A prescription for drug-free rivers: uptake of pharmaceuticals by a widespread streamside willow.

Following human excretion and limited removal with wastewater treatment, pharmaceuticals are accumulating in rivers worldwide. These chemicals can challenge the health of fish and aquatic organisms and since rivers provide drinking water sources, there is concern for cumulative exposure to humans. In this study, we discovered that sandbar willow (Salix exigua), a predominant riparian shrub along streams throughout North America, has the capacity to quickly remove pharmaceuticals from aqueous solutions. Our study tracked [3H]- or [14C]-labeled substances including 17α-ethynylestradiol (EE2), a synthetic estrogen in oral contraceptives; the antihypertensive, diltiazem (DTZ); and the anti-anxiety drug, diazepam (DZP); and for comparison, atrazine (ATZ), a root-absorbed herbicide. In growth chambers, willow saplings removed 40-80% of the substances from solutions in 24 h. Following uptake, the EE2 and DTZ were retained within the roots, while DZP and ATZ were partly passed on to the shoots. The absorbed EE2 was unextractable and apparently bound to the root tissue, while DTZ, DZP, and ATZ remained largely soluble (extractable). The uptake and translocation of the pharmaceuticals, reflected in the transpiration stream and root concentration factors, were reasonably predicted from their physicochemical properties, including octanol-water partitioning coefficients. These findings suggest the removal of pharmaceuticals as an unrecognized ecosystem service provided by riparian vegetation and especially the inundation tolerant sandbar willow. This encourages the conservation of riparian willows that line riverbanks, to remove pharmaceuticals and other contaminants. This phytoremediation also encourages the preservation of complex, braided channels and islands, which increase the extent of stream shorelines and riparian willows.

Green revolution trees: semidwarfism transgenes modify gibberellins, promote root growth, enhance morphological diversity, and reduce competitiveness in hybrid poplar.

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula × Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in most transgenic events; GA(20) and GA(8), in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations.

Hydrologic linkages between a climate oscillation, river flows, growth, and wood Δ13C of male and female cottonwood trees.

To investigate climatic influence on floodplain trees, we analysed interannual correspondences between the Pacific Decadal Oscillation (PDO), river and groundwater hydrology, and growth and wood (13)C discrimination (Δ(13)C) of narrowleaf cottonwoods (Populus angustifolia) in a semi-arid prairie region. From the Rocky Mountain headwaters, river discharge (Q) was coordinated with the PDO (1910-2008: r(2) = 0.46); this pattern extended to the prairie and was amplified by water withdrawal for irrigation. Floodplain groundwater depth was correlated with river stage (r(2) = 0.96), and the cottonwood trunk basal area growth was coordinated with current- and prior-year Q (1992-2008: r(2) = 0.51), increasing in the mid-1990s, and decreasing in 2000 and 2001. Annual Δ(13)C decreased during low-flow years, especially in trees that were higher or further from the river, suggesting drought stress and stomatal closure, and male trees were more responsive than females (-0.86 versus -0.43‰). With subsequently increased flows, Δ(13)C increased and growth recovered. This demonstrated the linkages between hydroclimatic variation and cottonwood ecophysiology, and we conclude that cottonwoods will be vulnerable to drought from declining river flows due to water withdrawal and climate change. Trees further from the river could be especially affected, leading to narrowing of floodplain forests along some rivers.

Lead Diversification 2: application to P38, gMTP and lead compounds.

Lead Diversification is a new technology platform developed at Pfizer for the functionalization of drug molecules using C-H activation. We describe its application to some drug programs such as P38 and gMTP and the development of some new plate based screens including a fluorination screen.

How trees thrive in a dry climate: diurnal and seasonal hydrology and water relations in a riparian cottonwood grove.

In semi-arid ecoregions, trees are restricted to river valley floodplains where river water supplements the limited precipitation. To characterize the associated diurnal and seasonal dynamics in hydrology and water relations, we studied narrowleaf cottonwoods (Populus angustifolia) along a prairie river in Canada. From June through August, the shallow soil moisture was depleted but moisture remained higher above the alluvial groundwater table, which dropped to 2.3 m along with river recession. Throughout the summer, with the daily rise in temperature and insolation, foliar stomatal conductance (gs) and transpiration (E) increased to midday and then fell, thus maintaining the midday leaf water potential (Ψmd) above ~-1.7 MPa. This Ψmd approximated the water potential associated with 12% loss of xylem conductivity due to cavitation for branches (P12); the Ψmd and P12 varied independently across eight trees, providing differences in relative hydraulic risk. Sap flux density (Fd) was measured with thermal dissipation probes, and revealed diurnal patterns similar to foliar E. In contrast to our expectation, the daily Fd maxima were consistent through the summer despite the seasonal recession in water supply. Canopy conductances (GS), derived from Fd, sapwood area and canopy area, declined with vapor pressure deficit (D) and fell slightly in late summer along with stomatal sensitivity, which reflects the magnitude of decrease in GS with increasing D. For spatial up-scaling, satellite-derived near-infrared reflectance of vegetation revealed the woodland phenology, with leaf expansion from May through June and gradual decline in photosynthetic productivity through the summer. Thus, the phreatophytic cottonwoods: (i) sustained substantial water use and productivity through the warm and dry summer, by (ii) accessing shallow soil moisture and then deeper alluvial groundwater, and (iii) providing diurnal stomatal regulation, to (iv) avoid xylem cavitation and (v) maintain fairly constant hydraulic conductance.

Streamside trees: responses of male, female and hybrid cottonwoods to flooding.

Cottonwoods, riparian poplars, are dioecious and prior studies have indicated that female poplars and willows can be more abundant than males in low-elevation zones, which are occasionally flooded. We investigated the response to flooding of clonal saplings of 12 male and 9 female narrowleaf cottonwoods (Populus angustifolia) grown for 15 weeks in a greenhouse, along with three females of a co-occurring native hybrid (Populus × jackii = Populus deltoides × Populus balsamifera). Three water-level treatments were provided, with substrate inundation as the flood treatment. In the non-flooded condition, the hybrids produced about four-fold more dry weight (DW) than the narrowleaf cottonwoods (P < 0.01). In both cottonwood taxa, flooding reduced stem height and DW, root and leaf area and weight, leaf chlorophyll and stomatal conductance (all P < 0.01). Inundation increased the foliar carbon-to-nitrogen ratio (+11%; P < 0.05) but did not significantly alter leaf water potential (mean -1.5 MPa), or foliar δ(13)C, which was lower in P. angustifolia (-32.8‰) than P. × jackii (-31.5‰; P < 0.05). Water level influenced the root distribution as roots were sparse in the saturated substrate and abundant in the capillary fringe above. The male and female P. angustifolia genotypes grew similarly with the favorable water levels, but the males tended to be more inhibited by flooding. Sapling DW of males was reduced by 56% compared with a 44% reduction for females (P = 0.1), and there were similar lower reductions for leaf, stem and root DW in females. These results demonstrate the inundation response of floodplain trees and suggest relative flood tolerance as: P. angustifolia female > P. angustifolia male > P. × jackii female. This indicates that narrowleaf cottonwoods are relatively flood tolerant and suggests that females are more flood tolerant than males. We propose the concept of 'strategic positioning', whereby the seed-producing females could be better adapted to naturally flooded, low-elevation streamside zones where seedling recruitment generally occurs.

2,4-Diaminopyridine delta-opioid receptor agonists and their associated hERG pharmacology.

A number of libraries were produced to explore the potential of 2,4-diaminopyridine lead 1. The resulting diaminopyridines proved to be potent and selective delta-opioid receptor agonists. Several rounds of lead optimisation using library chemistry identified compound 17 which went on to show efficacy in an electromyography model of neuropathic pain. The structure-activity relationship of the series against the hERG ion channel proved to be a key selectivity hurdle for the series.

Heterosis in poplar involves phenotypic stability: cottonwood hybrids outperform their parental species at suboptimal temperatures.

Heterosis or hybrid vigor is common in hybrid poplars, and to investigate its occurrence and physiological basis we compared narrowleaf cottonwoods, Populus angustifolia James, prairie cottonwoods, Populus deltoides Bartr. ex Marsh, and their native intersectional hybrids, P. × acuminata Rydb., from Alberta, Canada. Clonal replicates from 10 separate trees from each taxon were raised in growth chambers at different temperatures (T). Growth was similarly vigorous across the taxa at 20 and 24 °C, and morphological and physiological traits of the hybrids were generally intermediate between the parental species, or similar to the larger parent, demonstrating additive inheritance or dominance, respectively. Growth declined at 18 and 15 °C particularly in the parental species, and consequently hybrid vigor was displayed for root and especially leaf growth. Stomatal distributions and chlorophyll indices were intermediate in the hybrids and unaffected by T. Foliar nitrogen (N), net assimilation (Asat), stomatal conductance (gs) and transpiration (E) per unit of leaf area were lower in the hybrids, but the hybrids generally had larger leaf areas. Water-use efficiencies (Asat/gs) were similar across the taxa and reduced with warming, while nitrogen-use efficiencies (Asat/N) increased. δ13C was correlated with leaf mass per area, which varied across the taxa. Photosynthesis (Asat) was correlated with chlorophyll content index, N and/or gs in P. deltoides and the hybrids, but not in P. angustifolia, indicating different physiological limitations. We conclude that heterosis in P. × acuminata results from the compound benefits from multiple dominant traits, and superior growth particularly at suboptimal conditions. This indicates phenotypic stability or environmental adaptability, whereby heterozygosity provides metabolic diversity that allows hybrids to thrive across a broader environmental range.

Stomatal characteristics of riparian poplar species in a semi-arid environment.

Several native poplar species meet at the margins of their natural distributions in southern Alberta, Canada. In this semi-arid area, poplars are obligate riparian species but they occupy several intergrading ecoregions. Populus deltoides Bartr. ex Marsh predominates in the warmest and driest eastern prairie ecoregions; P. balsamifera L. occupies the cooler and wetter western parkland and montane ecoregions; and P. angustifolia James and hybrids between the species occur in the intermediate grassland ecoregions. We investigated stomatal characteristics of these poplars in 51 genotypes collected across the range of ecoregions and grown in a semi-arid common garden. Stomatal length differed among genotypes within species but did not differ among species, ranging from 19 to 22 microm. Total stomatal densities (adaxial plus abaxial) differed among genotypes within species but were similar among species (290-420 stomata mm(-2)). Single-surface stomatal densities differed among species and consequently, the ratio of adaxial:abaxial stomatal density also differed, ranging from 0.94 for P. deltoides to 0.27 for P. balsamifera, with intermediate stomatal density ratios in P. angustifolia and hybrids. In a subsequent study of a subset of the same genotypes, stomatal density was correlated with stomatal conductance (r2 = 0.75) and the conductance ratios differed among species in the same manner as the stomatal density ratios. We conclude that: (1) diverse poplar genotypes respond similarly to a semi-arid environment by producing comparatively small and dense stomata; (2) differences in stomatal density underlie differences in stomatal conductance and differences among species in stomatal density ratio or conductance ratio may reflect adaptation to climatic differences among ecoregions; and (3) there is substantial variation in stomatal characteristics within and among species and hybrids in this area that could be useful for the selection and breeding of poplars adapted to different climatic conditions.

Bud phenology and growth are subject to divergent selection across a latitudinal gradient in Populus angustifolia and impact adaptation across the distributional range and associated arthropods.

Temperate forest tree species that span large geographical areas and climatic gradients often have high levels of genetic variation. Such species are ideal for testing how neutral demographic factors and climate-driven selection structure genetic variation within species, and how this genetic variation can affect ecological communities. Here, we quantified genetic variation in vegetative phenology and growth traits in narrowleaf cottonwood, Populus angustifolia, using three common gardens planted with genotypes originating from source populations spanning the species' range along the Rocky Mountains of North America (ca. 1700 km). We present three main findings. First, we found strong evidence of divergent selection (Q ST > F ST) on fall phenology (bud set) with adaptive consequences for frost avoidance. We also found evidence for selection on bud flush duration, tree height, and basal diameter, resulting in population differentiation. Second, we found strong associations with climate variables that were strongly correlated with latitude of origin. More strongly differentiated traits also showed stronger climate correlations, which emphasizes the role that climate has played in divergent selection throughout the range. We found population × garden interaction effects; for some traits, this accounted for more of the variance than either factor alone. Tree height was influenced by the difference in climate of the source and garden locations and declined with increasing transfer distance. Third, growth traits were correlated with dependent arthropod community diversity metrics. Synthesis. Overall, we conclude that climate has influenced genetic variation and structure in phenology and growth traits and leads to local adaptation in P. angustifolia, which can then impact dependent arthropod species. Importantly, relocation of genotypes far northward or southward often resulted in poor growth, likely due to a phenological mismatch with photoperiod, the proximate cue for fall growth cessation. Genotypes moved too far southward suffer from early growth cessation, whereas those moved too far northward are prone to fall frost and winter dieback. In the face of current and forecasted climate change, habitat restoration, forestry, and tree breeding efforts should utilize these findings to better match latitudinal and climatic source environments with management locations for optimal future outcomes.

Higher photosynthetic capacity from higher latitude: foliar characteristics and gas exchange of southern, central and northern populations of Populus angustifolia.

Narrowleaf cottonwood (Populus angustifolia James) is an obligate riparian poplar that is a foundation species in river valleys along the Rocky Mountains, spanning 16° of latitude from southern Arizona, USA to southern Alberta, Canada. Its current distribution is fragmented, and genetic variation shows regional population structure consistent with the effects of geographic barriers and past climate. It is thus very well-suited for investigating ecophysiological adaptation associated with latitude. In other section Tacamahaca poplar species, genotypes from higher latitudes show evidence of short-season adaptation with foliar traits that contribute to higher photosynthetic capacity. We tested for similar adaptation in three populations of narrowleaf cottonwoods: from Arizona (south), Alberta (north) and Utah, near the centre of the latitudinal distribution. We propagated 20 genotypes from each population in a common garden in Alberta, and measured foliar and physiological traits after 3 years. Leaves of genotypes from the northern population had higher leaf mass per area (LMA), increased nitrogen (N) content and higher carotenoid and chlorophyll content, and these were associated with higher light-saturated net photosynthesis (Asat). In leaves of all populations the majority of stomata were abaxial, with the proportion of abaxial stomata highest in the southern population. Stomatal conductance (gs) and transpiration rates were higher in the northern population but water-use efficiency (Asat/gs) and leaf carbon isotope composition (δ(13)C) did not differ across the populations. These results (i) establish links between Asat and gs, N, chlorophyll and LMA among populations within this species, (ii) are consistent with the discrimination of populations from prior investigation of genetic variation and (iii) support the concept of latitudinal adaptation, whereby deciduous trees from higher latitudes display higher photosynthetic capacity, possibly compensating for a shorter and cooler growth season and reduced insolation.

Gibberellins in shoots and developing capsules of Populus species.

Extracts of stems of growing shoots of Populus deltoides and P. trichocarpa, and developing capsules of P. deltoides were analysed for gibberellins (GAs) by gas chromatography-mass spectrometry. The following known GAs were identified by comparison of their Kovats retention indices (KRIs) and mass spectra with those of standards: GA1, GA8, GA9, GA19, GA20, 16 beta,17-dihydro-17-hydroxy GA20, GA23, GA28, GA29, GA34, GA44, and GA97. Several of these have not been previously reported from Populus. In addition, two new GAs were identified as 12 beta-hydroxy GA53 (GA127) and 16 beta,17-dihydro-17-hydroxy GA53 and their structures were confirmed by partial synthesis. Evidence was found of 16,17-dihydro-16,17-dihydroxy GA9, 16,17-dihydro-16,17-dihydroxy GA12, 12-hydroxy GA14, and GA34-catabolite by comparison of mass spectra and KRIs with published data. Several putative GAs (hydroxy- and dihydroxy-GA12-like) were also found. The catabolites of active GAs or of key precursors, hydroxylated at C-2 in stems and either C-2, C-12, C-17, or C-16,17 in capsules, were the major proportion of the GAs.

Abscisic acid, phaseic acid and gibberellin contents associated with dormancy and germination in barley.

Analyses of abscisic acid (ABA), ent-kaurenoids and gibberellins (GAs) showed that there were major changes in the contents of these compounds associated with germination of after-ripened barley (Hordeum vulgare cv. Schooner and cv. Proctor) grain but not in hydrated dormant grain. Embryos from dormant and after-ripened dry grain contained similar amounts of ABA, of ent-kaurenoids and of GAs, determined by gas chromatography-mass spectrometry-selected ion monitoring. In embryos of after-ripened grain, ABA content decreased rapidly after hydration and ABA appeared to be metabolized (inactivated) to phaseic acid (PA) rather than diffusing into the endosperm or the surrounding medium as previously thought. Similar changes in ABA occurred in hydrated dormant grain during germination in darkness. Accumulation of ent-kaurenoids and GAs, including GA1, the first biologically active GA in the early 13-hydroxylation biosynthetic pathway, occurred to a much greater extent in after-ripened than in dormant grain and these changes occurred mainly after 18 h of hydration when ABA had already decreased and germination was occurring. The block in ent-kaurenoid and GA synthesis in dormant grain appeared to occur prior to ent-kaurene in the biosynthetic pathway. These results are consistent with the view that ABA is the primary effector of dormancy and that after-ripening involves the development of the ability to reduce the amount of ABA quickly following hydration. Accumulation of GAs does not appear to be causally related to loss of dormancy but it does appear to be related to germination.

Phytohormones and shoot growth in a three-generation hybrid poplar family.

Hybrid vigor for secondary growth in poplar has been linked with increased gibberellin (GA) concentration in cambial tissue, but the relationship between concentrations of GAs and hybrid vigor of primary growth has not been investigated. We explored associations between concentrations of GAs, abscisic acid (ABA) and indoleacetic acid (IAA) and shoot extension in a hybrid family of Populus deltoides Bartr. ex Marsh. (Clone ILL-129) and P. trichocarpa Torr. & Gray (Clone 93-968) and two F1 and 67 F2 genotypes. Rapidly elongating subapical internodes from shoots of 4-year-old clonal saplings were selected for hormone analysis. The F1 hybrids displayed heterosis (hybrid vigor) for primary growth as a result of the complementation of dominance for increased internode length from the P. trichocarpa parent and dominance for increased diameter from the P. deltoides parent. Internodes from the faster-elongating shoots of the P. trichocarpa parent had a fourfold higher concentration of bioactive GA1 and higher concentrations of GA20, GA44, GA29 and GA8 than the P. deltoides parent. However, the two fast-growing F1 hybrids had low concentrations of all five GAs, with concentrations similar to those of the slower-elongating P. deltoides parent. Concentrations of ABA and IAA were correlated with GA concentrations and there was thus no evidence of a consistent promoting or inhibiting effect of ABA or IAA within the F1 family. These results indicate that heterosis for internode growth was not primarily regulated by hormone concentration. The segregating population of F2 hybrids was analyzed to assess the possible utility of hormone screening as a selection tool. The internodes of most of the F2 hybrids were smaller than those of their F1 parents and the larger P. trichocarpa parent. Among the F2 hybrids, mature internode length varied fourfold, and was correlated with lengths of young, subapical internodes from which hormones were analyzed. In these internodes, GA1 concentrations were negatively correlated with length (r = 0.41), diameter (0.33) and mass (0.50). Indoleacetic acid concentration was negatively correlated only with diameter (r = 0.37) and positively correlated with GA1 (r = 0.46), whereas ABA concentration was not correlated with any parameter. Thus, in the F2 population, variation in hormone concentration was not primarily responsible for the variation in shoot growth, indicating that selection for individuals with high GA concentration may not be an effective strategy for identifying vigorous hybrid genotypes.

Transgenic modification of gai or rgl1 causes dwarfing and alters gibberellins, root growth, and metabolite profiles in Populus.

In Arabidopsis and other plants, gibberellin (GA)-regulated responses are mediated by proteins including GAI, RGA and RGL1-3 that contain a functional DELLA domain. Through transgenic modification, we found that DELLA-less versions of GAI (gai) and RGL1 (rgl1) in a Populus tree have profound, dominant effects on phenotype, producing pleiotropic changes in morphology and metabolic profiles. Shoots were dwarfed, likely via constitutive repression of GA-induced elongation, whereas root growth was promoted two- to threefold in vitro. Applied GA(3 )inhibited adventitious root production in wild-type poplar, but gai/rgl1 poplars were unaffected by the inhibition. The concentrations of bioactive GA(1) and GA(4) in leaves of gai- and rgl1-expressing plants increased 12- to 64-fold, while the C(19) precursors of GA(1) (GA(53), GA(44) and GA(19)) decreased three- to ninefold, consistent with feedback regulation of GA 20-oxidase in the transgenic plants. The transgenic modifications elicited significant metabolic changes. In roots, metabolic profiling suggested increased respiration as a possible mechanism of the increased root growth. In leaves, we found metabolite changes suggesting reduced carbon flux through the lignin biosynthetic pathway and a shift towards allocation of secondary storage and defense metabolites, including various phenols, phenolic glucosides, and phenolic acid conjugates.

Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature.

We identified a dwarf transgenic hybrid poplar (Populus tremula x Populus alba) after screening of 627 independent activation-tagged transgenic lines in tissue culture, greenhouse, and field environments. The cause of the phenotype was a hyperactivated gene encoding GA 2-oxidase (GA2ox), the major gibberellin (GA) catabolic enzyme in plants. The mutation resulted from insertion of a strong transcriptional enhancer near the transcription start site. Overexpression of the poplar GA2ox gene (PtaGA2ox1) caused hyperaccumulation of mRNA transcripts, quantitative shifts in the spectrum of GAs, and similarity in phenotype to transgenic poplars that overexpress a bean (Phaseolus coccineus) GA2ox gene. The poplar PtaGA2ox1 sequence was most closely related to PsGA2ox2 from pea (Pisum sativum) and two poorly known GA2oxs from Arabidopsis (AtGA2ox4 and AtGA2ox5). The dwarf phenotype was reversible through gibberellic acid application to the shoot apex. Transgenic approaches to producing semidwarf trees for use in arboriculture, horticulture, and forestry could have significant economic and environmental benefits, including altered fiber and fruit production, greater ease of management, and reduced risk of spread in wild populations.