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The transgenic Vip3A poplar plant confers high resistance against Hyphantria cunea Drury.

Poplar is severely damaged by Hyphantria cunea (fall webworm), which significantly reduces tree productivity. However, conventional pest management methods are largely ineffective against fall webworm infestation. In this study, we demonstrated that the Vip3A protein possesses high insecticidal activity against H. cunea by overexpressing a synthetic THI1-Vip3A gene in poplar plants. A dicot codon-optimized Vip3A gene, fused with the THI1 chloroplast signal peptide sequence, was chemically synthesized and introduced into the poplar cv. '741' genome via Agrobacterium-mediated transformation. PCR, RT-PCR, and ELISA analyses confirmed the integration and successful expression of the transgene at both the mRNA and protein levels. The Vip3A protein concentration in chloroplasts was approximately 4.8-fold higher than in the whole leaf extract, indicating that the Vip3A protein was successfully targeted to and accumulated within the chloroplasts by the THI1 signal peptide. Subsequently, four transgenic lines with high Vip3A expression were subjected to H. cunea infestation. Compared to wild-type plants, these four transgenic lines exhibited significantly higher resistance, resulting in pest mortality rates exceeding 95% and significantly reduced leaf damage. Together, these results indicate that Vip3A possesses high insecticidal activity against H. cunea. Therefore, transgenic THI1-Vip3A poplar plants can serve as valuable germplasm for breeding poplar cultivars with high resistance to H. cunea infestation.

Plants, Genetically Modified

Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.

Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.

Populus

Nutrikinetics and bioavailability of Promunel®, a standardized poplar-type propolis phenolic extract: a double-blinded, placebo-controlled, cross-over, randomized trial.

Brown poplar-type propolis has been recognized and used for centuries to help prevent upper respiratory tract infections (URTIs). However, the scarce, incomplete information in humans on the nutrikinetics and bioavailability of its phenolic constituents, combined with a lack of standardization in its phenolic content and profile pose major challenges to develop bioactive ingredients. Thus, the aim of this study was to establish the nutrikinetics and total bioavailability (NKBA) parameters of brown poplar-type propolis phenolics in humans using the Standardized Propolis Extract (SPE) Promunel®. To achieve this, a 48 h NBKA study was conducted following a double blinded, randomized, placebo-controlled, cross-over design in healthy humans (n = 10) with two doses of SPE (1X = 400 mg or 4X = 1600 mg). Phenolic compounds were detected, identified and quantified in the extract, plasma and urine through different LC-MS/UV technologies. The SPE used is a rich (304.44 ± 15.61 µmol mg-1) and diverse source of phenolic compounds (5 sub-families). A total of 63 and 85 phenolic metabolites were identified and quantified in plasma and urine, mostly in the form of glucuronides and sulfates. In plasma, phenolic metabolites reached Cmax (1.22 ± 0.20 for 1X and 4.80 ± 0.48 µM for 4X) after 1 h of SPE intake, while urinary excretion occurred mostly during the first 3 h after. The total net bioavailability of SPE phenolic compounds at 48 h was 57.16 ± 5.71% for 1X and 43.82 ± 6.77% for 4X. Generally, the data between SPE 1X and 4X were proportional, indicating that a higher dose does not substantially modulate total net bioavailability. Overall, our data shows that brown poplar-type SPE phenolic compounds are highly bioavailable in the form of cinnamic acid and flavonoid conjugates, and that these compounds are rapidly absorbed and eliminated through the urine. Our results suggest that, for a sustained presence in circulation, brown poplar-type propolis supplements should be consumed more than once a day.

Humans

A Leucine-Rich Repeat Receptor-Like Protein Associated with a QTL for Septoria Stem Canker in Populus trichocarpa × Populus deltoides Hybrid Poplar.

The fungal plant pathogen Sphaerulina musiva (Ascomycota) causes Septoria stem canker, the most economically damaging disease of Populus plantations in North America, yet the genetic determinants of host resistance remain uncharacterized in hybrid poplar. Using an inoculation experiment with the 52-124 pseudo-backcross family of Populus trichocarpa × Populus deltoides (TD × D) hybrid poplar, a single significant QTL was identified on Chromosome 16 (LOD = 4.93) associated with both stem canker count and disease severity score. Transcriptomic analysis of two resistant and two susceptible genotypes across a 72-hour infection time course identified a single differentially expressed gene within the QTL candidate gene window: Podel.16G125900, a putative leucine-rich repeat receptor-like protein (LRR-RLP) with homology to receptor-like protein 33 in Arabidopsis thaliana. Podel.16G125900 is located 3001 bp (0.019 cM) upstream of the QTL peak and showed a strong infection-induced upregulation in susceptible genotype 852 (log2 fold-change = 20.47) and higher baseline expression in resistant genotypes relative to susceptible genotypes across all infection time points, consistent with a resistance mechanism in which expression level contributes to the degree of resistance conferred. Two P. trichocarpa homologs were not differentially expressed and differ substantially in sequence content, suggesting the resistance function is specific to the resistant P. deltoides lineage. These findings identify Podel.16G125900 as a strong candidate gene underlying quantitative resistance mechanisms modulating Septoria stem canker resistance in the 52-124 family of TD × D hybrid poplar and provide a target for future functional validation and marker-assisted resistance breeding.

Disease Resistance

Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in sycamore and poplar.

During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.

Arabinose

Tonoplast sucrose transporter SUT4-dependent sugar partitioning modulates phenological transitions and reproductive success in poplar.

Climate uncertainty is intensifying the need for greater plasticity in carbohydrate reserve utilization to support winter survival and spring growth in woody perennials. In poplar, the single-copy SUT4, which encodes a tonoplast-localized sucrose transporter, and the SUT5/SUT6 genome duplicates, which encode plasma membrane-localized transporters, are expressed year-round, with SUT4 showing the highest expression during cool seasons. Given its role in vacuolar sucrose efflux and winter-predominant expression, SUT4 may play a key role in modulating seasonal carbohydrate dynamics. While SUT4-knockdown and knockout effects have been studied under greenhouse conditions, their impact under field conditions remains unexplored. Here, we report a field-based study comparing CRISPR knockout mutants of winter-expressed SUT4 and SUT5/SUT6 in Populus tremula × alba. We show that sut4, but not sut5/6, mutants exhibited earlier autumn leaf senescence, delayed spring bud flush, reduced stem growth, and altered sugar partitioning in winter xylem and bark relative to controls. After 2 years in the field, all genotypes flowered before leaf flush in early spring; however, sut4 mutants produced sterile ovules despite developing normal-looking catkins. Metabolic profiling revealed disrupted sucrose and raffinose dynamics in elongating sut4 catkins. This was accompanied by transcriptomic signatures of elevated stress and downregulation of proanthocyanidin biosynthesis and circadian clock genes. These findings highlight the critical role of SUT4 in coordinating sugar allocation, stress responses, and seasonal development in poplar.

Populus

[Inhalation of an extract from the leaf buds of the deltoid poplar during the early period following tonsillectomy].

To prevent inflammatory processes in the mucous membrane of the upper respiratory tract during early periods after tonsillectomy (2-7 days) the authors practiced inhalations of the extract of Eastern poplar leaf buds in 40 patients with chronic tonsillitis after the operation. The use of the extract of Eastern poplar leaf buds at early periods after tonsillectomy promotes abating of pains, speedy cicatrization of postoperative wounds, decrease of reactive inflammation in the pharynx and improvement of the general condition in the patients.

Adolescent

Factors underlying a latitudinal gradient in the S/G lignin monomer ratio in natural poplar variants.

The chemical composition of wood plays a pivotal role in the adaptability and structural integrity of trees. However, few studies have investigated the environmental factors that determine lignin composition and its biological significance in plants. Here, we examined the lignin syringyl-to-guaiacyl (S/G) ratio in members of a Populus trichocarpa population sourced from their native habitat and conducted a genome wide association study to identify genes linked to lignin formation. Our results revealed many significant associations, suggesting that lignin biosynthesis is a complex polygenic trait. Additionally, we found an increase in the S/G ratio from northern to southern geographic origin of the trees sampled, along with a corresponding metabolic and transcriptional reprogramming of xylem cell wall biosynthesis. Further molecular analysis identified a mutation in a cell wall laccase genetically associated with higher S/G ratios that predominate in trees from warmer lower latitudes. Collectively, our findings suggest that lignin heterogeneity arises from an evolutionary process enabling poplar adaptation to different climatic challenges.

Populus

Parent-of-origin effects on allelic expression bias in interspecific poplar hybrids.

In hybrid plants, phenotypic outcomes are governed by interactions between the two parental genomes. However, the mechanisms underlying the interplay of divergent regulatory networks from these genomes remain poorly understood. In this study, we compared gene-level and allele-specific expression patterns, as well as differentially enriched pathways between F₁ and complex backcross (CBC) lines derived from a natural interspecific hybrid population of Populus fremontii (Pf) and P. angustifolia (Pa). Metabolic differences between Pf and Pa which exhibit low and high levels respectively of phenylpropanoid-derived condensed tannins were leveraged. Using individualized transcriptome references, differential expression and clustering analyses revealed CBC-biased and F₁-biased expression for genes involved in phenylpropanoid metabolism and photosynthesis, respectively. Biased expression of these genes at the allele level was also observed in F1. At the whole-transcriptome level, Pa-biased genes predominated in F₁ hybrids, and Pa alleles displayed more conserved expression patterns than Pf alleles across examined samples. Further analyses indicated that allelic expression bias was significantly associated with parental origin, which could be driven by sequence variations in cis-regulatory elements and differences in CpG island length. Our findings demonstrate strong parent-of-origin effects on divergent regulatory networks governing gene expression in poplar hybrids and provide clues for strategic parental selection tailored to specific metabolic pathways of interest.

cis-regulation

Comparative transcriptomics uncovers poplar and fungal genetic determinants of ectomycorrhizal compatibility.

Ectomycorrhizal symbiosis supports tree growth and is crucial for nutrient cycling and temperate and boreal ecosystems functioning. The establishment of functional ectomycorrhiza (ECM) first requires the association of compatible partners. However, host and fungal genetic determinants governing mycorrhizal compatibility are unknown. To identify such factors in poplar and its fungal associates, we mined existing and de novo tree and fungal transcriptional datasets. We identified co-expressed genes enabling ECM symbiosis at early and mature stages of the interaction. These sets of genes can be divided into general fungal-sensing and ECM-specific components. We highlight the importance of fungal modulation of plant JA-related defenses and the regulation of secretory pathways for ECM compatibility, including upregulation of key fungal small secreted proteins, the downregulation of plant secreted peroxidases, and the downregulation of plant cell wall remodeling proteins concomitantly with the upregulation of fungal glycosyl hydrolases acting on pectin. Not only gene regulation, but also its temporal scale and dynamics seem to play a crucial role for mycorrhizal compatibility. The expression profile of the host Common Symbiosis Pathway and nutrient transporters was also studied, revealing constitutive levels of expression and moderate upregulation in compatible ECM interactions. Overall, these results underscore the importance of novel biological functions during the establishment of ECM symbiosis, help us gain insights into the molecular events determining mycorrhiza compatibility, and serve as a data-rich transcriptomic resource to open new research questions in the field.

Mycorrhizae

Novel Mycoparasitic Mechanisms and Colonization Patterns on Poplar Revealed by GFP Tagging of the Biocontrol Fungus Clonostachys reniana.

Clonostachys rosea has long been the primary model for studying mycoparasitism within its genus; however, the potential of other species remains largely unexplored. In this study, we established a PEG-CaCl2-mediated protoplast transformation system for Clonostachys reniana. Our results demonstrate for the first time that this species is amenable to genetic manipulation and produces transformants with genetic stability, providing a reliable platform for functional genomic research in this fungus. Using green fluorescent protein tagging, we achieved stable transformants that retained wild-type physiological traits. Crucially, our data indicated that C. reniana utilizes a distinct mycoparasitic mechanism, which differs from the well-established sequential process of adhesion, coiling, and lysis seen in C. rosea. Confocal and scanning electron microscopy revealed that C. reniana, following initial coiling and invasive structure formation, penetrates the host hyphae of Botryosphaeria dothidea. It then grows longitudinally within the hyphal lumen, absorbing nutrients and eliminating the pathogen from the inside. Furthermore, C. reniana hyphae can colonize the intercellular spaces of the stem periderm in 84K poplar (Populus alba × P. glandulosa) while displaying a distinct tissue-specific behavior in the roots by forming a mantle on the root surface resembling that of ectomycorrhizae. These findings establish C. reniana as a highly promising secondary model species within the Clonostachys genus. By uncovering a novel "internal-consumption" mycoparasitic mode, this study expands our understanding of the ecological diversity of biocontrol fungi and provides a powerful genetic toolset for future functional genomic research.

Clonostachys reniana

Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. II. Intracellular hyphae and vesicles.

Intracellular hyphae and vesicles in mycorrhizal roots of yellow poplar were examined by electron microscopy. An investing layer of host wall material and cytoplasm enclosed the endophyte within the cells. Young developing hyphae contained abundant cytoplasm and few vacuoles. As hyphae matured, they became highly vacuolated and accumulated carbohydrate (glycogen) and lipid reserves. Mature vesicles were engorged with lipid droplets, possessed a trilaminate wall and were also enclosed by host wall material and cytoplasm. Compared with uninfected cells, infected cortical cells showed an increase in cytoplasmic volume, enlarged nuclei, and a reduction of starch reserves. Host nuclei were always proximal to the hyphae during hyphal development and deterioration. While other cytoplasmic components of infected and uninfected cells were comparable large electron-dense bodies occurred in vacuoles of most cells containing hyphae. Deterioration of intracellular hyphae occurred throughout the samples examined. Septa separated functional and degenerating portions of the hyphae. Hyphal deterioration involved degeneration and ultimate disappearance of fungal cytoplasm as well as collapse of hyphal walls. Based on these observations, the authors hypothesize that deterioration of the endophyte may release significant quantities of mineral nutrients, via hyphal contents, which are absorbed by the host.

Cell Wall

Cytological evidence supporting the inclusion of poplar mosaic virus in the carlavirus group of plant viruses.

The filamentous particles of poplar mosaic virus (PopMV) were detected in palisade, mesophyll and parenchyma cells of systemically infected leaves of Nicotiana clevelandii and N. megalosiphon. The particles were sometimes found scattered throughout the cytoplasm, but more often they occurred loosely associated within ovoid aggregates (up to 3.5 x 1.5 mum) adjacent to chloroplasts and/or mitochondria. PopMV did not induce the formation of 'pinwheel' inclusions, nor other conspicuous ultrastructural or cytopathic effects in infected cells. These observations provide additional evidence that PopMV is a Carlavirus and not a Potyvirus nor a mite-borne virus. PopMV particles were also detected occasionally within plasmodesmata.

Chloroplasts

[Natural pollution of continental waters by the antioxygenic effect of phytomelanin genesis in aqueous extract of autumn leaves, especially poplars].

Phenolase-acid phenol systems are present in variable amounts, variable proportion and variable water solubility in the autumn leaves of trees and bushes. The oxidative polycondensations into phytomelanins caused by these systems confer antioxygen properties to aqueous macerations of senescent leaves, which are sometimes very intense. They induce therefore a biochemical natural pollution of continental waters. The Poplars proved to be particularly deleterious.

Melanins

A single-nucleus and spatial transcriptomic atlas of poplar leaves reveals the regulation of leaf polarity and cuticle deposition.

Leaf adaxial-abaxial polarity is fundamental for plant morphogenesis and environmental adaptation through asymmetric cell differentiation. Emerging evidence reveals dorsoventral metabolic gradients act downstream of transcriptional networks to fine-tune cellular specialization. While conserved transcription factors (e.g., HD-ZIP III and KANADI) establish initial polarity, the molecular networks driving position-specific cellular differentiation and their integration with metabolic adaptation remain unclear. Leveraging single-nucleus and spatial transcriptomics, we resolve major cell classes (mesophyll, epidermal, and vascular-associated) and their adaxial-abaxial subtypes, revealing dorsoventral polarity in transcriptional profiles and metabolic pathways. Adaxial cells are enriched in phenylpropanoid/flavonoid biosynthesis, while abaxial cells show preferential activation of stress and hormone signaling. Notably, we identify MYC2 as a key regulator of adaxial cuticle biosynthesis, binding to promoters of lipid biosynthetic and transport genes (e.g., CER10 and LTPG1) and promoting cuticle thickening. Our study uncovers how positional identity shapes transcriptional and metabolic polarity in leaves, with MYC2 emerging as a central regulator coordinating organ-specific adaptations. These findings provide insights into the spatial regulation of plant development and stress resilience, offering potential strategies for engineering stress-tolerant woody crops.

Plant Leaves

Resistance of Populus davidiana × P. bolleana overexpressing cinnamoyl-CoA reductase gene to Lymantria dispar larvae.

Lignin is a crucial defense phytochemical against phytophagous insects. Cinnamoyl-CoA reductase (CCR) is a key enzyme in lignin biosynthesis. In this study, transgenic Populus davidiana × P. bolleana overexpressing the PdbCCR gene were generated via Agrobacterium-mediated transformation. Successful integration of PdbCCR into the poplar genome was confirmed by PCR amplification and quantitative reverse transcription PCR (qRT-PCR). The lignin content in the transgenic poplar leaves was significantly higher than that in the wild poplar, and after L. dispar larvae fed on the transgenic poplar, the CCR activity was clearly induced. The L. dispar larvae grew slowly after feeding on transgenic poplar and the laccase, cellulase and three detoxifying enzymes were induced compared with larvae after feeding on wild-type poplar. The bioassay further revealed that transgenic poplar plants overexpressing PdbCCR showed a high level of resistance to L. dispar larvae. These results confirmed that PdbCCR is a candidate gene for breeding insect resistant poplar.

Populus