTs to function as physiological antioxidants, preventing oxidative and cellular damage in response to drought and UV-B irradiation. Chlorophyll fluorescence was used to monitor photosystem II performance, and both hydrogen peroxide and malondialdehyde content was assayed as a measure of oxidative damage. Transgenic MYB-overexpressing poplar (Populus tremula × P. tremuloides) with high CT content showed reduced photosystem damage and lower hydrogen peroxide and malondialdehyde content after drought and UV-B stress. This antioxidant effect of CT was observed using two different poplar MYB CT regulators, in multiple independent lines and different genetic backgrounds. Additionally, low-CT MYB134-RNAi transgenic poplars showed enhanced susceptibility to drought-induced oxidative stress. UV-B radiation had different impacts than drought on chlorophyll fluorescence, but all high-CT poplar lines displayed reduced sensitivity to both stresses. Our data indicate that CTs are significant defences against oxidative stress. The broad distribution of CTs in forest systems that are exposed to diverse abiotic stresses suggests that these compounds have wider functional roles than previously realized.

Factors Affecting Foliar Oregonin and Condensed Tannin in Red Alder (Alnus rubra Bong.): Phytochemicals Implicated In Defense Against Western Tent Caterpillar (Malacosoma californicum Packard).

Past work shows a significant negative correlation between foliar oregonin concentration and western tent caterpillar (Malacosoma californicum Packard) feeding on red alder (Alnus rubra Bong.). Above an oregonin threshold of 20% leaf dry weight, little feeding by caterpillars is observed. Concentrations of defensive chemicals are influenced by plant genotype, environmental conditions, insect feeding, and the interactions of these factors. Our objective was to measure the effects of nitrogen (N) availability and wounding on foliar oregonin and condensed tannin concentrations in red alder genotypes. One-year-old seedlings from 100 half-sib red alder families were treated with two levels of ammonium nitrate (NH4NO3) for two growing seasons in a common garden. In the second year, leaves from 50 families from the fertilization experiment were used in a bioassay feeding experiment to determine the effects of N fertilization and genotype on WTC damage, and to identify a subset of 20 families with a range of damage to analyze for phytochemical composition. In separate experiments, wound-induction treatments were conducted outdoors and, in a greenhouse using the N treated trees in their third and fourth year, respectively. Foliar condensed tannin, oregonin and N concentrations were measured and ranked among the plant genotypes, and between the two N treatments and two wounding treatments. Results showed that oregonin and condensed tannin concentrations varied among the alder genotypes. Leaf N concentration was negatively correlated with concentration of oregonin. Neither of the measured phenolic compounds responded to wounding. The results suggest that red alder foliar oregonin and condensed tannin are likely constitutive defenses that are largely determined by genotype, and that the negative correlation of defense compounds with plant internal N status holds in this N-fixing tree.

Saprotrophic and ectomycorrhizal fungal sporocarp stoichiometry (C†:†N†:†P) across temperate rainforests as evidence of shared nutrient constraints among symbionts.

Quantifying nutritional dynamics of free-living saprotrophs and symbiotic ectomycorrhizal fungi in the field is challenging, but the stoichiometry of fruiting bodies (sporocarps) may be an effective methodology for this purpose. Carbon (C), nitrogen (N) and phosphorus (P) concentrations of soils, foliage and 146 sporocarp collections were analyzed from 14 Pseudotsuga menziesii var. menziesii stands across a podzolization gradient on Vancouver Island (Canada). N and P concentrations were considerably higher in saprotrophic fungi. Fungal N% increased with soil N content at a greater rate for saprotrophs than ectomycorrhizal fungi, while fungal P% of saprotrophs was more constrained. Fungal N : P was more responsive to soil N : P for ectomycorrhizal fungi (homeostatic regulation coefficient 'H' = 2.9) than saprotrophs (H = 5.9), while N : P of ectomycorrhizal fungi and host tree foliage scaled almost identically. Results underscore the role of ectomycorrhizal fungi as nutrient conduits, supporting host trees, whereas saprotrophs maintain a greater degree of nutritional homeostasis. Site nutrient constraints were shared in equal measure between ectomycorrhizal fungi and host trees, particularly for P, suggesting neither partner benefits from enhanced nutrition at the expense of the other. Sporocarp stoichiometry provides new insights into mycorrhizal relationships and illustrates pervasive P deficiencies across temperate rainforests of the Pacific Northwest.

Evolutionary classification of ammonium, nitrate, and peptide transporters in land plants.

BACKGROUND: Nitrogen uptake, reallocation within the plant, and between subcellular compartments involves ammonium, nitrate and peptide transporters. Ammonium transporters are separated into two distinct families (AMT1 and AMT2), each comprised of five members on average in angiosperms. Nitrate transporters also form two discrete families (NRT1 and NRT2), with angiosperms having four NRT2s, on average. NRT1s share an evolutionary history with peptide transporters (PTRs). The NRT1/PTR family in land plants usually has more than 50 members and contains also members with distinct activities, such as glucosinolate and abscisic acid transport. RESULTS: Phylogenetic reconstructions of each family across 20 land plant species with available genome sequences were supplemented with subcellular localization and transmembrane topology predictions. This revealed that both AMT families diverged prior to the separation of bryophytes and vascular plants forming two distinct clans, designated as supergroups, each. Ten supergroups were identified for the NRT1/PTR family. It is apparent that nitrate and peptide transport within the NRT1/PTR family is polyphyletic, that is, nitrate and/or peptide transport likely evolved multiple times within land plants. The NRT2 family separated into two distinct clans early in vascular plant evolution. Subsequent duplications occurring prior to the eudicot/monocot separation led to the existence of two AMT1, six AMT2, 31 NRT1/PTR, and two NRT2 clans, designated as groups. CONCLUSION: Phylogenetic separation of groups suggests functional divergence within the angiosperms for each family. Distinct groups within the NRT1/PTR family appear to separate peptide and nitrate transport activities as well as other activities contained within the family, for example nitrite transport. Conversely, distinct activities, such as abscisic acid and glucosinolate transport, appear to have recently evolved from nitrate transporters.

Shoot excision has no effect on net flux of protons, ammonium or nitrate in seedling roots of a conifer and three crop species.

A high-flux region, 5mm from the root tips of seedlings of coastal Douglas-fir (Pseudotsuga menziesii), soybean (Glycine max), zucchini (Cucurbita pepo) and pea (Pisum sativum), was monitored using a microelectrode ion flux measurement system, for changes in the net fluxes of H(+), NH(4)(+) and NO(3)(-) in response to shoot removal. In all species, careful excision of the seedling shoot had no significant effect on the net fluxes of H(+), NH(4)(+) or NO(3)(-) measured 5mm from the root tip. Experiments were carried out for up to 80min after shoot removal, and no temporal interactions were noted.

Relating nutritional and physiological characteristics to growth of Pinus radiata clones planted on a range of sites in New Zealand.

Six clones of radiata pine with known differences in growth rate were examined for clonal nutritional characteristics and for physiological determinants of clonal growth rate. We compared growth, foliar characteristics and nutrient, ¹³C and ¹5N concentration data for the six clones in 4- to 6-year-old field trials planted over a range of nutritionally contrasting sites. These data were also compared with growth, nutrient uptake and remobilization, foliar characteristic and gas exchange data from intensive physiological glasshouse experiments using 1- and 2-year-old plants of the same clones. Significant genotype x environment interactions in our field experiments conducted over strong nutritional gradients allowed us to identify radiata pine clones with consistent, superior growth and nutritional characteristics and clones that may be suited to particular site conditions. Our results suggest that the opportunity exists to exploit clone x site variation for site-specific clonal deployment and planting of fast-growing clones could be accompanied by planting of clones able to take relative advantage of site nutritional characteristics. Faster tree growth was not strongly related to any physiological characteristic, and the factors influencing growth rate differed among clones. The fastest-growing clone had consistent, high uptake of all nutrients, high fascicle weights and high water-use efficiency.

Induction of acid phosphatase transcripts, protein and enzymatic activity by simulated herbivory of hybrid poplar.

Herbivory and wounding upregulate a large suite of defense genes in hybrid poplar leaves. A strongly wound- and herbivore-induced gene with high similarity to Arabidopsis vegetative storage proteins (VSPs) and acid phosphatase (AP) was identified among genes strongly expressed during the poplar herbivore defense response. Phylogenetic analysis showed that the putative poplar acid phosphatase (PtdAP1) gene is part of an eight-member AP gene family in poplar, and is most closely related to a functionally characterized soybean nodule AP. Unlike the other poplar APs, PtdAP1 is expressed in variety of tissues, as observed in an analysis of EST data. Following wounding, the gene shows an expression profile similar to other known poplar defense genes such as protease inhibitors, chitinase, and polyphenol oxidase. Significantly, we show for the first time that subsequent to the wound-induction of PtdAP1 transcripts, AP protein and activity increase in extracts of leaves and other tissues. Although its mechanism of action is as yet unknown, these results suggest in hybrid poplar PtdAP1 is likely a component of the defense response against leaf-eating herbivores.

Ion fluxes across the pitcher walls of three Bornean Nepenthes pitcher plant species: flux rates and gland distribution patterns reflect nitrogen sequestration strategies.

Nepenthes pitcher plant species differ in their prey capture strategies, prey capture rates, and pitcher longevity. In this study, it is investigated whether or not interspecific differences in nutrient sequestration strategy are reflected in the physiology and microstructure of the pitchers themselves. Using a non-invasive technique (MIFE), ion fluxes in pitchers of Nepenthes ampullaria Jack, Nepenthes bicalcarata Hook.f., and Nepenthes rafflesiana Jack were measured. Scanning electron microscopy was also used to characterize the distribution of glandular and other structures on the inner pitcher walls. The results demonstrate that nutrient sequestration strategy is indeed mirrored in pitcher physiology and microstructure. Species producing long-lived pitchers with low prey capture rates (N. ampullaria, N. bicalcarata) showed lower rates of NH(4)(+) uptake than N. rafflesiana, a species producing short-lived pitchers with high capture rates. Crucially, species dependent upon aquatic commensals (N. ampullaria, N. bicalcarata) actively manipulated H(+) fluxes to maintain less acid pitcher fluid than found in 'typical' species; in addition, these species lacked the lunate cells and epicuticular waxes characteristic of 'typical' insectivorous congeners. An unexpected finding was that ion fluxes occurred in the wax-covered, non-glandular zones in N. rafflesiana. The only candidates for active transport of aqueous ions in these zones appear to be the epidermal cells lying beneath the lunate cells, as these are the only sites not visibly coated with epicuticular waxes.

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.

Ammonium and nitrate uptake, nitrogen productivity and biomass allocation in interior spruce families with contrasting growth rates and mineral nutrient preconditioning.

Four full-sib families of interior spruce (Picea glauca (Moench) Voss) x Picea engelmanii Parry ex Engelm.) with contrasting growth rates (two fast-growing and two slow-growing families) were grown aeroponically with either a 2% relative nitrogen addition rate or free access to nitrogen. Fast-growing families showed greater plasticity in allocating biomass to shoots at high nitrogen supply and to roots at low nitrogen supply than slow-growing families. Compared with the slow-growing families, short-term net ammonium uptake rate measured with an ion selective electrode was significantly greater in fast-growing families at high ammonium supply, but not at low supply. Net nitrate uptake showed the same trend, but differences among families were not significant. Results indicate that differences in seedling growth rate are partly a result of physiological differences in net nitrogen uptake efficiency and nitrogen productivity.