Assembly and Characterization of the Mitochondrial Genome of Fallopia aubertii (L. Henry) Holub.

BACKGROUND: Fallopia aubertii (L. Henry) Holub is a perennial semi-shrub with both ornamental and medicinal value. The mitochondrial genomes of plants contain valuable genetic traits that can be utilized for the exploitation of genetic resources. The parsing of F. aubertii mitochondrial genome can provide insight into the role of mitochondria in plant growth and development, metabolism regulation, evolution, and response to environmental stress. METHODS: In this study, we sequenced the mitochondrial genome of F. aubertii using the Illumina NovaSeq 6000 platform and Nanopore platform. We conducted a comprehensive analysis of the mitochondrial genome of F. aubertii, which involved examining various aspects such as gene composition, repetitive sequences, RNA editing sites, phylogeny, and organelle genome homology. To achieve this, we employed several bioinformatics methods including sequence alignment analysis, repetitive sequence analysis, phylogeny analysis, and more. RESULTS: The mitochondrial genome of F. aubertii has 64 genes, including 34 protein-coding genes (PCGs), three rRNAs, and 27 tRNAs. There were 77 short tandem repeat sequences detected in the mitochondrial genome, five tandem repeat sequences identified by Tandem Repeats Finder (TRF), and 50 scattered repeat sequences observed, including 22 forward repeat sequences and 28 palindrome repeat sequences. A total of 367 RNA coding sites were predicted in PCGs, with the highest number (33) found within ccmB. Ka/Ks values estimated for mitochondrial genes of F. aubertii and three closely related species representing Caryophyllales were less than 1 for most of the genes. The maximum likelihood evolutionary tree showed that F. aubertii and Nepenthes ×ventrata are most closely related. CONCLUSIONS: In this study, we obtained basic information on the mitochondrial genome of F. aubertii and this study investigated repeat sequences and homologous segments, predicted RNA editing sites, and utilized the Ka/Ks ratio to estimate the selection pressure on mitochondrial genes of F. aubertii. We also discussed the systematic evolutionary position of F. aubertii based on mitochondrial genome sequences. Our study revealed variations in the sequence and structure of mitochondrial genomes in Caryophyllales. These findings are of great significance for identifying and improving valuable plant traits and serve as a reference for future molecular studies of F. aubertii.

Fallopia japonica and Fallopia × bohemica extracts cause ultrastructural and biochemical changes in root tips of radish seedlings.

Japanese knotweed (Fallopia japonica) and Bohemian knotweed (Fallopia × bohemica) are invasive plants that use allelopathy as an additional mechanism for colonization of the new habitat. Allelochemicals affect the growth of roots of neighboring plants. In the present study, we analyze the early changes associated with the inhibited root growth of radish seedlings exposed to aqueous extracts of knotweed rhizomes for 3 days. Here, we show that cells in the root cap treated with the knotweed extracts exhibited reduced cell length and displayed several ultrastructural changes, including the increased abundance of dilated ER cisternae filled with electron-dense material (ER bodies) and the accumulation of dense inclusions. Moreover, mitochondrial damage was exhibited in the root cap and the meristem zone compared to the non-treated radish seedlings. Furthermore, malfunction of the intracellular redox balance system was detected as the increased total antioxidative capacity. We also detected increased metacaspase-like proteolytic activities and, in the case of 10% extract of F. japonica, increased caspase-like proteolytic activities. These ultrastructural and biochemical effects could be the reason for the more than 60% shorter root length of treated radish seedlings compared to controls.

The exploration of neuraminidase inhibitory activity on Fallopia denticulata, an ethnic herb in China.

This study was designed to explore the bioactive ingredients in the extracts of Fallopia denticulata (C.C. Huang) Holub, a medicinal plant grown in China, which exhibits the best neuraminidase (NA) inhibition activity. Three fractions of ethyl acetate, ethanol, and water were tested on NA inhibition assay, and the best one was conducted by ultra-performance liquid chromatography-time-of-flight mass spectrometry in the negative and positive modes to analyze the metabolic components. The results revealed the identification of the following 21 compounds: 3 organic acids, 11 flavonoids, 1 coumarin, and 6 others, such as ß-daucosterol, gallic acid, and syringic acid, of which 12 compounds were discovered for the first time in F. denticulata. In addition, we used the molecular docking technique to support the anti-NA activity of each compound in the best extract. The results confirmed that the two better bioactive compounds were (-)-epicatechin gallate and (+)-catechin. Therefore, F. denticulata could be used as a potential material for new anti-influenza drugs.

Responses of the species complex Fallopia × bohemica to single-metal contaminations to Cd, Cr or Zn: growth traits, metal accumulation and secondary metabolism.

Plant responses to heavy metals and their storage constitute a crucial step to understand the environmental impacts of metallic trace elements (MTEs). In controlled experiments, we previously demonstrated the tolerance and resilience of Japanese knotweed to soil artificial polymetallic contamination. Using the same experimental design, we tested here the effect of three individual MTEs on Fallopia × bohemica performance traits. Rhizome fragments from three different sites (considered as distinct morphotypes) were grown in a greenhouse for 1 month on a prairial soil artificially contaminated with either Cd, Cr (VI) or Zn at concentrations corresponding to relatively highly polluted soils. Our results confirmed the high tolerance of Bohemian knotweed to metal stress, though, plant response to MTE pollution was dependant on MTE identity. Bohemian knotweed was stimulated by Cr (VI) (increased root and aerial masses), did not display any measurable change in performance traits under Cd at the high dose of 10 mg kg-1, and uptook all MTEs in its rhizome, but only Zn was transferred to its aerial parts. We also highlighted changes in root secondary metabolism that were more accentuated with Zn, including the increase of anthraquinone, stilbene and biphenyl derivatives. These results compared to multi-contamination experiments previously published suggest complex interactions between metals and plant, depending principally on metal identity and also suggest a potential role of soil microbes in the interactions.

Landscape perceptions and social representations of Fallopia spp. in France.

Choices have to be made to manage invasive species because eradication often is not possible. Both ecological and social factors have to be considered to improve the efficiency of management plans. We conducted a social study on Fallopia spp., a major invasive plant taxon in Europe, including (1) a survey on the perception of a landscape containing Fallopia spp. using a photoquestionnaire and (2) an analysis of the social representations of Fallopia spp. of managers and users in one highly invaded area and one less invaded area. The respondents to the photoquestionnaire survey appreciated the esthetics of the landscapes less when tall Fallopia spp. were present. Few people were able to identify and name the plant, and this knowledge negatively affected the appreciation of the photos containing Fallopia spp. The central core of the social representation of Fallopia spp. was composed of the invasive status of the plant, its density, and its ecological impacts. The peripheral elements of the representation depended on the people surveyed. The users highlighted the natural aspect whereas the managers identified the need for control. In the invaded area, the managers qualified the species as "unmanageable," whereas the species was qualified as "foreign" in the less invaded area. Those results provide insights that have to be included when objectives of management plans of these species are selected.

Polyploidy and introgression in invasive giant knotweed (Fallopia sachalinensis) during the colonization of remote volcanic islands.

Invasive giant knotweed (Fallopia sachalinensis) is native to northeastern Asia. In Korea, F. sachalinensis is confined to two volcanic islands, Ullung and Dok islands, where it occurs as dodecaploids (2n = 132). We investigated the molecular variation in 104 accessions from 94 populations of F. sachalinensis and its relatives throughout their native range to elucidate the origin of these island populations. All F. sachalinensis plants on Ullung and Dok islands were uniquely dodecaploid, whereas other populations were tetraploid (2n = 44). Among the 39 cpDNA haplotypes identified, the accessions from these islands shared two unique haplotypes, and were resolved as a well-supported monophyletic clade. However, this clade was sister to a clade comprising F. japonica accessions from southwestern Japan and separated from the clade comprising F. sachalinensis from other areas; this relationship is inconsistent with morphological evidence. The monophyly of the F. sachalinensis populations on Ullung and Dok islands suggests a single colonization event. The progenitor was likely from Japan, where it possibly captured F. japonica var. japonica cpDNA via introgression. The Ullung Island populations subsequently differentiated through polyploidization and mutations post-introduction. Our results also indicate that giant knotweed in Europe and North America likely originated from northern Japan and/or Sakhalin Island.

Isolation and Determination of Phenolic Glycosides and Anthraquinones from Rhizomes of Various Reynoutria Species.

Giant knotweeds of the genus Reynoutria (syn. Fallopia)-Reynoutria japonica, Reynoutria sachalinensis, and a hybrid of them, Reynoutria x bohemica-are noxious invasive plants in Europe and North America. R. japonica is a traditional East Asian (Japan and China) drug (Polygoni cuspidati rhizoma). Recently, it has been included in European Pharmacopoeia as one of the traditional Chinese medicinal herbs. In this study, a reversed-phase high performance liquid chromatography method with diode array detector and time-of-flight mass spectrometry was developed and validated for the profiling of rhizomes from European invasive populations and Polygoni cuspidati rhizoma purchased in China. Twenty-five compounds were identified, mainly stilbenes, anthraquinones, flavan-3-ols, and phenylpropanoid esters. Tatariside B, hydropiperoside, vanicoside C, a new compound (3,6-O-di-p-coumaroyl)-ß-fructofuranosyl-(2 → 1)-(2'-O-acetyl-6'-O-feruloyl)-ß-glucopyranoside) were reported for the first time in these raw materials. Six compounds from three phytochemical classes-stilbenes: piceid and resveratrol; anthraquinones: emodin and physcion; hydroxycinnamic sucrose esters: vanicosides A and B-were quantified using the validated method. R. japonica from China contained twice as many stilbenoids than samples from Poland (piceid 14.83 mg/g dm vs. 7.45 mg/g and resveratrol 1.29 mg/g vs. 0.65 mg/g). R. sachalinensis rhizomes contained lower quantities of anthraquinones and no detectable stilbenes, which together with higher amounts of hydroxycinnamic glycosides makes it easily distinguishable from the other two. The phytochemical profile of R. x bohemica was intermediate between the two parent species.

A Survey of Genetic Variation and Genome Evolution within the Invasive Fallopia Complex.

The knotweed taxa Fallopia japonica, F. sachalinensis and their interspecific hybrid F. × bohemica are some of the most aggressive invaders in Europe and North America and they are serious threats to native biodiversity. At the same time, they constitute a unique model system for the creation of hybrids and studies of the initiation of evolutionary processes. In the presented study, we focused on (i) examining genetic diversity in selected populations of three Fallopia taxa in the invaded (Poland) and native ranges (Japan), (ii) establishing genome size and ploidy levels and (iii) identifying ribosomal DNA (rDNA)-bearing chromosomes in all of the taxa from the invaded range. We found that the genetic diversity within particular taxa was generally low regardless of their geographical origin. A higher level of clonality was observed for the Polish populations compared to the Japanese populations. Our study suggests that the co-occurrence of F. sachalinensis together with the other two taxa in the same stand may be the source of the higher genetic variation within the F. × bohemica hybrid. Some shift towards the contribution of F. japonica alleles was also observed for selected F. × bohemica individuals, which indicates the possibility of producing more advanced generations of F. × bohemica hybrids. All of the F. sachalinensis individuals were hexaploid (2n = 6x = 66; 2C = 6.01 pg), while those of F. japonica were mostly octoploid (2n = 8x = 88; 2C = 8.87 pg) and all of the F. × bohemica plants except one were hexaploid (2n = 6x = 66; 2C = 6.46 pg). Within the chromosome complement of F. japonica, F. sachalinensis and F. × bohemica, the physical mapping of the rDNA loci provided markers for 16, 13 and 10 chromosomes, respectively. In F. × bohemica, a loss of some of rDNA loci was observed, which indicates the occurrence of genome changes in the hybrid.

Comparative Histological and Phytochemical Study of Fallopia species.

Fallopia species which belong to the Polygonaceae family have several data related to their use in the Asian herbal medicine. In this work, some histological and phytochemical parameters of Fallopia japonica, F. sachalinensis, and F. x bohemica were analysed and compared. Rhizome and leaf samples were collected before, during, and after the flowering period at 3 habitats in Szombathely and 4 habitats in Baranya County, Hungary. The main histological characteristics of the stem, leaf and petiole were studied by light microscopy in cross section. Total tannin and anthraquinone contents were determined according to the official methods of the Hungarian Pharmacopoeia VIIIth (equal to the European Pharmacopoeia 6th). No species-specific markers were found in any plant part. In the rhizome, the highest tannin content was measured in Japanese knotweed, followed by Bohemian and giant knotweed in each period. The tannin content measured in each plant was higher in the leaves than in the rhizomes except F. japonica. The rhizome of F. japonica had the highest anthraquinone content before the flowering period, followed by F. x bohemica and F. sachalinensis. According to earlier and our preliminary data, Fallopia taxa are of great therapeutic promise in the future.

Cytochrome P450 CYP71AT96 catalyses the final step of herbivore-induced phenylacetonitrile biosynthesis in the giant knotweed, Fallopia sachalinensis.

The giant knotweed Fallopia sachalinensis (Polygonaceae) synthesizes phenylacetonitrile (PAN) from L-phenylalanine when infested by the Japanese beetle Popillia japonica or treated with methyl jasmonate (MeJA). Here we identified (E/Z)-phenylacetaldoxime (PAOx) as the biosynthetic precursor of PAN and identified a cytochrome P450 that catalysed the conversion of (E/Z)-PAOx to PAN. Incorporation of deuterium-labelled (E/Z)-PAOx into PAN emitted from the leaves of F. sachalinensis was detected using gas chromatography-mass spectrometry. Further, using liquid chromatography-tandem mass spectrometry, we detected the accumulation of (E/Z)-PAOx in MeJA-treated leaves. These results showed that (E/Z)-PAOx is the biosynthetic precursor of PAN. MeJA-induced mRNAs were analysed by differential expression analysis using a next-generation sequencer. Of the 74,329 contigs obtained from RNA-seq and de novo assembly, 252 contigs were induced by MeJA treatment. Full-length cDNAs encoding MeJA-induced cytochrome P450s CYP71AT96, CYP82AN1, CYP82D125 and CYP715A35 were cloned using 5'- and 3'-RACE and were expressed using a baculovirus expression system. Among these cytochrome P450s, CYP71AT96 catalysed the conversion of (E/Z)-PAOx to PAN in the presence of NADPH and a cytochrome P450 reductase. It also acted on (E/Z)-4-hydroxyphenylacetaldoxime and (E/Z)-indole-3-acetaldoxime. The broad substrate specificity of CYP71AT96 was similar to that of aldoxime metabolizing cytochrome P450s. Quantitative RT-PCR analysis showed that CYP71AT96 expression was highly induced because of treatment with MeJA as well as feeding by the Japanese beetle. These results indicate that CYP71AT96 likely contributes the herbivore-induced PAN biosynthesis in F. sachalinensis.

Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI.

Identification of B-type procyanidins in Fallopia spp. involved in biological denitrification inhibition.

Nitrogen (N) is considered as a main limiting factor in plant growth, and nitrogen losses through denitrification can be responsible for severe decreases in plant productivity. Recently, it was demonstrated that Fallopia spp. is responsible for biological denitrification inhibition (BDI) through the release of unknown secondary metabolites. Here, we investigate the secondary metabolites involved in the BDI of Fallopia spp. The antioxidant, protein precipitation capability of Fallopia spp. extracts was measured in relation to the aerobic respiration and denitrification of two bacteria (Gram positive and Gram negative). Proanthocyanidin concentrations were estimated. Proanthocyanidins in extracts were characterized by chromatographic analysis, purified and tested on the bacterial denitrification and aerobic respiration of two bacterial strains. The effect of commercial procyanidins on denitrification was tested on two different soil types. Denitrification and aerobic respiration inhibition were correlated with protein precipitation capacity and concentration of proanthocyanidins but not to antioxidant capacity. These proanthocyanidins were B-type procyanidins that inhibited denitrification more than the aerobic respiration of bacteria. In addition, procyanidins also inhibited soil microbial denitrification. We demonstrate that procyanidins are involved in the BDI of Fallopia spp. Our results pave the way to a better understanding of plant-microbe interactions and highlight future applications for a more sustainable agriculture.