Physiological and molecular responses in phosphorus-hyperaccumulating Polygonum species to high phosphorus exposure.

Phosphorus (P)-hyperaccumulators for phytoextraction from P-polluted areas generally show rapid growth and accumulate large amounts of P without any toxicity symptom, which depends on a range of physiological processes and gene expression patterns that have never been explored. We investigated growth, leaf element concentrations, P fractions, photosynthetic traits, and leaf metabolome and transcriptome response in amphibious P-hyperaccumulators, Polygonum hydropiper and P. lapathifolium, to high-P exposure (5 mmol L-1), with 0.05 mmol L-1 as the control. Under high-P exposure, both species demonstrated good growth, allocating more P to metabolite P and inorganic P (Pi) accompanied by high potassium and calcium. The expression of a cluster of unigenes associated with photosynthesis was maintained or increased in P. lapathifolium, explaining the increase in net photosynthetic rate and the rapid growth under high-P exposure. Metabolites of trehalose metabolism, including trehalose 6-phosphate and trehalose, were sharply increased in both species by the high-P exposure, in line with the enhanced expression of associated unigenes, indicating that trehalose metabolic pathway was closely related to high-P tolerance. These findings elucidated the physiological and molecular responses involved in the photosynthesis and trehalose metabolism in P-hyperaccumulators to high-P exposure, and provides potential regulatory pathways to improve the P-phytoextraction capability.

Polygonum cillinerve polysaccharide inhibits transmissible gastroenteritis virus by regulating microRNA-181.

Transmissible gastroenteritis virus (TGEV) is an important pathogen capable of altering the expression profile of cellular miRNA. In this study, the potential of Polygonum cillinerve polysaccharide (PCP) to treat TGEV-infected piglets was evaluated through in vivo experiments. High-throughput sequencing technology was employed to identify 9 up-regulated and 17 down-regulated miRNAs during PCP-mediated inhibition of TGEV infection in PK15 cells. Additionally, miR-181 was found to be associated with target genes of key proteins in the apoptosis pathway. PK15 cells were treated with various concentrations of PCP following transfection with miR-181 mimic or inhibitor. Real-time PCR assessed the impact on TGEV replication, while electron microscopy (TEM) and Hoechst fluorescence staining evaluated cellular functionality. Western blot analysis was utilized to assess the expression of key signaling factors-cytochrome C (cyt C), caspase 9, and P53-in the apoptotic signaling pathway. The results showed that compared with the control group, 250 µg/mL PCP significantly inhibited TGEV gRNA replication and gene N expression (P < 0.01). Microscopic examination revealed uniform cell morphology and fewer floating cells in PCP-treated groups (250 and 125 µg/mL). TEM analysis showed no typical virus structure in the 250 µg/mL PCP group, and apoptosis staining indicated a significant reduction in apoptotic cells at this concentration. Furthermore, PCP may inhibit TGEV-induced apoptosis via the Caspase-dependent mitochondrial pathway following miR-181 transfection. These findings provide a theoretical basis for further exploration into the mechanism of PCP's anti-TGEV properties.

Molecular cloning and in silico analysis of chalcone isomerase from Polygonum minus.

BACKGROUND: Chalcone isomerase (CHI; EC 5.5.1.6) is one of the key enzymes in the flavonoid biosynthetic pathway that is responsible for the intramolecular cyclization of chalcones into specific 2S-flavanones. METHODS AND RESULTS: In this study, the open reading frame (ORF) of CHI was successfully isolated from the cDNA of Polygonum minus at 711-bp long, encoding for 236 amino acid residues, with a predicted molecular weight of 25.4 kDa. Multiple sequence alignment and phylogenetic analysis revealed that the conserved residues (Thr50, Tyr108, Asn115, and Ser192) in the cleft of CHI enzyme group active site are present in PmCHI protein sequence and classified as type I. PmCHI comprises more hydrophobic residues without a signal peptide and transmembrane helices. The three-dimensional (3D) structure of PmCHI predicted through homology modeling was validated by Ramachandran plot and Verify3D, with values within the acceptable range of a good model. PmCHI was cloned into pET-28b(+) plasmid, expressed in Escherichia coli BL21(DE3) at 16 °C and partially purified. CONCLUSION: These findings contribute to a deeper understanding of the PmCHI protein and its potential for further characterization of its functional properties in the flavonoid biosynthetic pathway.

Antiviral activities of Polygonum perfoliatum L. extract and related phenolic acid constituents against hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is an important public health problem. Polygonum perfoliatum L. is a traditional medicinal herb and has been reported to have pharmacological activities such as anti-inflammatory, antibacterial, and antiviral. In this study, the antiviral activities and mechanisms of Polygonum perfoliatum L. extract against HBV and the effective components were investigated. The results showed that the total extract of Polygonum perfoliatum L. reduced the levels of HBV e antigen (HBeAg) secretion and the viral covalently closed circular DNA (CCC DNA) formation, but had little or no negative effects on viral capsid assembly and pregenomic RNA packaging. Further fractionation showed that the water extract (WE) fraction exerted comparable anti-HBV activities with the total extract, especially in inhibiting the CCC DNA formation and HBeAg production, indicating that the effective antiviral components are mainly distributed in this fraction. Further study showed that the phenolic acids constituents, protocatechuic acid, and gallic acid, but not ethyl caffeate, which is reported enriched in the WE fraction, showed strong anti-HBV activities in inhibiting viral core DNA synthesis, CCC DNA formation, and HBeAg production. These results suggested that the Polygonum perfoliatum L. total extract and the related phenolic acids like protocatechuic acid and gallic acid could inhibit HBV replication and also indicated the potential utility of Polygonum perfoliatum L. and related constituents as sources of novel antivirals against HBV.

Plastid and mitochondrial phylogenomics reveal correlated substitution rate variation in Koenigia (Polygonoideae, Polygonaceae) and a reduced plastome for Koenigia delicatula including loss of all ndh genes.

Koenigia, a genus proposed by Linnaeus, has a contentious taxonomic history. In particular, relationships among species and the circumscription of the genus relative to Aconogonon remain uncertain. To explore phylogenetic relationships of Koenigia with other members of tribe Persicarieae and to establish the timing of major evolutionary diversification events, genome skimming of organellar sequences was used to assemble plastomes and mitochondrial genes from 15 individuals representing 13 species. Most Persicarieae plastomes exhibit a conserved structure and content relative to other flowering plants. However, Koenigia delicatula has lost functional copies of all ndh genes and the intron from atpF. In addition, the rpl32 gene was relocated in the K. delicatula plastome, which likely occurred via overlapping inversions or differential expansion and contraction of the inverted repeat. The highly supported but conflicting relationships between plastome and mitochondrial trees and among gene trees complicates the circumscription of Koenigia, which could be caused by rapid diversification within a short period. Moreover, the plastome and mitochondrial trees revealed correlated variation in substitution rates among Persicarieae species, suggesting a shared underlying mechanism promoting evolutionary rate variation in both organellar genomes. The divergence of dwarf K. delicatula from other Koenigia species may be associated with the well-known Eocene Thermal Maximum 2 or Early Eocene Climatic Optimum event, while diversification of the core-Koenigia clade associates with the Mid-Miocene Climatic Optimum and the uplift of Qinghai-Tibetan Plateau and adjacent areas.

Flower, seed, and fruit development in three Tunisian species of Polygonum: Implications for their taxonomy and evolution of distyly in Polygonaceae.

Polygonum is the largest genus of Polygonaceae and 5 species are reported in Tunisia. In order to characterized flower, seed, and fruit development in Polygonum, flower and fruit of Polygonium equisetiforme (var. graecum and peyerinhoffi), P. aviculare and P. maritimum, collected from Tunisia, were examined. Flowers are composed of five oblong tepals. P. equisetiforme and P. aviculare have whitish-pink distylous flowers with dimorphism of style, filament and anther height, pollen diameter and stigma size. In contrast, P. maritimum shows white homostylous flowers. The floral vasculature showed that the tepals are inserted in one whorl and their traces arise independently in 3+2 manner. The eight stamens are arranged in a 5+3 manner and the staminal bundles arise independently in the two whorls. The epidermis and endothecium cells width were higher in P. maritimum and the lowest endothecium width was observed in P. aviculare. Polygonum aviculare and P. equisetiforme showed circular pollen with shallow colpi and trilobite pollen shape with deep colpi, while P. maritimum rarely showed shallow colpi. The ovule is anatropous with basal placentation in P. equisetiforme and P. aviculare and apical placentation in P. maritimum. The young seed coat was formed by an endotesta with thick-walled cells, a mesotesta and exotesta with thin-walled cells and a tegmen composed of radially elongated cells. The fruits of the studied species are trigonous with ovate-lanceolate shape. In P. aviculare, the exocarp is thicker compared to the two other species, in P. equisetiforme, the mature exocarp consists of smaller rectangular cells with narrow cavities, and in P. maritimum showed a thinner exocarpIn conclusion, P. equisetiforme and P. aviculare are a typically distylous species from the morphological point of view and we discussed the significance of heterostyly in Polygonaceae. From this first morpho-anatomical study of Polygonum species in North Africa, we can conclude mainly that there is no significant difference between P. equisetiforme var. graecum and var. peyerinhoffi supporting a taxonomic grouping of these two varieties.

Transgenerational effects of parental light environment on progeny competitive performance and lifetime fitness.

Plant and animal parents may respond to environmental conditions such as resource stress by altering traits of their offspring via heritable non-genetic effects. While such transgenerational plasticity can result in progeny phenotypes that are functionally pre-adapted to the inducing environment, it is unclear whether such parental effects measurably enhance the adult competitive success and lifetime reproductive output of progeny, and whether they may also adversely affect fitness if offspring encounter contrasting conditions. In glasshouse experiments with inbred genotypes of the annual plant Polygonum persicaria, we tested the effects of parental shade versus sun on (a) competitive performance of progeny in shade, and (b) lifetime reproductive fitness of progeny in three contrasting treatments. Shaded parents produced offspring with increased fitness in shade despite competition, as well as greater competitive impact on plant neighbours. Inherited effects of parental light conditions also significantly altered lifetime fitness: parental shade increased reproductive output for progeny in neighbour and understorey shade, but decreased fitness for progeny in sunny, dry conditions. Along with these substantial adaptive and maladaptive transgenerational effects, results show complex interactions between genotypes, parent environment and progeny conditions that underscore the role of environmental variability and change in shaping future adaptive potential. This article is part of the theme issue 'The role of plasticity in phenotypic adaptation to rapid environmental change'.

Tissue-specific and intracellular localization of indican synthase from Polygonum tinctorium.

The plant Polygonum tinctorium produces the secondary metabolite indican (indoxyl-ß-D-glucoside), a precursor of the blue dye indigo. P. tinctorium synthesizes indican through the actions of the UDP-glucosyltransferase (UGT), indican synthase. Herein, we partially purified an indican synthase from the leaves and subsequently performed peptide mass fingerprinting analysis. Consequently, we identified a fragment that was homologous to a UDP-glucosyltransferase 72B (UGT72B) family member. We named it PtIgs (P. tinctoriumindoxyl-ß-D-glucoside synthase) and obtained the full-length cDNA using rapid amplification of the cDNA ends. The primary structure of PtIGS, which PtIgs encoded, showed high identity with indican synthases (ItUGT1 and ItUGT2) from Indigofera tinctoria (Inoue et al., 2017). Moreover, in expression analyses of P. tinctorium, PtIGS mRNA was virtually found only in the leaves, was most highly expressed in the 1st leaves, and decreased with leaf age. Because PtIGS expression tended to reflect indican contents and synthesis activities, we concluded that PtIGS functions as an indican synthase in plant cells. To examine intracellular localization of PtIGS, crude leaf extracts were separated into cytosol and microsome fractions, and found PtIGS in the cytosol and in microsome fractions. Furthermore, microsomal PtIGS was soluble in the presence of detergents and urea and was strongly associated with membranes. Finally, we confirmed endoplasmic reticulum (ER) membrane localization of PtIGS using ultracentrifugation with a sucrose density gradient. These data suggest that PtIGS interacts with some kind of proteins on ER membranes to certainly carry out a delivery of substrate.

Functional Characterisation of New Sesquiterpene Synthase from the Malaysian Herbal Plant, Polygonum Minus.

Polygonum minus (syn. Persicaria minor) is a herbal plant that is well known for producing sesquiterpenes, which contribute to its flavour and fragrance. This study describes the cloning and functional characterisation of PmSTPS1 and PmSTPS2, two sesquiterpene synthase genes that were identified from P. minus transcriptome data mining. The full-length sequences of the PmSTPS1 and PmSTPS2 genes were expressed in the E. coli pQE-2 expression vector. The sizes of PmSTPS1 and PmSTPS2 were 1098 bp and 1967 bp, respectively, with open reading frames (ORF) of 1047 and 1695 bp and encoding polypeptides of 348 and 564 amino acids, respectively. The proteins consist of three conserved motifs, namely, Asp-rich substrate binding (DDxxD), metal binding residues (NSE/DTE), and cytoplasmic ER retention (RxR), as well as the terpene synthase family N-terminal domain and C-terminal metal-binding domain. From the in vitro enzyme assays, using the farnesyl pyrophosphate (FPP) substrate, the PmSTPS1 enzyme produced multiple acyclic sesquiterpenes of ß-farnesene, α-farnesene, and farnesol, while the PmSTPS2 enzyme produced an additional nerolidol as a final product. The results confirmed the roles of PmSTPS1 and PmSTPS2 in the biosynthesis pathway of P. minus, to produce aromatic sesquiterpenes.

Employing a biochemical protecting group for a sustainable indigo dyeing strategy.

Indigo is an ancient dye uniquely capable of producing the signature tones in blue denim; however, the dyeing process requires chemical steps that are environmentally damaging. We describe a sustainable dyeing strategy that not only circumvents the use of toxic reagents for indigo chemical synthesis but also removes the need for a reducing agent for dye solubilization. This strategy utilizes a glucose moiety as a biochemical protecting group to stabilize the reactive indigo precursor indoxyl to form indican, preventing spontaneous oxidation to crystalline indigo during microbial fermentation. Application of a ß-glucosidase removes the protecting group from indican, resulting in indigo crystal formation in the cotton fibers. We identified the gene coding for the glucosyltransferase PtUGT1 from the indigo plant Polygonum tinctorium and solved the structure of PtUGT1. Heterologous expression of PtUGT1 in Escherichia coli supported high indican conversion, and biosynthesized indican was used to dye cotton swatches and a garment.

Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.

KEY MESSAGE: Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

DNA methylation mediates genetic variation for adaptive transgenerational plasticity.

Environmental stresses experienced by individual parents can influence offspring phenotypes in ways that enhance survival under similar conditions. Although such adaptive transgenerational plasticity is well documented, its transmission mechanisms are generally unknown. One possible mechanism is environmentally induced DNA methylation changes. We tested this hypothesis in the annual plant Polygonum persicaria, a species known to express adaptive transgenerational plasticity in response to parental drought stress. Replicate plants of 12 genetic lines (sampled from natural populations) were grown in dry versus moist soil. Their offspring were exposed to the demethylating agent zebularine or to control conditions during germination and then grown in dry soil. Under control germination conditions, the offspring of drought-stressed parents grew longer root systems and attained greater biomass compared with offspring of well-watered parents of the same genetic lines. Demethylation removed these adaptive developmental effects of parental drought, but did not significantly alter phenotypic expression in offspring of well-watered parents. The effect of demethylation on the expression of the parental drought effect varied among genetic lines. Differential seed provisioning did not contribute to the effect of parental drought on offspring phenotypes. These results demonstrate that DNA methylation can mediate adaptive, genotype-specific effects of parental stress on offspring phenotypes.

Evolutionary potential for increased invasiveness: High-Performance Polygonum cespitosum genotypes are competitively superior in full sun.

PREMISE OF STUDY: The presence of genetic variation for traits that contribute to ecological range expansion can provide the potential for introduced taxa to evolve greater invasiveness. Genotypes that contribute to the spread of introduced range populations must have the ability to maintain fitness under changing environmental stress and competitive intensity. Previously, we identified a subset of genotypes in populations of the invasive annual Polygonum cespitosum that express consistently high reproductive fitness in diverse (shaded, dry, and resource-rich) conditions. Here, we investigated whether these broadly adaptive (High-Performance) genotypes also show a competitive advantage over conspecifics in full sun and/or shade. METHODS: We grew a population-balanced sample of 13 High-Performance and 13 'Control' genotypes in intraspecific competitive arrays, comprising all four possible combinations of High-Performance vs. Control target plants and competitive backgrounds, in both full sun and shaded glasshouse environments. KEY RESULTS: In full sun, High-Performance genotypes (1) better maintained growth and reproductive output despite competition and (2) more strongly suppressed growth and reproduction of target plants. However, genotypes did not differ significantly in shade. CONCLUSIONS: Competitive superiority in open conditions may contribute to increasing predominance of these broadly adapted genotypes in introduced-range Polygonum cespitosum populations, and hence to the evolution of greater invasiveness. This study provides insight into the role of genotypic variation for ecological traits in the range expansion of a contemporary plant invader. It also highlights how such variation can be differently expressed in alternative environments (gene by environment interaction).

Limits to Future Adaptation in the Invasive Plant Polygonum cespitosum: Expression of Functional and Fitness Traits at Elevated CO2.

For organisms to adapt to future environments, they must both evolve appropriate functional responses and phenotypically express those responses under future climatic and CO2 conditions. We examined these 2 components of future adaptation in an invasive annual plant (Polygonum cespitosum) by performing a "resurrection" experiment under field conditions simulating a future environment. Resurrection experiments reveal recent evolution by comparing genotypes from natural populations sampled across a multigeneration interval. We collected genotypes from the same 3 North American populations in 1994 and 2005 and raised inbred lines from these collections under free air CO2 enrichment to examine functional and fitness traits expressed in hot, dry conditions at both ambient and elevated CO2 (N = 295 plants). The species has rapidly evolved in its introduced range to increase photosynthetic rate (collection year effect P ≤ 0.011) and delay senescence (P = 0.017) under full-sun, dry field conditions, but these adaptive changes were not expressed when the field environment included elevated CO2 (within-treatment year effect P ≥ 0.20 for both traits). Populations showed different levels of reproductive output and its genetic variance in these novel, stressful conditions. These findings illustrate constraints on evolutionary adaptation to predicted future conditions at both the species and population levels.

An ideal weed: plasticity and invasiveness in Polygonum cespitosum.

The introduced Asian plant Polygonum cespitosum has only recently become invasive in northeastern North America, spreading into sunny as well as shaded habitats. We present findings from a multiyear case study of this ongoing species invasion, drawing on field environmental measurements, glasshouse plasticity and resurrection experiments, and molecular genetic (microsatellite) data. We focus in particular on patterns of individual phenotypic plasticity (norms of reaction), their diversity within and among populations in the species' introduced range, and their contribution to its potential to evolve even greater invasiveness. Genotypes from introduced-range P. cespitosum populations have recently evolved to express greater adaptive plasticity to full sun and/or dry conditions without any loss of fitness in shade. Evidently, this species may evolve the sort of "general-purpose genotypes" hypothesized by Herbert Baker to characterize an "ideal weed." Indeed, we identified certain genotypes capable of extremely high reproductive output across contrasting conditions, including sunny, shaded, moist, and dry. Populations containing these high-performance genotypes had consistently higher fitness in all glasshouse habitats; there was no evidence for local adaptive differentiation among populations from sunny, shaded, moist, or dry sites. Norm of reaction data may provide valuable insights to invasion biology: the presence of broadly adaptive, high-performance genotypes can promote a species' ecological spread while providing the fuel for increased invasiveness to evolve.

Genetic diversity and population structure in Polygonum cespitosum: insights to an ongoing plant invasion.

Molecular markers can help elucidate how neutral evolutionary forces and introduction history contribute to genetic variation in invaders. We examined genetic diversity, population structure and colonization patterns in the invasive Polygonum cespitosum, a highly selfing, tetraploid Asian annual introduced to North America. We used nine diploidized polymorphic microsatellite markers to study 16 populations in the introduced range (northeastern North America), via the analyses of 516 individuals, and asked the following questions: 1) Do populations have differing levels of within-population genetic diversity? 2) Do populations form distinct genetic clusters? 3) Does population structure reflect either geographic distances or habitat similarities? We found low heterozygosity in all populations, consistent with the selfing mating system of P. cespitosum. Despite the high selfing levels, we found substantial genetic variation within and among P. cespitosum populations, based on the percentage of polymorphic loci, allelic richness, and expected heterozygosity. Inferences from individual assignment tests (Bayesian clustering) and pairwise FST values indicated high among-population differentiation, which indicates that the effects of gene flow are limited relative to those of genetic drift, probably due to the high selfing rates and the limited seed dispersal ability of P. cespitosum. Population structure did not reflect a pattern of isolation by distance nor was it related to habitat similarities. Rather, population structure appears to be the result of the random movement of propagules across the introduced range, possibly associated with human dispersal. Furthermore, the high population differentiation, genetic diversity, and fine-scale genetic structure (populations founded by individuals from different genetic sources) in the introduced range suggest that multiple introductions to this region may have occurred. High genetic diversity may further contribute to the invasive success of P. cespitosum in its introduced range.

Functional characterization of sesquiterpene synthase from Polygonum minus.

Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of ß -sesquiphellandrene.

Sexual reproduction of Japanese knotweed (Fallopia japonica s.l.) at its northern distribution limit: new evidence of the effect of climate warming on an invasive species.

PREMISE OF THE STUDY: In response to climate warming, plant species may shift their distribution northward, but such a process is slow and hard to detect. Alternatively, phenological changes (earlier flowering) are expected as first adaptations for populations located near their distribution limit. That could be the case for the invasive Japanese knotweed (Fallopia japonica s.l., including the hybrid Bohemian knotweed F. ×bohemica). We hypothesized that climate warming now allows the species to produce viable seeds in the northernmost populations. METHODS: Seeds were collected along a 550 km long transect in Quebec, Canada, and tested for germination. The genetic diversity of a population was determined using polymorphic microsatellite markers to verify whether the species is actually producing new individuals through sexual reproduction. KEY RESULTS: Japanese knotweed produces, in Quebec, a large number of seeds with a high germination rate (up to 93%). The geographical limit for viable seed production in North America has been extended to Quebec City, about 500 km north of the formerly reported limit. Bohemian knotweeds are genetically diverse, while true Japanese knotweeds all share a common multilocus genotype. This suggests that Bohemian knotweed stands mostly arose from seed, while true Japanese knotweeds result only from the propagation of rhizome or stem fragments. CONCLUSIONS: The effect of climate change is already palpable on the phenology of invasive plant species at their northern distribution limit. Bohemian knotweed, which until recently was rare in Quebec, could rapidly spread in the near future with the help of an additional diaspore type (seeds).

Production of anthraquinones, phenolic compounds and biological activities from hairy root cultures of Polygonum multiflorum Thunb.

Polygonum multiflorum Thunb. is a highly important medicinal plant producing anthraquinones (emodin and physcion) and phenolic compounds which has pharmaceutical use. In vitro seedling explants such as roots, internodals, nodals and leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed roots were induced from internodals and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 30 g/l sucrose showed highest accumulation of biomass (99.05 g/l FW [fresh weight] and 10.95 g/l DW [dry weight]) and highest production of anthraquinones content (emodin 211.32 µg/g DW and physcion 353.23 µg/g DW) were observed at 20 days. Nearly 9.5-fold increment of biomass was evident in suspension cultures at 20 days of culture and hairy root biomass produced in suspension cultures possessed 3.7- and 3.5-fold higher content of emodin and physcion, respectively, when compared with the untransformed control roots. MS basal liquid medium was superior for the growth of hairy roots and production of anthraquinones compared with other culture media evaluated (SH, B5 and N6), with MS-basal liquid medium supplemented with 30 g/l sucrose was optimal for secondary metabolite production. A total of 23 polyphenolic compounds were identified and quantified from P. multiflorum untransformed and hairy roots, which includes hydroxybenzoic acids, hydroxycinnamic acids, flavonols and other groups of phenolic compounds. The ultra-performance liquid chromatography (UPLC) analysis of the phenolic compounds profile revealed that pyrogallol, hesperidin, naringenin and formononetin were higher in hairy roots compared to untransformed roots. The total phenolics, flavonoids content, antioxidant and antimicrobial activity was high in hairy roots compared to untransformed roots. This is the first report for the production of anthraquinones (emodin and physcion), phenolic compounds and biological activities from hairy root cultures of P. multiflorum.

[Study on sequence characterized amplified region (SCAR) markers of Polygonum capitatum].

OBJECTIVE: To establish sequence characterized amplified region markers of Polygonum capitatum. METHOD: The random primer was screened through RAPD to obtain the specific RAPD marker band, and the band was separated, extracted, cloned and sequenced. The specific primers were designed for conventional PCR reaction on the basis of the specific band, and the SCAR marker was acquired. RESULT: Screening from 50 RAPD primer, only C29 primer had 2 specific bands could distinguish P. capitatum from P. nepalense, then 4 pairs of specific primers were designed based on the 2 sequences of RAPD marker bands, and only 1 pair primer (Z1-2) was successfully converted into SCAR marker after repeated tests. CONCLUSION: The Z1-2 primer, could be used as an effective SCAR mark to identify Z300 DNA for P. capitatum. The SCAR mark was established and can be used as a molecular marker to distinguish P. capitatum from P. nepalense