Molecular bases for the stronger plastic response to high nitrate in the invasive plant Xanthium strumarium compared with its native congener.

MAIN CONCLUSION: High expressions of nitrate use and photosynthesis-related transcripts contribute to the stronger plasticity to high nitrate for the invader relative to its native congener, which may be driven by hormones. Strong phenotypic plasticity is often considered as one of the main mechanisms underlying exotic plant invasions. However, few studies have been conducted to investigate the related molecular mechanisms. Here, we determined the differences in the plastic responses to high nitrate between the invasive plant X. strumarium and its native congener, and the molecular bases by transcriptome analysis and quantitative real-time PCR validation. Our results showed that the invader had higher plasticity of growth, nitrogen accumulation and photosynthesis in responses to high nitrate than its native congener. Compared with its congener, more N utilization-related transcripts, including nitrate transporter 1/peptide transporter family 6.2 and nitrate reductase 1, were induced by high nitrate in the root of X. strumarium, improving its N utilization ability. More transcripts coding for photosynthetic antenna proteins were also induced by high nitrate in the shoot of X. strumarium, enhancing its photosynthesis. Hormones may be involved in the regulation of the plastic responses to high nitrate in the two species. Our study contributes to understanding the molecular mechanisms underlying the stronger plasticity of the invader in responses to high nitrate, and the potential function of plant hormones in these processes, providing bases for precise control of invasive plants using modern molecular techniques.

[Multiplex PCR method for simultaneous identification of Xanthii Fructus and its adulterants].

The purpose of this study is to establish a molecular method to identify Xanthii Fructus and two adulterants, the fruits of Xanthium mongolicum and X. italicum. Xanthii Fructus is the fruit of X. sibiricum, which is a Chinese herbal medicine used clinically to treat allergic rhinitis. The fruits of X. mongolicum and X. italicum have strong morphological similarities with Xanthii Fructus, while their safety of medication cannot be guaranteed. The genomes of X. sibiricum, X. mongolicum, and X. italicum were sequenced, which generated sequences of 2.21, 2.24, and 2.54 Gb, respectively. Based on the 76 specific contigs screened out by BLASTN and Bowtie 2, the corresponding primers were designed by Primer 5.0. Three pairs of primers with stable amplification efficiency and good reproducibility were screened out to establish a multiplex PCR method based on the PCR amplification results. Further, the annealing temperature, the amount of DNA template, the number of cycles, different DNA polymerases, and different PCR thermal cyclers were optimized. Fragments of 262 bp and 458 bp from X. sibiricum, 260, 454, and 927 bp from X. mongolicum, and 260 bp and 926 bp from X. italicum were amplified under the following conditions: the annealing temperature of 52 ℃, 35 cycles, 30 ng template DNA. Then, the established method was used to detect 18 samples of X. sibiricum, 17 samples of X. mongolicum, and 12 samples of X. italicum. The results showed that all the samples had positive results, which were consistent with the morphological identification results, thus proving the stability and reliability of the established method. Combining genome sequencing technology and multiplex PCR method to identify Xanthii Fructus and its adulterants can not only obtain the difference in genetic background but also facilitate the design of reliable primers. The multiplex PCR have high specificity and repeatability, providing a new method for the molecular identification of Xanthii Fructus.

Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum.

Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been reported, the remaining GAOs corresponding to various STLs' biosynthesis pathways remain unidentified. In this study, 68,199 unigenes were studied in a de novo transcriptome assembly of X. sibiricum fruits. By comparison with previously published GAO sequences, two candidate X. sibiricum GAO gene sequences, XsGAO1 (1467 bp) and XsGAO2 (1527 bp), were identified, cloned, and predicted to encode 488 and 508 amino acids, respectively. Their protein structure, motifs, sequence similarity, and phylogenetic position were similar to those of other GAO proteins. They were most strongly expressed in fruits, according to a quantitative real-time polymerase chain reaction (qRT-PCR), and both XsGAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two XsGAO genes were cloned into the expression vector for eukaryotic expression in Saccharomyces cerevisiae, and the enzyme reaction products were detected by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) methods. The results indicated that both XsGAO1 and XsGAO2 catalyzed the two-step conversion of germacrene A (GA) to GAA, meaning they are unlike classical GAO enzymes, which catalyze a three-step conversion of GA to GAA. This cloning and functional study of two GAO genes from X. sibiricum provides a useful basis for further elucidation of the STL biosynthesis pathway in X. sibiricum.

Characteristics of the completed chloroplast genome sequence of Xanthium spinosum: comparative analyses, identification of mutational hotspots and phylogenetic implications.

BACKGROUND: The invasive species Xanthium spinosum has been used as a traditional Chinese medicine for many years. Unfortunately, no extensive molecular studies of this plant have been conducted. RESULTS: Here, the complete chloroplast (cp) genome sequence of X. spinosum was assembled and analyzed. The cp genome of X. spinosum was 152,422 base pairs (bp) in length, with a quadripartite circular structure. The cp genome contained 115 unique genes, including 80 PCGs, 31 tRNA genes, and 4 rRNA genes. Comparative analyses revealed that X. spinosum contains a large number of repeats (999 repeats) and 701 SSRs in its cp genome. Fourteen divergences (Π > 0.03) were found in the intergenic spacer regions. Phylogenetic analyses revealed that Parthenium is a sister clade to both Xanthium and Ambrosia and an early-diverging lineage of subtribe Ambrosiinae, although this finding was supported with a very weak bootstrap value. CONCLUSION: The identified hotspot regions could be used as molecular markers for resolving phylogenetic relationships and species identification in the genus Xanthium.

Genetic diversity and genetic differentiation of invasive weed Xanthium italicum in China.

Xanthium italicum is an aggressive weed found worldwide. Despite several ecological, morphological, and physiological research on its invasion mechanism, the mechanism of its successful invasion has not been revealed from the viewpoint of population genetics. Thus, we aimed to evaluate the genetic variation within and among populations of the alien invasive weed X. italicum in China, and to provide a theoretical basis for its invasion mechanism. For that, we employed inter-simple sequence repeat (ISSR) markers to explore the genetic diversity and genetic differentiation of 185 individuals sampled from 10 populations. Eight selected primers yielded a total of 76 bright and discernible bands. X. italicum showed an intermediate genetic diversity at the population level (percentage of polymorphic loci (PPL) = 60.26%, Nei's genetic diversity (H) = 0.2098, Shannon's information index (I) = 0.3129). However, the genetic diversity at the species level was significantly high (PPL = 100%; H = 0.3673; I = 0.5425). The coefficient of gene differentiation (GST, 41.4%) and analysis of molecular variance showed that genetic differentiation mainly occurred within populations. The estimated gene flow (Nm, 0.7085) and Mantel test indicated that genetic differentiation in the populations may primarily come from genetic drift and anthropogenic activities. Our results revealed the high genetic diversity of X. italicum, which may help explain its invasion success in China. This knowledge may contribute to the efforts for decreasing and eventually stopping X. italicum invasion in China.

Xanthium italicum est une plante envahissante trouvée dans le monde entier. En dépit de quelques recherches écologiques, morphologiques et physiologiques à propos de son mécanisme d'invasion, le mécanisme de son invasion réussie n'a pas encore été révélé du point de vue de la génétique démographique. Donc, nous avons visé à évaluer la variation génétique au sein de et parmi les populations de plante exotique envahissante X. italicum en Chine, et à offrir une base théorique à son mécanisme d'invasion. À cet effet, nous avons employé des marqueurs des répétitions de séquences inter-simples (ISSR) afin d'explorer la diversité génétique et la différenciation génétique de 185 individus échantillonnés à partir de 10 populations. Huit amorces sélectionnées ont donné un total de 76 bandes brillantes et perceptibles. X. italicum a montré une diversité génétique intermédiaire au niveau de la population (pourcentage de loci polymorphe (PPL) = 60.26%, la diversité génétique de Nei (H) = 0.2098, l'indice d'information de Shannon (I) = 0.3129). Toutefois, la diversité génétique était considérablement élevée au niveau des espèces (PPL = 100% ; H = 0.3673 ; I = 0.5425). Tant le coefficient de différenciation génétique (GST, 41.4%) que l'analyse de la variance moléculaire ont reflété que la différenciation génétique se produisait principalement au sein des populations. En fonction du flux génétique estimé (Nm, 0.7085) et du test Mantel, la différenciation génétique au sein des populations pourrait provenir principalement de la dérive génétique et des activités anthropiques. Nos résultats ont révélé la haute diversité génétique de X. italicum, cela peut aider à expliquer son succès d'invasion en Chine. Ces connaissances pourraient contribuer à la réduction et à l'arrêt éventuel de l'invasion de X. italicum en Chine.

Metabolomic differences between invasive alien plants from native and invaded habitats.

Globalization facilitated the spread of invasive alien species (IAS), undermining the stability of the world's ecosystems. We investigated the metabolomic profiles of three IAS species: Chromolaena odorata (Asteraceae) Datura stramonium (Solanaceae), and Xanthium strumarium (Asteraceae), comparing metabolites of individual plants in their native habitats (USA), to their invasive counterparts growing in and around Kruger National Park (South Africa, ZA). Metabolomic samples were collected using RApid Metabolome Extraction and Storage (RAMES) technology, which immobilizes phytochemicals on glass fiber disks, reducing compound degradation, allowing long-term, storage and simplifying biochemical analysis. Metabolomic differences were analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) of samples eluted from RAMES disks. Partial Least Squares-Discriminant Analysis (PLS-DA) of metabolomes of individual plants allowed statistical separation of species, native and invasive populations of each species, and some populations on the same continent. Invasive populations of all species were more phytochemically diverse than their native counterparts, and their metabolomic profiles were statistically distinguishable from their native relatives. These data may elucidate the mechanisms of successful invasion and rapid adaptive evolution of IAS. Moreover, RAMES technology combined with PLS-DA statistical analysis may allow taxonomic identification of species and, possibly, populations within each species.

Phylogenetic Analysis and Molecular Characterization of Xanthium sibiricum Using DNA Barcoding, PCR-RFLP, and Specific Primers.

The fruits of Xanthium sibiricum have been widely used in traditional Chinese medicine for the treatment of nasal sinusitis and headaches. The genus Xanthium (cocklebur) is a taxonomically complex genus. Different taxonomic concepts have been proposed, some including several species, others lumping the different taxa in a few extremely polymorphic species. Due to the morphological similarities between species, the correct authentication of X. sibiricum is very difficult. Therefore, we established a polymerase chain reaction-restriction fragment length polymorphism method and diagnostic PCR based on nuclear internal transcribed spacer and chloroplast trnQ-rps16 barcodes to differentiate X. sibirium from related species.Results from the phylogenetic analyses based on sequence information from four marker regions (plastidal psbA-trnH and trnQ-rps16 and nuclear ITS and D35) support those taxonomic concepts accepting a reduced number of species, as four to five major clades are revealed in the phylogenetic reconstructions. X. sibiricum, together with some accessions from closely related taxa, is always supported as monophyletic, constituting a well-defined genetic entity. Allele-specific primer pairs for ITS and trnQ-rps16 were designed to amplify diagnostic products from the genomic DNA of X. sibiricum. Specific PCR in combination with digestion using the restriction enzyme MseI allowed for the identification of X. sibiricum by producing specific restriction patterns. The results demonstrate that the applied techniques provide effective and accurate authentication of X. sibiricum.

Identification and Functional Characterization of Sesquiterpene Synthases from Xanthium strumarium.

Xanthium strumarium synthesizes various pharmacologically active sesquiterpenes. The molecular characterization of sesquiterpene biosynthesis in X. strumarium has not been reported so far. In this study, the cDNAs coding for three sesquiterpene synthases (designated as XsTPS1, XsTPS2 and XsTPS3) were isolated using the X. strumarium transcriptome that we recently constructed. XsTPS1, XsTPS2 and XsTPS3 were revealed to have primary activities forming germacrene D, guaia-4,6-diene and germacrene A, respectively, by either ectopic expression in yeast cells or purified recombinant protein-based in vitro assays. Quantitative real-time PCRs and metabolite analysis for the different plant parts showed that the transcript abundance of XsTPS1-XsTPS3 is consistent with the accumulation pattern of their enzymatic products, supporting their biochemical functions in vivo. In particular, we discovered that none of the XsTPS2 product, guaia-4,6-diene, can be detected in one of the X. strumarium cultivars used in this study (it was named the Hubei-cultivar), in which a natural deletion of two A bases in the XsTPS2 cDNA disrupts its activity, which further confirmed the proposed biochemical role of XsTPS2 in X. strumarium in vivo.

Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

The medicinal plant Xanthium strumarium L. (X. strumarium) is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs) are a class of 21-24 nucleotide (nt) non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

Correlation between the genetic diversity and variation of total phenolic acids contents in Fructus Xanthii from different populations in China.

Fructus Xanthii (Cang-Er-Zi) is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. For the effective quality control of its medicinal values, reflected by chemical variation patterns, in addition to the relationship with genetic diversity, analyses based on UV spectrophotometry, HPLC fingerprinting and inter-simple sequence repeat (ISSR) molecular markers were carried out, involving 16 Xanthium populations from different locations in China. The HPLC data showed considerable variation of chemical constituents among the 16 Xanthium populations, and they were classified to three chemotypes by hierarchical clustering analysis. Abundant genetic diversity was detected among the Xanthium populations, which were also clustered into three groups based on their ISSR data and varied according to different species. Combining the genetic divergence and chemical differences showed an important result that, in the two chemotypes, the higher contents of total phenolic acids (TPA) in Fructus Xanthii showed greater genetic diversity (I). We suggest that genetic diversity affects the contents of TPA. Since variable phenolic acid contents may affect therapeutic efficacy, it is important to point out that combining the use of genetic base with chemotype will help control the favourable chemotypes and breed new cultivars with more desirable chemical constituents.